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Syracuse University SURFACE Biology - Theses College of Arts and Sciences 5-2013 Structural and Functional Studies on the Role of the Retinoblastoma Binding Protein Five (RbBP5) in the Histone Methyltransferase Activity of the Mixed Lineage Leukemia (MLL1) Core Complex Melody Sanders Syracuse University Follow this and additional works at: http://surface.syr.edu/bio_thesis Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Biology Commons Recommended Citation Sanders, Melody, "Structural and Functional Studies on the Role of the Retinoblastoma Binding Protein Five (RbBP5) in the Histone Methyltransferase Activity of the Mixed Lineage Leukemia (MLL1) Core Complex" (2013). Biology - Theses. Paper 1. This Thesis is brought to you for free and open access by the College of Arts and Sciences at SURFACE. It has been accepted for inclusion in Biology Theses by an authorized administrator of SURFACE. For more information, please contact [email protected]. ABSTRACT Cells employ elaborate mechanisms to introduce structural and chemical variation into chromatin in the form of covalent post-translational modifications. Covalent modifications of histones contribute to the dynamic states of chromatin structure that govern nearly all of DNAcoupled processes such as transcription, replication, and repair. The mechanism by which covalent modifications of histones contribute to these activities remains a central question to understanding genome regulation and its dysfunction in human disease. Although there is extensive literature documenting the identification of many of the enzymes that place histone modifications, far less is known about how the enzymes are targeted and how their enzymatic activities are regulated. The long-term goal of the work in this thesis seeks to understand the mechanisms underlying the accessibility of genes in chromatin. In particular, this study focuses on identifying the underlying molecular mechanisms involved in the regulation of one such element of variation in chromatin, the methylation of lysine four on histone H3. Histone H3 lysine 4 methylation (H3K4me) is an evolutionarily conserved epigenetic mark that is correlated with transcriptional activation in eukaryotes. H3K4 methylation has been shown to be important for a number of biological processes including gene expression and DNA replication. This mark is mainly catalyzed by a group of enzymes that contain an evolutionarily conserved SET domain. The evolutionarily conserved SET domain was originally named for its presence in three Drosophila melanogaster proteins: the position effect variegation modifier SU(VAR)3-9, the polycomb group protein E(z), and the trithorax group protein (TRX) . In vertebrates, the Mixed Lineage Leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3K4 methyltransferases. The catalytic activity of MLL1 is regulated by a conserved group of proteins that include the Tryptophan-Aspartate-repeat protein-5 (WDR5), the retinoblastoma-binding protein-5 (RbBP5) and the absent small homeotic-2-like protein (Ash2L). The focus of this investigation is the RbBP5 component of the MLL1 core complex. The RbBP5 subunit of the MLL1 core complex has been shown to be required for enzymatic activity and disruption of RbBP5 is frequently observed in patients with malignant primary brain tumors. To gain insight into the functional role of RbBP5 in the regulation of the enzymatic activity of the MLL1 core complex, mutations targeting individual residues in a highly conserved stretch of amino acid residues in RbBP5 were generated. Biochemical and biophysical analyses of the variant proteins were utilized to assess the functional role of each amino acid on the intrinsic properties of RbBP5 alone or when assembled within the context of the entire complex. These studies identify several conserved aromatic residues and one acidic residue in RbBP5 required for interaction with MLL1 and the overall dimethyltransferase activity of the complex. The residues identified constitute a previously uncharacterized MLL1 interaction motif located in RbBP5. Therefore, this study provides important insights into how the RbBP5 subunit of the MLL1 core complex contributes to its overall enzymatic activity. Understanding the role that RbBP5 plays in facilitating proper H3K4 methylation may provide insight into how misregulation of RbBP5 can lead to brain tumor genesis. Structural and Functional Studies on the Role of the Retinoblastoma Binding Protein Five (RbBP5) in the Histone Methyltransferase Activity of the Mixed Lineage Leukemia (MLL1) Core Complex By Melody Sanders B.S. 2009 Syracuse University THESIS Submitted in partial fulfillment of the requirements for the degree Masters of Science in the Graduate School of Syracuse University May 2013 © Copyright 2013 Melody Sanders All Rights Reserved ACKNOWLEDGEMENTS It gives me great pleasure in expressing my gratitude to all those people who have supported me and had their contribution in making this thesis possible. However, any attempt to list the people and opportunities to which my life has been richly blessed would be like trying to count the stars in the heavens. Therefore for anyone that I may have left out, of course, the responsibility is entirely my own. I express my profound sense of reverence and gratitude to my principal investigator and advisor Dr. Michael Cosgrove. You have been patient and encouraging in times of new ideas and difficulties. I have considered you a steady influence throughout my graduate career. Your ability to select and to approach compelling research problems, your high scientific standards, and your passion for science have been indispensable to my growth not only as a scientist but also as a young woman. It is also my fortune to gratefully acknowledge Dr. Gina Lee Glauser. This feat would not have been possible without you. Thank you doesn’t seem sufficient but it is said with appreciation and respect for your support, encouragement, care, understanding, and precious guidance. Dr. Laila Kobrossy and Dr. Anamika Patel, words fail me when expressing my sincere love and appreciation. My deepest gratitude goes toward the two of you. I will always remember the generosity and encouragement the two of you expressed to me in the midst of tragedy. A long journey is easier travelled together. To Anamika, you are and will remain my best role model for a scientist, mentor, and friend. Last, but certainly not least I would like to thank Dr. Dean Stith with help towards my graduate and postgraduate affairs. I would also like to thank as well the members of my thesis v committee, Dr. Ramesh Raina, Dr. Stewart Loh, and Dr. Phil Borer for insightful discussions and suggestions. I would like to make special mention to Dr. Raina, for his constant guidance, support, motivation, and an ever-ready helping attitude during the completion of my degree. vi DEDICATION I would like to dedicate this thesis to my beautiful family, with special regards to my boys. I know times are hard for us right now and our futures are uncertain, but God willing I promise to make a way. Always remember to work hard, pride yourselves on self-discipline, and unrelenting devotion and determination on the things you want to see happen. Most importantly, love one another and NEVER stop dreaming. I would also like to dedicate this thesis to Michelle and Peter. Thank you for opening up your home to me when I had nowhere else to go. This thesis could never have reached its completion without the safe and peaceful place in your home and in your hearts that you gave me. With Love, Mel vii TABLE OF CONTENTS ABSTRACT……………………………………………………………..i TITLE PAGE……………………...……...…………………………….iii COPYRIGHT NOTICE………………………………………………...iv ACKNOWLEDGEMENTS………………………………………..........v TABLE OF CONTENTS……………………………………………...vii LIST OF FIGURES……………………………………………………..x LIST OF TABLES……………………………………………............xiii CHAPTER 1: INTRODUCTION…………………………….……….1 BACKGROUND AND SIGNIFICANCE ……….……………….2 RATIONAL OF STUDY………………………………………...25 CHAPTER 2: A CONSERVED PATCH OF AMINO ACIDS IN RbBP5 REQUIRED FOR INTERACTION WITH THE SET DOMAIN OF MLL IN THE MLL1 CORE COMPLEX……………………………………………………............29 TITLE PAGE…..……………………….…….………………….29 INTRODUCTION……...………………………………………..30 EXPERIMENTAL PROCEDURES….………………………….35 RESULTS………...…...…………………………………………39 viii DISCUSSION……….....………………………………………...82 CHAPTER 3: BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF TWO STABLE SUBDOMAINS IDENTIFIED IN THE RETINOBLASTOMA BINDING PROTEIN FIVE BY LIMITED PROTEOLYSIS.…………………..………………………………….87 TITLE PAGE…..……………………….…….………………….87 INTRODUCTION……...………………………………………..88 EXPERIMENTAL PROCEDURES….………………………….90 RESULTS………………………………………………………..93 DISCUSSION…………………………………………………..102 OVERALL CONCLUSIONS AND FUTURE PROSPECTIVES……………………………………………............104 APPENDIX..........................................................................................106 BIOGRAPHICAL DATA…………………………………………..138 ix LIST OF FIGURES FIGURE 1.1. Schematic Diagram of the effects of histone acetylation on chromatin structure……………………………………………………………………………………………4 FIGURE 1.2. Generic model of chromatin remodeling by ATP dependent protein machineries………………………………………………………………………………………..6 FIGURE 1.3. Schematic illustration of the members of the MLL1 core complex……………......................................................................................................................13 FIGURE 1.4. Structural analysis of a RbBP5 peptide bound to WDR5…………………………21 FIGURE 2.1. Schematic representation of the human retinoblastoma binding protein five (RbBP5) showing the domain architecture of recombinantly expressed full-length RbBP5……………………………………………………………………………………………32 FIGURE 2.2. Schematic representation of the human retinoblastoma binding protein five (RbBP5) showing the domain architecture of recombinantly expressed full-length and truncated RbBP5 constructs used in this investigation……………………………………………………..41 FIGURE 2.3 ClustalW2 multiple sequence alignment of the predicted globular domain of RbBP5 showing only a subgroup of residues, and high throughput methyltransferase assays showing activity of the RbBP5 protein with mutations in conserved residues identified in its predicted globular region…………………………………………………………………………………...46 FIGURE 2.4. Biophysical characterization of wild-type and mutant RbBP5 proteins by analytical ultracentrifugation………………………………………………………………………………..50 FIGURE 2.5. Biophysical characterization of wild-type and mutant RbBP5 proteins by circular dichroism………………………………………………………………………………………...52 FIGURE 2.6 MALDI-TOF mass spectrometry spectra of MWRAD and MWAD complexes………………………………………………………………………………………..54 FIGURE 2.7. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry………………………………………………………………...58 FIGURE 2.8. Biophysical characterization of wild-type WRAD and WR(W329A)AD proteins by analytical ultracentrifugation…………………………………………………………………….61 FIGURE 2.9. Biophysical characterization of wild-type WRAD and WR(F332A)AD proteins by analytical ultracentrifugation…………………………………………………………………….62 FIGURE 2.10. Biophysical characterization of wild-type WRAD and WR(E347A)AD proteins by analytical ultracentrifugation…………………………………………………………………….63 FIGURE 2.11. Biophysical characterization of wild-type MWRAD and MWR(W329A)AD proteins by analytical ultracentrifugation…………………………………………………………………65 x FIGURE 2.12. Biophysical characterization of wild-type MWRAD and MWR(F332A)AD proteins by analytical ultracentrifugation…………………………………………………………………66 FIGURE 2.13. Biophysical characterization of wild-type MWRAD and MWR(F336A)AD proteins by analytical ultracentrifugation…………………………………………………………………67 FIGURE 2.14. Biophysical characterization of wild-type MWRAD and MWR(E347A)AD proteins by analytical ultracentrifugation…………………………………………………………………68 FIGURE 2.15. Biophysical characterization of wild-type MWR(W329A)AD protein by analytical ultracentrifugation………………………………………………………………………………..69 FIGURE 2.16. Biophysical characterization of wild-type and mutant RbBP5 proteins by circular dichroism…………………………………………………………………………………………71 FIGURE 2.17. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry………………………………………………………………...73 FIGURE 2.18. Biophysical characterization of wild-type MWRAD, MWR(W329F)AD, and MWR(W329Y)AD by analytical ultracentrifugation……………………………………………….75 FIGURE 2.19. Biophysical characterization of wild-type MWRAD, and MWR(W329R)AD, by analytical ultracentrifugation…………………………………………………………………….76 FIGURE 2.20. Biophysical characterization of RbBP5 wild-type and RbBP5 (V375A) and RbBP5 (V377A) proteins……………………………………………………………………………………79 FIGURE 2.21. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry………………………………………………………………...80 FIGURE 2.22. Biophysical characterization of WDR5-RbBP5 (V375A) and WDR5-RbBP5(V377A) interactions……………………………………………………………………………………….81 FIGURE 2.23. Model for multiple methylation by the MLL1 core complex……………………86 FIGURE 3.1. Schematic representation of the human retinoblastoma binding protein five (RbBP5) showing the domain architecture of recombinantly expressed full-length RbBP5……………………………………………………………………………………………89 FIGURE 3.2. Limited proteolysis of full-length RbBP5………………………………...............95 FIGURE 3.3. Biophysical characterization of wild-type RbBP5 (#1-495) proteins by analytical ultracentrifugation………………………………………………………………………………..96 FIGURE 3.4. Biophysical characterization of RbBP5 wild-type and RbBP5 (#1-495) proteins by circular dichroism………………………………………………………………………………..97 FIGURE 3.5. Biophysical characterization of RbBP5 (#1-337) by analytical ultracentrifugation………………………………………………………………………………..98 xi FIGURE 3.6. Biophysical characterization of RbBP5 (#1-337) by circular dichroism………………………………………………………………………………………...99 FIGURE 3.7. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry……………………………………………………………….100 xii LIST OF TABLES Table 1.1 Histone posttranslational modification types and the residues modified…………………………………………………………………………………………...7 Table 2.1 RbBP5 mutations resulting in decreased enzymatic activities………………………..48 Table 2.2 Summary of sedimentation coefficients derived from sedimentation velocity analyses of the Retinoblastoma Binding Protein Five (RbBP5) expressed with mutations in various positions or expressed as truncated constructs…………………………………………………...51 Table 2.3 Summary of Rate Constants derived from globally fitting experimental data……………………………………………………………………………………………….59 Table 2.4 Summary of Rate Constants derived from globally fitting experimental data……………………………………………………………………………………………….74 Table 3.1 Summary of Rate Constants derived from globally fitting experimental data……………………………………………………………………………………...............101 xiii Chapter One: Introduction 1 BACKGROUND AND SIGNIFICANCE: 1.1 The Chromatin Landscape Eukaryotes have evolved elaborate mechanisms to regulate the expression of genes by packaging the genome into chromatin. The fundamental repeating unit of chromatin, the nucleosome core particle, consists of approximately 147 bp of genomic DNA wrapped around a histone octamer, containing one tetramer of histones H3 and H4 and two H2A-H2B dimers (Kornberg, 1974). Nucleosomes are packaged into progressively higher order structures to ultimately form chromosomes. In its extended form, chromatin can be visualized as an array of nucleosomes or “beads on a string”, but in the nucleus of the cell, the chromatin fiber undergoes extensive degrees of folding resulting in increasing degrees of chromatin compaction (Schwarz and Hansen, 1994). Since the recognition of chromatin structure as a repeating unit of DNA wrapped around histones, it has been suggested that its function extends beyond merely just a storage vehicle for DNA. The discovery that nucleosomes impede transcription in vitro (Wolffe and Hayes, 1999), in addition to experiments in vivo showing that deletions of histones or their basic tails elicited specific effects on gene expression, provided a glimpse of chromatin’s importance in the regulation of the expression of genetic programs (Allison, 1996; Hayes and Wolffe, 1992; Lee et al., 1993). It has now become widely recognized that the structure of chromatin largely affects DNA-templated processes such as DNA transcription, replication, recombination, and repair. Within this realm, access to DNA must be tightly regulated to allow sequence-specific DNA binding factors, chromatin regulators, and the general transcription machinery to bind. 2 As stated above, the packaging of the DNA into nucleosomes affects all stages of DNAtemplated processes. Therefore, eukaryotes employ proteins, often in complexes, that modify chromatin through the highly coordinated introduction or removal of post-translational modifications on histone proteins to regulate access to the underlying genome- resulting in the correct establishment and propagation of gene expression patterns. Each of the core histones that make up the nucleosome are predominantly globular with the exception of their N-terminal tails, which are unstructured and protrude from the lateral surface of the histone octamer. One striking feature of the flexible N-terminal tails of histones is that they serve as platforms that can be targeted by enzymes for the introduction or removal of distinct chemical moieties known as covalent posttranslational modifications. Post-translational modifications themselves can significantly affect the degree of chromatin compaction by creating generally more condensed “heterochromatic” or more open “euchromatic’ regions that impact the procession of chromosome processes such as transcription. Currently, there are two characterized mechanisms for how modifications affect chromatin structure. One is that chromatin packaging is altered directly (either by change in electrostatic charge or through internucleosomal contacts), to open or close the DNA polymer (Figure 1.1), thus controlling access of DNA-binding proteins such as transcription factors (Bannister and Kouzarides, 2011; Berger, 2007; Gardner et al., 2011; Jenuwein and Allis, 2001; Kouzarides, 2002; Wolffe and Hayes, 1999). The second mechanism posits that the attached chemical moieties alter the nucleosome surface to promote or occlude the association of chromatin binding modules (Kouzarides, 2007). Histone acetylation and 3 Figure 1.1. Schematic diagram of the effects of histone acetylation on chromatin structure. The thin protruding blue lines represent the amino-terminal tails of histones, and the winding black lines represent DNA. The lower panel depicts a more “open” structure for acetylated chromatin. Conversely, histone deacetylation results in condensed, transcriptionally inactive chromatin (upper panel). 4 phosphorylation are two examples of post-translational modifications that likely weaken histoneDNA contacts, leading to chromatin de-repression (Figure 1.1). Although difficult to demonstrate in vivo, considering only the electrostatic requirements for folding of the chromatin fiber, histone acetylation through the neutralization of positive charge, and histone phosphorylation through addition of a negative charge would likely cause decondenstation of the chromatin polymer (Brownell and Allis, 1996; Schwarz and Hansen, 1994). Histone modifications can also indirectly affect chromatin structures by serving as marks for the recruitment of enzymatic machineries that remodel the nucleosome and/or chromatin structure (Suganuma and Workman, 2011) (Figure 1.2). These protein complexes are recruited to modifications and bind via specific domains. Lysine acetylation was first discovered to be recognized by bromodomains, methylation by chromodomains and PHD finger domains, and phosphorylation is recognized by 14-3-3 domains in proteins (Dhalluin et al., 1999; Li et al., 2006; Muslin et al., 1996; Sims and Reinberg, 2006). Today we know that more than a dozen modifications exists on histone tails, including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP-ribosylation, proline isomerization, citrullination, propionlyation, and glycosylation (Table 1.1), most of which are recognized to govern the structure of chromatin and play a direct role in some aspect of gene regulation or genomic function (Gardner et al., 2011). This remarkable diversity of potential histone modification patterns provides cells with an enormous combinatorial potential for the precise regulation of gene expression (Suganuma and Workman, 2011). This potential can be summarized by the histone code hypothesis. In its original form, this hypothesis states that “multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions” (Allis et al., 2000). 5 FIGURE 1.2. Generic model of chromatin remodeling by ATP dependent protein machineries. The binding of the remodeling complex is ATP dependent. Upon the addition of ATP the conformation of the nucleosomes (depicted as gray spheres) results in changes of histone-DNA contacts. Remodeling of the chromatin fiber can lead to histone eviction or nucleosome sliding. 6 Table 1.1 Histone posttranslational modification types and the residues modified. Modification types Residue(s) modified Acetylation Lysine Phosphorylation Serine/threonine Methylation Lysine/arginine Ubiquitylation Lysine Sumoylation Lysine ADP-ribosylation Lysine Citrullination Arginine Butyrylation Lysine Propionlyation Lysine Glycosylation Serine/threonine 7 The chromatin-modifying enzymes that facilitate alterations to the chromatin landscape by placing, interpreting, or removing modifications have recently been more generally referred to as writers, readers, and erasers, respectively (Gardner et al., 2011). At defined points, writers place marks on defined histone residues, which are interpreted by readers possessing specialized domains that aid the progression of a specific functional outcome in the cell. At a time when such signaling needs to be terminated, based on the requirements of the cell, proteins known as “erasers” are recruited to their defined targets to remove the mark, thereby ending the associated functional consequence (Gardner et al., 2011; Jenuwein and Allis, 2001; Strahl and Allis, 2000). Admittedly, the situation is by nature vastly more complicated as recent studies suggest that there is no strict functional outcome of a given modification as many of these marks have several, seeming conflicting roles (Rando, 2012; Smith and Shilatifard, 2010). 1.2 H3K4 methylation As described above, the concept of the histone code hypothesis suggests that information relevant to gene expression is embedded within chromatin in the form of covalent histone modifications. Methylation of lysine residues provides an example of the histone code hypothesis. Until recently, insights into histone lysine methylation and its functional consequences significantly lagged behind, despite its discovery in 1964 (Murray, 1964). However, it is now apparent that lysine methylation can create a binding site for proteins that can alter the local properties of chromatin, constituting an important epigenetic indexing system resulting in the “activation” or repression” of specific genes, or large chromosomal regions depending on the context and extent of the modification (mono-, di-, and trimethylationdiscussed below) (Ruthenburg et al., 2007) . Given that histone H3 lysine 4 methylation (H3K4me) is a hallmark of actively transcribed genes, and its importance in many different 8 biological processes, understanding the enzymes that mediate, interpret, and remove this modification have been the focus of several investigations. H3K4 methylation is an evolutionarily conserved epigenetic mark predominantly linked to transcriptional activation in eukaryotes (Eissenberg and Shilatifard, 2010; Kouzarides, 2002; Ruthenburg et al., 2007; Sims et al., 2003). Adding an additional layer of complexity to epigenetic regulation, the epsilon amino group of lysine 4 can be mono-, di-, or tri-methylated resulting in distinct functional consequences. This is emphasized by genome-wide chromatin immunoprecipitation experiments that demonstrate that the degree of lysine methylation is different in distinctive genomic contexts. For example, in S. cerevisiae, lysine 4 is trimethylated in the promoter region of actively transcribed genes, dimethylation is enriched in the open reading frames, and monomethylation is enriched in the 3 ‘ ends of genes (Pokholok et al., 2005). This H3K4 localization pattern holds true in higher eukaryotes, with the additional observation that nucleosomes in distal enhancer sequences are enriched in monomethyl lysine (Barski et al., 2007). In lower eukaryotes, lysine 4 monomethylation is associated with transcriptional silencing. These studies suggest that the degree of H3K4 methylation is a highly regulated process. Interestingly, eukaryotes have evolved a panoply of highly conserved enzymes whose function appears to precisely regulate the degree and extent of H3K4 methylation. H3K4 methylation is mainly deposited by a class of enzymes that share an evolutionarily conserved SET domain. The exception to this rule is the WRAD enzyme, a recently discovered novel methyltransferase, which is structurally unrelated to SET domain proteins (Patel et al., 2009; Patel et al., 2011). The SET domain was first recognized as a conserved motif present in three Drosophila melanogaster proteins: a modifier of position-effect variegation, Suppressor of 9 variegation 3-9 (Suv(var)3-9), the Polycomb-group chromatin regulator, Enhancer of zeste (E(z)), and the trithorax-group chromatin regulator, Trithorax (Trx) (Dillon et al., 2005). The function of the SET-domain proteins is to transfer a methyl group from S-adenosyl-L-methionine to the amino group of a lysine residue on the histone and in some cases non-histone proteins, leaving a methylated lysine residue and the cofactor product. Seven main families of SETdomain proteins are known, all of which differ with respect to their substrate specificity, processivity, and the presence of additionally associated domains (Dillon et al., 2005; Jenuwein et al., 1998; Rea et al., 2000). While there are several SET domain enzymes, members of the SET1 family share the virtues that they all methylate H3K4 and interact with an evolutionarily conserved core complex of proteins that function together to control the degree of H3K4 methylation (Cosgrove and Patel, 2010; Crawford and Hess, 2006; Dillon et al., 2005; Dou et al., 2006; Jenuwein et al., 1998; Rea et al., 2000; Southall et al., 2009). The first H3K4 methyltransferase to be identified was S. cerevisiae SET1p (Miller et al., 2001; Roguev et al., 2001). In mammals, the number of H3K4 methyl writers is much greater, as there are over six SET1-related proteins including Set1a, Set1b, and four members of the Mixed Lineage Leukemia (MLL) family, all of which are capable of catalyzing the methylation of H3K4. MLL1, the human homologue of the Drosophila protein Trithorax, has been the most intensively studied because of its involvement in genetic rearrangements that occur in infant acute leukemias and other therapy related malignancies. 10 1.3 MLL1 The MLL gene located in the human genome at chromosome 11, band q23, was initially identified through its recurring involvement in reciprocal translocations found in numerous cases of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (Ernst et al., 2002; Hess, 2004; Slany, 2009). MLL1 was later shown to encode a major H3K4- specific histone methyltransferase enzyme that functions to maintain gene expression during development and hematopoiesis (Ernst et al., 2002; Milne et al., 2005; Ono et al., 2005). Despite the important biological role of MLL1 and its involvement in human leukemias, the underlying molecular details for the regulation of the histone methyltransferase activity of MLL1 remain poorly understood. The MLL1 protein is a member of the SET1 family of H3K4 methyltransferases that functions to maintain gene expression during development and hematopoiesis (Hess et al., 1997; Milne et al., 2002;). The most well studied target genes of MLL1 encompass the homeobox transcription factors, better known as HOX genes, which are important for specifying segment identity and cell fate during metazoan development (Milne et al., 2002; Milne et al., 2005). These genes also play a role in leukemogenesis, which is why they are important in the context of MLL’s role in cancer (Ernst et al., 2002; Guenther et al., 2005; Hess, 2004; Milne et al., 2005; Ono et al., 2005; Slany, 2009; Yokoyama et al., 2004; Yu et al., 1995). A unique pattern of H3K4 methylation has been observed in HOX gene clusters, where large continuous regions of H3K4 methylation are observed spanning multiple genes and intergenic regions (Guenther et al., 2005). Genetic studies in mice have demonstrated that MLL1-null mutations result in embryonic lethality and are associated with multiple development defects and hematopoietic abnormalities (Yu et al., 1995). 11 1.4 Mechanistic Determinants of Multiple Lysine Methylation Catalyzed by the MLL1 Core Complex Like most histone modifying enzymes, MLL1 exists in a multi-protein complex and regulates the degree of H3K4 methylation (Cosgrove and Patel, 2010). Although the subunit composition of SET1 family members varies to some degree, each SET1 family member is capable of interacting with a conserved core group of proteins that include the WD-40 repeat protein-5 (WDR5), the Retinoblastoma binding protein-5 (RbBP5), the Absent small homeotic 2like protein (Ash2L), and Dumpy 30 (DPY30), which is required for distinct states of H3K4 methylation (Figure 1.3) (Cao et al., 2010; Cosgrove and Patel, 2010; Crawford and Hess, 2006; Dou et al., 2006; Patel et al., 2009; Ruthenburg et al., 2007; Southall et al., 2009). Because of the central role of H3K4 methylation in transcriptional regulation, understanding how different H3K4 methylation states are established and maintained is crucial for understanding how MLL1 is misregulated. Recent research suggests several models for the establishment and maintenance of the degree of methylation by the SET1 family of histone lysine methyltransferases. One model suggests that multiple lysine methylation is achieved by the successive addition of a methyl group catalyzed by distinct histone lysine methyltransferases (Patel et al., 2011). In this model the sequential addition of methyl groups is catalyzed by distinct enzymes that differ in their abilities to use unmodified, mono-, and di-methylated peptide histones as substrates- a phenomenon known as product specificity. 12 FIGURE 1.3. Schematic illustration of the members of the MLL1 core complex. WDR5, RbBP5, Ash2L, and DPY30 form a core configuration of proteins that are capable of interacting with MLL1 to form the MLL1 core complex required for di-methyltransferase activity. Also denoted in this schematic diagram is the interaction between Arginine 3765 (labeled R) in MLL1 and WDR5. The motif was previously shown to be required for the assembly and di-methyltransferase activity of the MLL1 core complex (Patel et al., 2008). 13 Consistent with this hypothesis, several SET domain enzymes have been discovered that display different product specificities toward histone lysine residues. Biochemical and structure-function studies suggest that the product specificity of SET domain enzymes is governed by the presence of a tyrosine or phenylalanine at a conserved position in the SET domain active site, called the “Phe/Tyr switch” position (Collins et al., 2005; Couture et al., 2008; Dillon et al., 2005; Patel et al., 2011; Southall et al., 2009). In general, SET domain enzymes are predicted to be lysine monomethyltransferases based on the presence of a tyrosine at the switch position, whereas in most di- and tri-methyltransferases, a phenylalanine or another hydrophobic amino acid is located in this site. SET1- related enzymes possessing tyrosine in the switch position generally have smaller active sites allowing them to only catalyze mono-methylation. In contrast, SET1related enzymes possessing a phenylalanine in the switch position have larger active sites that can accommodate the rotation of pre-methylated substrates, therefore allowing a processive mechanism (Collins et al., 2005; Couture et al., 2008; Dillon et al., 2005; Patel et al., 2011; Southall et al., 2009). Although mutagenesis experiments have validated this Phe/Tyr switch hypothesis for several SET domain containing enzymes, enzymes from the SET1 family appear to contradict this rule. Interestingly, all of the SET1 family enzymes posses a tyrosine in the critical switch position, which is predicted to limit their enzymatic activity to that of a monomethyltransferases (Collins et al., 2005; Cosgrove and Patel, 2010; Couture et al., 2008; Dillon et al., 2005; Southall et al., 2009) However, mono-, di-, and tri-methylation have been attributed to SET1 family complexes in vivo and in vitro (Cao et al., 2010; Cosgrove and Patel, 2010; Crawford and Hess, 2006; Dou et al., 2006; Eissenberg and Shilatifard, 2010; Miller et al., 2001; Ruthenburg et al., 2007). These early observations laid the groundwork for a large body of subsequent studies to resolve this paradox. It is postulated that the product specificity of the 14 SET1 family enzymes is regulated by proteins that bind to and alter the conformation of the catalytic SET domain active site, in turn altering their ability to catalyze the addition of each methyl group to the lysine side chain. In an effort to identify the minimal MLL1 core complex components necessary for mono, di-, and tri-methylation, Roeder and colleagues (Dou et al., 2006) developed a baculovirus system to co-express and immunopurify recombinant components from insect cells. In this system, results suggest that the minimal complex required for mono-, di-, and tri-methylation includes a core configuration of proteins comprised of the 180kDa MLL-C terminal fragment, WDR5, RbBP5, and Ash2L. Notably, they observed that optimal methyltransferase activity was contingent upon MLL-C terminal associations with the other structural components and that the MLL-C terminus alone could not efficiently catalyze H3K4 methylation (Dou et al., 2006). MLL1 subcomplexes that lacked any of the other three components had compromised methyltransferase activity. Omission of either RbBP5 or Ash2L resulted in substantial loss of H3K4 activity, whereas the absence of WDR5 resulted in complete loss of H3K4 methylation. With respect to the degree of H3K4 methylation in the absence of RbBP5 or Ash2L, H3K4 trimethylation was completely abolished, H3K4 dimethylation was significantly decreased, while monomethylation remained unchanged. Surprisingly, they were also able to show through these studies that the WDR5-RbBP5-Ash2L sub-complex associates with the MLL1 SET domain, but can exist independently of the catalytic subunit, providing a structural platform that can associate with the SET domains of different MLL-family members (Dou et al, 2006). These results are consistent with the idea that the WDR5-RbBP5-Ash2L sub-complex regulates the product specificity of the MLL1 C-terminal SET domain. The versatility of this reconstitution system allowed for the structure-function analysis of the MLL1 core complex, and provided insights into 15 the regulation of its enzymatic activity. Since the same group of proteins are conserved from yeast to humans, the conclusions from this study can be generalized for other members of the SET1 family of methyltransferases. These studies along with others have lead to a model by which the degree of H3K4 methylation is regulated by these components that interact with MLL to induce a conformation change in the active site therefore regulating the product specificity of the MLL1 SET domain (Couture et al., 2008; Crawford and Hess, 2006; Dou et al., 2006; Southall et al., 2009). In support of the conformation change hypothesis, a separate independent group was able to demonstrate that inclusion of equimolar amounts of either Ash2L or RbBP5 significantly promotes methyltransferase activity of a minimal MLL1 SET domain construct in vitro (Cao et al., 2010). In spite of these studies, the molecular details that describe how MLL1 catalyzes multiple lysine methylation remained elusive until recently. This in part stemmed from the fact that the intrinsic product specificity of the isolated SET domain remained unknown because of no observable catalytic activity without the presence of interacting proteins (Cao et al., 2010; Dou et al., 2006; Southall et al., 2009). To resolve these issues, an in vitro model system was developed by Cosgrove and colleagues that allowed for the identification of the protein structural features that are responsible for regulation of H3K4 methylation by the human MLL1 core complex. Using this system, Cosgrove and colleagues were able to establish that the intrinsic product specificity of an isolated MLL1 SET domain is indeed that of an H3K4 mono-methyltransferase consistent with the prediction of the “Phe/Tyr switch” hypothesis (Cheng and Zhang, 2007; Collins et al., 2005; Cosgrove and Patel, 2010; Couture et al., 2008; Dillon et al., 2005). Mutagenesis of conserved tyrosine 3942 to a phenylalanine in the switch position of the MLL SET domain alters the product specificity of MLL1 to that of a tri-methyltransferase (Patel et al., 16 2009). In addition, in the absence of interacting proteins, the isolated MLL1 SET domain is a slow mono-methyltransferase (Patel et al., 2009). However, when the MLL1 SET domain is assembled with a complex containing WDR5, RbBP5, Ash2L, and DPY30, robust dimethyltransferase activity is readily observed. At first glance, these results appear to support the hypothesis that the MLL1 SET domain interacting proteins regulate the product specificity of the MLL1 SET domain, however further experimental analyses suggests a different mechanism. Recall that one model for regulation of multiple lysine methylation is that it is achieved by allosteric control of a single SET domain. Therefore one explanation for the results obtained above is that a protein-interaction induced conformational change alters the position of Tyr3942 in the MLL1 SET domain active site, like what occurs when this tyrosine is replaced with phenylalanine. However, the kinetic behavior observed with the assembled MLL1 core complex is substantially different from the behavior that would be anticipated from the simple movement of Tyr3942 in the MLL1 SET domain active site. First, while the Y3942F MLL1 SET domain readily trimethylates H3K4, the assembled MLL1 core complex only di-methylates H3K4 in assays (Patel et al., 2009). Secondly, despite the ability of the Y3492A enzyme to readily catalyze mono-, di-, and tri- methylation of H3 peptides in vitro, the rate constants of methylation by the wild-type and the Y3942 MLL1 SET domain are similar. In contrast, the rate constant for the reaction catalyzed by the MLL1 core complex increases 600-fold when compared to the MLL1 SET domain fragment alone. Taken together, these results suggest that the rate limiting step for di-methylation by the MLL1 core complex is distinct from that of the monomethylation activity of the isolated MLL1 SET domain. Even more critically, it is important to note that single turnover kinetic experiments reveal that the mechanism of dimethylation by the MLL1 core complex involves the transient accumulation of a 17 monomethylated intermediate, while no such intermediate is observed with the Y3942F MLL1 SET domain (Patel et al., 2009). This transient accumulation of monomethylated species in the reaction catalyzed by the MLL1 core complex implies that the monomethylated peptide could potentially be released from the MLL1 SET domain active site before dimethylation occurs. Encouraged to understand this phenomena further, Cosgrove and colleagues proposed an alternative hypothesis that suggests that in order for multiple lysine methylation to occur, each methyl group is added by a distinct methyltransferase. Unexpectedly, it was shown that the nonSET domain components of the MLL1 core complex possess a previously unrecognized histone methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex (Patel et al., 2009; Patel et al., 2011). In addition, it was shown that the non-SET domain components of the MLL1 core complex (WDR5, RbBP5, Ash2L, and DPY30 or WRAD) possess monomethyltransferase activity in the absence of the MLL1 SET domain (Patel et al., 2011). Because the WRAD components lack homology to a conserved SET or DOT1-like methyltransferase fold, this enzyme constitutes a novel previously uncharacterized methyltransferase (Patel et al., 2011). Like the activity of other known lysine methyltransferases, the WRAD enzyme is zinc dependent, inhibited by the co-factor product S-adensoyl homocysteine, and displays Michaelis-Menten kinetics (Patel et al., 2011). This work has opened up a whole new area of investigation involving a core configuration of proteins that have already been shown to be intimately involved in several other cellular processes including differentiation, transcription, multicellular development, and cancer (Ang et al., 2011; Bralten et al., 2010; Gopal et al., 2012; Hsu et al., 1995; Hsu and Meyer, 1994; Jiang et al., 2011; Riemenschneider et al., 1999; Riemenschneider et al., 2003; South et al., 2010; Stoller et al., 2010; Vardanyan et al., 2008; Wan et al., 2012; Wysocka et al., 2005). Further studies are thus 18 necessary to determine the catalytic motif in this enzyme and what role, this enzyme may play in oncogenesis. In light of the above findings the subject of the work in this thesis focuses on the RbBP5 component of the MLL1 core complex. 1.5 The Retinoblastoma Binding Protein Five is a Critical Component of the MLL1 Core Complex The Retinoblastoma Binding Protein Five (RbBP5) was originally identified through direct screening of cDNA expression libraries to isolate distinct clones of cellular proteins that bind to the retinoblastoma protein, perhaps one of the best studied tumor suppressors (Classon and Harlow, 2002; Classon and Settleman, 2000; Ferreira et al., 1998; Lai et al., 1999; Magnaghi-Jaulin et al., 1998; Paggi et al., 1996; Suryadinata et al., 2011; Sutcliffe et al., 2000; Williams et al., 2006). This report demonstrated that the human RbBP5 gene encodes a protein of approximately 66kDa that localizes in the nucleus (Saijo et al., 1995). Although currently no three dimensional structure of the full length human retinoblastoma binding protein exists, sequence analysis, domain predictions, and homology modeling of RbBP5 predicts that this protein is composed of a N-terminal seven blade β-propeller domain, a short hinge region consisting of a stretch of conserved residues, and a C-terminal domain of unknown function (please see chapter 2: Figure 2.1). Since its discovery, human RbBP5 is now established as a well known conserved component of several multisubunit histone H3 lysine four methyltransferases, one representative example being the Mixed Lineage Leukemia-1 (MLL1) core complex. More recently, misregulation of this protein has been implicated in malignant glioblastomas (Riemenschneider et al., 2003). Within the MLL1 core complex, RbBP5 exists in a multisubunit core configuration 19 of proteins including MLL1, WDR5, Ash2L, and DPY30. The essential role of the RbBP5 subunit in regulating the MLL1 core complex’s methyltransferase activity has been demonstrated with reports showing that the deletion or down regulation this core component protein leads to an overall reduction in observable levels of methyltransferase activity, especially di- and trimethylation of H3K4 (Cao et al., 2010; Crawford and Hess, 2006; Dou et al., 2006; Patel et al., 2009). Additionally, initial studies on the MLL1 core complex revealed through a detailed biophysical analyses on the organization and assembly of the MLL1 core complex, that the RbBP5 component of the MLL1 core complex forms important interactions with both the WDR5 and Ash2L subunits of the complex (Patel et al., 2009). Later reports have confirmed these interactions, and have mapped the domains in RbBP5 responsible for these contacts. As a whole, these studies have shown that the C terminus, a region outside of RbBP5’s WD40 repeat domain is responsible for its interaction with WDR5 and Ash2L. 1.6 RbBP5 Binds WDR5 Using a Segment Neighboring its β-propeller domain Subsequent to the discovery of WDR5 as a binding partner for the RbBP5 protein within the MLL1 core complex, several groups sought to map the minimal structural features of RbBP5 mediating this interaction. Based on pull-down experiments from two independent laboratories, it has been demonstrated the RbBP5’s interaction with WDR5 can be precisely mapped to a short 20 FIGURE 1.4. Structural analysis of a RbBP5 peptide bound to WDR5. The WDR5 molecule is shown as a ribbon representation (purple) and the bound RbBP5 peptide is shown (green stick with transparent surface). The RbBP5 peptide-binding mode reveals that the peptide binds in an extended fashion. (The structural representation was generated in Chimera using PDB coordinates 2XL2.) 21 segment of the unstructured C-terminal tail segment in RbBP5 (Avdic et al., 2011; Odho et al., 2010). To gain insight into the molecular basis of this interaction, crystals were obtained of WDR5 and a synthetic RbBP5 peptide containing a small segment of residues responsible for binding to WDR5. Structural analysis of the RbBP5 peptide-binding mode reveals that the peptide binds in an extended fashion on the opposite face of WDR5 from the canonical binding site that has been shown to accommodate the WDR5 interaction motif of MLL1 (Figure1.4). Closer inspection of this structure reveals that the RbBP5 binding site on WDR5 has strong hydrophobic character that can accommodate two residues Val-375 and Val-377 present on the RbBP5 binding motif (Odho et al., 2010). When these two residues are mutated to glutamic acid, binding to WDR5 shown by immunoprecipitation experiments is severely compromised (Odho et al., 2010). To investigate the effect of the addition of RbBP5 in the presence and absence of WDR5 on MLL1 SET domain mediated methyltransferase activity, a series of methyltransferase activity assays were carried out with the MLL1 SET domain using unmodified histone H3 peptide as a substrate. In addition, the ability of the RbBP5 proteins containing glutamic acid substitutions in Valine residues 375 and 377 shown to be required for interaction with WDR5 were also tested for their ability to further stimulate MLL1 SET domain mediated activity in the presence and absence of WDR5. Previous studies have shown that MLL1 alone is slow monomethyltransferases and the addition of WDR5 does not significantly enhance MLL1 SET domain enzymatic activity. Consistent with previous results, the addition of WDR5 alone did not significantly enhance MLL1 SET domain catalyzed methyltransferase activity. However, when the RbBP5 wild-type protein was added, a small increase in activity of the MLL1 SET domain is observed (Odho et al., 2010). Significantly, the addition of both WDR5 and RbBP5 resulted in a 22 much higher level of activity and enhancement. In a second separate series of methyltransferase assays, carried out with the MLL1 SET and the RbBP5 proteins containing glutamic acid substitutions in Valine residues at positions 375 and 377, the mutations alone did not interfere with the ability of these proteins to further stimulate MLL1 methyltransferase activity when compared to the stimulatory effect of the wild-type RbBP5 protein (Odho et al., 2010). Interestingly enough, in the presence of WDR5, previous enhancement of MLL1 methyltransferase activity was lost with the RbBP5 variant proteins. Taken together, these results suggest, that WDR5 may act as a structural scaffold for the assembly of the MLL1 core complex by bridging interactions between RbBP5 and MLL1, to allow for RbBP5 mediated stimulation of MLL1 SET domain methyltransferase activity. Consistent with this hypothesis, other studies have demonstrated that RbBP5’s β-propeller domain and a short region of the tail was sufficient enough to stimulate methyltransferase activity of the MLL1 SET domain (Avdic et al., 2011). 1.7 The Ash2L SPRY Domains Binds a Stretch of Acidic Residues in RbBP5 In addition to binding WDR5, RbBP5 forms another important interaction with the Ash2L subunit within the MLL1 core complex. Recent reports have show that the SPRY domain of Ash2L recognizes an acidic stretch of residues of RbBP5. It has been hypothesized that this interaction between RbBP5 is more than likely mediated by electrostatic contacts (Chen et al., 2011). In support of this hypothesis, previous data has shown that substitution of Arginine 343 in Ash2L is enough to completely abolish its interaction with RbBP5. Not to mention, the mutation of several other positively charged residues has resulted in the weakening of these contacts, although to variable extents (Chen et al., 2011). 23 1.8 Studies in Yeast Reveal that the RbBP5 Homolog Swd1 is Required for SET Protein Stability, Histone Methylation and Gene Expression Many reports have suggested that RbBP5 of the MLL1 core complex is capable of establishing direct contacts with the MLL1 SET domain to stimulate its intrinsic methyltransferase activity. In spite of these studies, there has yet to be any study that identifies the surfaces on MLL1 and RbBP5 required for this interaction, until recently. One recent study on the homologous yeast Complex of Proteins Associated with SET1 (COMPASS) has shed many important insights into the regulation of methyltransferase activity by MLL1 complex members, specifically by Swd1 (RbBP5). In defining the mechanism of interaction between SET1 and Swd1 (RbBP5), Briggs and colleagues have shown that two patches of acidic residues found in the C-terminal domain of Swd1, is important for maintaining SET1 protein levels and H3K4 methylation in vivo. Likewise, they were also able to identify that deletion of a basic patch of residues on the conjugate protein SET1, abrogates the interactions between SET1 and SWD1. Furthermore, the deletion of either the acidic or basic patches in either protein, resulted in severe growth defects, loss of telomere silencing, and decreased gene expression. Additionally, this study shows that this acidic patch of Swd1 is also conserved in human RbBP5, and is necessary for protein–protein interactions between SETd1a1 and RbBP5 (Mersman et al., 2012). Additional experiments indicate that RbBP5 is capable of forming a direct interaction to the nSET domain of SET1, but does not form a direct interaction with MLL1. Based on these results, although the complex members between human and yeast may be evolutionarily conserved, the assembly and mode of regulation may differ. For example, although work in this study suggests that Swd1 can interact with SET1 in the absence of other complex components, work done on MLL1 shows that the human homolog of Swd1, (RbBP5) requires WDR5 to stably 24 interact with MLL1. Additionally, studies suggest that human SET1 complex components are prerequisite for the stability of SET1A and SET1B protein levels, however, this does not appear to be the case for MLL1. Taken together, the data suggest that although the SET1A/B and MLL1 exists in complexes along side an evolutionarily conserved core configuration of protein, the manner in which they interact and assemble into multisubunit complexes to regulate H3K4 methylation are likely distinct. Rational of Study Although there have been several reports focused on RbBP5’s interaction with other members of the MLL1 core complex, a detailed characterization of RbBP5 is still lacking. In particular, relatively little is known about the contribution of RbBP5 in facilitating and/or regulating H3K4 methylation within the context of the MLL1 core complex. Several lines of evidence have emerged that suggests that the RbBP5 component of the MLL1 core complex is involved in the catalytic mechanism of H3K4 methylation mediated by the MLL1 core complex (Avdic et al., 2011; Cao et al., 2010; Crawford and Hess, 2006; Dou et al., 2006; Odho et al., 2010; Patel et al., 2009; Southall et al., 2009). Several studies have also suggested that RbBP5 may directly interact with MLL1 SET domain to enhance methyltransferase activity (Avdic et al., 2011; Cao et al., 2010; Crawford and Hess, 2006; Dou et al., 2006; Odho et al., 2010; Patel et al., 2009; Southall et al., 2009). In addition, RbBP5 is a member of WRAD, a novel methyltransferase whose enzymatic activity has only recently been reported (Patel et al., 2011). This WRAD sub-complex of the MLL1 core complex is able to bind MLL1 and stimulate its methyltransferase activity. Lastly, it has also been reported in an independent study that only an Ash2L/RbBP5 heterodimer is required for weak methyltransferase activity (Cao et al., 2010). Collectively, these studies suggest that RbBP5 is important for proper H3K4 methylation. In 25 spite of this body of work, the underlying mechanism of exactly how the RbBP5 protein contributes to these methyltransferases activities remains undefined. Therefore, additional biochemical and structural studies are pivotal in order to understand how RbBP5 contributes to H3K4 methylation. Therefore, to better understand the role of RbBP5 in H3K4 methylation, the work in this thesis seeks to address the central question of what are the protein structural features of RbBP5 that are required for the di-and tri-methyltransferase activity of the MLL1 core complex. The RbBP5 protein could be playing a number of roles in the MLL1 core complex including: 1. RbBP5 is serving as a bridge to bring other components of the complex together; 2.RbBP5 is interacting with MLL1 to stimulate SET domain activity; 3. RbBP5 binds the H3 histone substrate or cofactor SAM; or 4. RbBP5 is part of a shared active site within the WRAD subcomplex. By taking a structure-function approach the research in the following chapters of this thesis seeks to identify novel protein structural features of RbBP5 responsible for the enzymatic of the MLL1 core complex. By defining the role of RbBP5 we hope to further delineate the underlying mechanism of H3K4. In order to study the role of RbBP5 in H3K4 methylation by the MLL1 core complex, extensive domain mapping experiments on the full-length human RbBP5 protein were performed by Anamika Patel to identify the minimal domain requirements for enzymatic activity. These studies reveal that the minimal domain of RbBP5 required to recapitulate methyltransferase activity consists of residues 323-402 (Patel, unpublished data). Close inspection of the residues in the sequence of the minimal domain of RbBP5 (323-402) reveals a highly conserved stretch of amino acids with no known function. In light of these findings, to probe the functional role of each of these residues in MLL1 core complex mediated methyltransferase activity, point 26 mutations were introduced into conserved residues in this domain. Once mutations were introduced into the RbBP5 protein and confirmed, high-throughput methyltransferase enzymatic assays were carried out to identify mutated residues that resulted in an overall decrease in the dimethyltransferase activity of the MLL1 core complex. Further validation of residues resulting in reduced activity were verified through quantitative MALDI-TOF methyltransferase assays. Out of these screens, residues W329, F332, F336, and E347 were identified and confirmed to have reduced di-methyltransferase activity within the MLL1 core complex. Further experiments demonstrate that these residues are required for interaction with the MLL1 SET domain. Although numerous studies have suggested that RbBP5 is required for full activity of the complex, to date there have been no reports on the inherent structural features required for protein-protein interactions and enzymatic activity involving RbBP5. Taken together, these studies constitute the identification a novel structural motif in RbBP5 required for interaction with MLL1 within the MLL1 core complex and consequently the overall dimethyltransferase activity of the complex. This work is detailed in chapter 2, and encompasses the major body of work done for this thesis. Furthermore, previously determined Valine residues 375 and 377 in RbBP5 identified to be important for the interaction with WDR5 were further characterized and confirmed taking a more stringent biophysical and kinetic approach. This work is described at the end of chapter two, and provides further validation of the WDR5-RbBP5 interaction motif. The third chapter of this thesis, reports on the identification and characterization of two stable sub -domains of RbBP5 identified using limited proteolysis experiments to facilitate highresolution structure determination of RbBP5. Currently, there is no three-dimensional structure of full-length RbBP5, and although several attempts were made to try to crystallize the fulllength protein using the hanging drop vapor diffusion method and sparse matrix screening, no 27 crystals have emerged from crystallization trials. Using limited proteolysis to identify flexible regions in RbBP5 that may prevent this protein from crystallization, I have succeeded in identifying two potentially well -ordered regions of RbBP5 containing residues 1-495 and 1-337. The identity of these domains was confirmed utilizing N-terminal sequencing and their exact masses were confirmed via electrospray ionization mass spectrometry. Although no crystals have emerged from early crystallization trials of these newly identified stable subdomains in RbBP5, this study illustrates alternative and varied approaches in studying the role of a protein without the availability of a three-dimensional structure. The final part of this thesis provides an overview of the body of work and proposed future directions in light of the findings of these studies. 28 CHAPTER TWO: A Conserved Patch of Amino Acids in RbBP5 is Required for Interaction with the SET domain of MLL1 in the MLL1 Core Complex 29 INTRODUCTION: The correct establishment and propagation of genetic programs in eukaryotes depends in part on enzymes that are responsible for the deposition and removal of covalent posttranslational modifications on histone proteins. The methylation of histone H3 at lysine 4 (H3K4) is a wellstudied epigenetic mark required for the recruitment of enzymatic machineries that maintain transcriptionally permissible states of chromatin. This mark is mainly catalyzed by a group of enzymes that contain an evolutionarily conserved Suppressor of Variegation, Enhancer of Zeste, and Trithorax (SET) domain (Ruthenburg et al., 2007). Abnormalities in H3K4 methylating enzymes of the SET1 family have been observed in various cancers (Bhaumik et al., 2007; Chi et al., 2010) the most prominent example resulting from aberrations of the Mixed Lineage Leukemia gene. Studies on the archetypal member of the SET1 family, human MLL1, have shed light on several mechanistic determinants underlying the mode of regulation for these enzymes. Of specific interest, immunoprecipitation experiments have shown that unlike other SET domain enzymes, members of the SET1 family are found in a multisubunit core configuration of proteins comprised of WDR5, RbBP5, Ash2L, and DPY30 (WRAD). Growing bodies of studies have highlighted the important role that WRAD plays in the overall methyltransferase activity of the core complex. The key common finding of these studies is that deletion or down regulation of a core complex protein leads to an overall reduction in the observed level of methyltransferase activity (Avdic et al., 2011; Cao et al., 2010; Cosgrove and Patel, 2010; Crawford and Hess, 2006; Dou et al., 2006; Odho et al., 2010; Patel et al., 2009; Southall et al., 2009). More importantly, it has been demonstrated that although WDR5 by itself does not stimulate methyltransferase activity of the MLL1 SET domain in vitro, in contrast, both RbBP5 and Ash2L 30 contribute to the overall catalytic activity. When stoichiometric amounts of RbBP5 are added to a MLL1 SET domain construct and WDR5, a small 2-fold increase in the overall reaction rate in observed, when compared to the reaction catalyzed by MLL1 alone (Chi et al., 2010; Patel et al., 2009). When Ash2L was added to the MLL1 SET domain construct along with WDR5 and RbBP5, a considerable increase in the overall reaction rate was observed when compared with that of the MLL1 SET domain protein alone (Chi et al., 2010; Patel et al., 2009). Although several reports have emerged on the Ash2L subunit and its contribution to this activity, far less is known about the contribution of RbBP5 in facilitating and/or regulating H3K4 methylation within the context of the MLL1 core complex. The retinoblastoma binding proteins five (RbBP5) was originally discovered because it is capable of directly binding the Retinoblastoma protein (pRb), the protein product of the Retinoblastoma gene. The Rb family members are essential regulators of cell cycle progression. Studies of the retinoblastoma gene (Rb) have shown that its protein product (pRb) acts to restrict cell proliferation, inhibit apoptosis, and promote cell differentiation (Lai et al., 1999; MagnaghiJaulin et al., 1998; Paggi et al., 1996). The RbBP5 protein contains a predicted B-propeller domain followed by a short hinge region, and a less conserved C-terminus (Figure 2.1). Recently, this protein has garnered much attention because of its presence as a conserved core component of the MLL1 core complex. The RbBP5 subunit of the MLL1 core complex has been shown to be required for enzymatic activity and disruption of RbBP5 has been frequently 31 FIGURE 2.1. Schematic representation of the human retinoblastoma binding protein five (RbBP5) showing the domain architecture of recombinantly expressed full-length RbBP5. Full-length RbBP5 contains 538 amino acid residues with a WD40 repeat domain (residues 14322) depicted in pink, followed by a predicted globular domain with a stretch of highly conserved residues shown in blue. This predicted globular region has no sequence homology to any know protein. Additionally, RbBP5 has a predicted disordered C-terminus shown in white. 32 observed in patients with malignant primary brain tumors (Bralten et al., 2010). Importantly, the absence of RbBP5 results in the loss of H3K4 dimethylation in vitro and the loss of H3K4 diand tri- methylation in vivo (Dou et al., 2006). However, the exact functional role of RbBP5 in MLL1 core complex H3K4 dimethyltransferase activity is not well understood. One of the possible means by which RbBP5 functions within the MLL1 core complex is that it may be directly involved in the H3K4 di- and trimethylation reaction. This is supported by experiments in vivo that demonstrate that RNAi knockdown of RbBP5 in HeLa cells severely impairs the ability of the MLL1 core complex to catalyze H3K4 di- and trimethylation (Dou et al., 2006). This study is further corroborated by experiments in vitro that show there is no detectable H3K4 di-methylation activity in the absence of RbBP5 by the MLL1 core complex (Dou et al., 2006). Not to mention, several other lines of evidence suggest that the RbBP5 subunit could be directly interacting with the SET domain of MLL1 to stimulate enzymatic activity (Odho et al., 2010; Patel et al., 2009). Collectively, these findings convey the important functional role RbBP5 takes within MLL1 core complex. In spite of these studies, the molecular details underlying RbBP5’s catalytic influence on the MLL1 core complex activity have yet to be determined. In this thesis, this research seeks to understand the mechanism behind RbBP5 mediated stimulation of the MLL1 core complex methyltransferase activity. By comprehensive mapping of RbBP5 we identify a minimal domain of RbBP5 required for enzymatic activity within the MLL1 core complex. Within this domain sequence analysis uncovers a highly conserved stretch of amino acids that exists among 19 homologs of the human Retinoblastoma binding protein-5 (RbBP5) subunit of the MLL1 core complex. The organisms range for yeast to humans, along with plants and animals. To gain insight into the functional role of this conserved stretch of residues, mutations targeting individual residues in this highly conserved stretch were generated. 33 Biochemical and biophysical analyses of the protein with various mutations in conserved residues were utilized to assess the functional role each may endow on the intrinsic properties of RbBP5 alone or when assembled within the context of the entire complex. Importantly, we find that mutation of several conserved aromatic residues and one acidic residue in RbBP5 results in a significant reduction in the overall dimethyltransferase activity of the MLL1 core complex. Additionally we find that these residues when mutated diminish WRAD’s ability to interact with MLL1. The residues identified constitute a previously uncharacterized novel MLL1 interaction motif located in RbBP5. Taken together, our study provides fundamental insights into how the RbBP5 subunit of the MLL1 core complex contributes to the overall enzymatic activity of WRAD and the MLL1 core complex. Understanding the role that RbBP5 plays in facilitating proper H3K4 methylation may provide insight into how misregulation of RbBP5 leads to its association with brain tumorigenesis and progression. In addition, understanding the functions of H3K4 methyltransferases and their interacting partners will be key in deciphering the “epigenetic code” and how this mark contributes to fundamental regulatory and cell developmental fate decisions. 34 EXPERIMENTAL PROCEDURES Small Scale Expression of RbBP5 Variants For High-throughput Assays: RbBP5 wild-type and mutant plasmid DNA were transformed into Escherichia coli Rosetta II plysS cells. Transformation reactions were plated on LB plates containing 50ug/mL carbenicillin and 20ug/mL chloramphenicol and incubated at 37°C overnight. Isolated single colonies were picked from wild-type and mutant plates the following day and inoculated in 5mL of Terrific Broth, containing 50ug/mL carbenicillin and 20ug/mL chloramphenicol. Cultures were allowed to grow overnight at 37°C. The next day, 50uL of overnight cultures were added to 5mL of Terrific Broth containing 50ug/mL of carbenicillin. The cultures were incubated with shaking for 2 hours at 37°C. After 2 hours, the cells were induced with 750uM isopropyl-1-thio-D-galactopyranoside. Small scale cultures were then placed on a shaker at 15 °C and grown overnight. The following day, the small scale cultures were spun down and the pellets were stored at -80 °C until further use. For Small Scale lysis: 50 mL of lysis buffer (50 mM Tris (pH 7.4) 300 mM Sodium Chloride (NaCl), 3 mM dithiothreitol (DTT) was prepared. One EDTA free protease inhibitor tablet, 0.1mM phenylmethylsulfonyl fluoride (PMSF), and 750uM sarkosyl was added to the 50 mL buffer preparations. A 100uL aliquot of freshly prepared lysis buffer cocktail was removed from the solutions and used to resuspend the crude pellet. To the sample cell lysis mixture 10uL of 10X Bug Buster, followed by the addition of 2.5uL DNAse 1 was added. The samples were then placed on a rotator at 4 °C, and cells were allowed to lyse with gentle turning for approximately 4 hours. Cell lysis mixtures were spun down at 4 °C in a micro centrifuge at 10,000 rpm for 20 35 minutes. The supernatant was removed and placed in a carefully labeled tube. The enzyme mixture was stored at -80°C until use. Protein expression and purification: The RbBP5 gene was purchased from open bio systems and sub-cloned into a pHis parallel vector, which encodes a Tobacco Etch Virus (TEV) cleavage Nterminal 6x-His fusion tag. Versions of full-length human RbBP5 (1-538) and RbBP5 constructs were overexpressed in Escherichia coli (Rosetta II, Novagen), by growing cells containing a given plasmid at 37°C in Terrific Broth medium containing 50ug/mL carbenicillin. The temperature was then lowered to 15 °C and cells were induced for 16–18 hours with 750uM isopropyl-1-thio-D-galactopyranoside (IPTG). Cells were harvested, re-suspended in a lysis buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 3 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, and EDTA-free protease inhibitor mixture (RocheApplied Science)), lysed with a microfluidizer cell disrupter, and clarified by centrifugation. Supernatants containing the RbBP5 His-tagged proteins were purified by nickel affinity chromatography (HisTrap column, GE Healthcare). For the first step of purification, the crude lysate was passed through a HisTrap column (GE healthcare) containing nickel beads. The bound 6x-His-RbBP5 was eluted from the column using a linear gradient of elution buffer containing 500mM imidazole. The peak fractions that contained RbBP5 were collected, pooled, and then dialyzed against column buffer that contained 50 mM Tris pH 7.4, 300mM NaCl, 30 mM Imidazole, and 3mM DTT at 4°C to remove the excess imidazole and to cleave the 6X-His tag in the presence of TEV (Tobacco Etch Virus) protease. The dialysis buffer was changed three times. For the second step of purification, the dialyzed protein was passed through the His-Trap column and the flow through fractions that contained the untagged version of RbBP5 were pooled and combined. As a final step of purification, the protein was passed through a gel filtration column (Superdex 36 200TM GE Healthcare) pre-equilibrated with the sample buffer containing 20mM Tris (pH 7.5), 300mM NaCl, 1mM Tris (2-carboxyethyl) phosphine (TCEP), and 1µM zinc chloride. All other proteins used in methyltransferase assays were purified as previously described. Mutagenesis and Oligonucleotide Primers: Point mutations were introduced into RbBP5 using the QuickChange site directed mutagenesis kit (Stratagene). Plasmids were sequenced to verify the presence of the intended mutations and the absence of additional mutations. Integrated DNA Technologies synthesized oligonucleotide primers were used in the mutagenesis. 3 [H] methyltransferase assay: Radiolabelled methyltransferase assays were conducted by combining 4µM of enzyme with 500 µM histone H3 peptide containing residues 1-20 (with GGK-Biotin on the C-terminus) and one microcurrie of 3H-methyl-S-adenosyl-methionine (3HSAM, GE Healthcare) in 50mM Tris, pH 8.5, 200mM NaCl, 3mM DTT, 5mM MgCl2, and 5% glycerol. The reactions were incubated at 15 °C for 8 hours, stopped by the addition of SDSloading buffer to 1X, and separated by SDS-PAGE on a 4-12% gradient gel (Invitrogen). The gel then was soaked in an autoradiography enhancer solution (Enlightning, Perkin Elmer), dried, and exposed to film at -80°C for 24 hours to 5 days. Western Blotting : Equal volumes of RbBP5 crude lysates were loaded in the wells of an SDSPAGE gel along with molecular weight markers. SDS -PAGE were performed on RbBP5 crude lysates and the proteins were transferred from the gel to a nitrocellulose membrane. Membranes were blocked with a blocking solution containing 5% nonfat dry dairy milk in a 1X phosphate buffered saline solution with tween 20 (0.1 %) (PBST) at room temperature with gentle agitation for 1 hour. Blocking solution was decanted and the membrane was incubated with primary antibody for 2 hours with gentle agitation (RbBP5 anti-body purified from Bethyl laboratories; working dilution 1:1000). After the membrane was incubated with the primary antibody, the 37 membranes were washed with 1X (PBST 0.1 %) for 10 minutes 3 times. The membrane was then incubated with a secondary antibody (Pierce goat anti-rabbit; working dilution 1:5,000) with gentle agitation for 1 hour. Membranes were washed again with 1X PBST 0.1% for 10 minutes three times. After washing the membranes, membranes were placed in weigh boats and a fresh chemilumescence solution was prepared by adding 1mL of SuperSignal Peroxide Solution and 1 mL of SuperSignal Luminol Enhancer directly on the membranes. The solution was distributed evenly. The weigh boat was then covered with foil and incubated in signal solution for 2 minutes. After incubation, membrane edges were bloated on paper towels to soak up excess signal solution. Human RbBP5 was detected with chemilumescence at various exposures. MALDI-TOF mass spectrometry methyltransferase assay: Mass spectrometry assays were conducted as previously reported; 7 µM of enzyme was incubated with 250 µM s-adenosylmethionine (SAM) and 10 µM histone H3 peptide (1-20) at 15 °C in 50mM TrisCl pH 9.0, 200mM NaCl, 3 mM DTT, and 5% glycerol. The reactions were quenched at various time points by the addition of trifluoroacetic acid to 0.5%. The quenched samples were diluted 1:4 with αcyano-4-hydroxycinnamic acid. MALDI TOF mass spectrometry was performed on a Bruker AutoFlex mass spectrometer (State University of New York, ESF) operated in reflectron mode. Final spectra were averaged from 100 shots/position at 10 different positions. Analytical Ultracentrifugation: Analytical ultracentrifugation experiments were carried out using a Beckman Coulter ProteomeLabTM XL-A analytical ultracentrifuge equipped with absorbance optics and a 4-hole An-50 Ti analytical rotor. Sedimentation velocity experiments were carried out at 10°C and 50,000 rpm (200,000 x g) using 3 mm two sector charcoal-filled Epon centerpieces with quartz windows. For each sample, 300 scans were collected with the time interval between scans set to zero. Protein samples in 20mM Tris (pH 7.5), 300mM NaCl, 1mM 38 TCEP and 1µM ZnCl2 were run at various concentrations. Sedimentation boundaries were analyzed by the continuous distribution (c(s)) method using the program SEDFIT. The program SEDNTERP version 1.09 was used to correct the experimental S-value (s*) to standard conditions at 20°C in water (S20,w) and to calculate the partial specific volume of each protein. Circular Dichroism Spectroscopy: CD spectra were collected on an AVIV Model 420 ds spectropolarimeter equipped with a Neslab CFT-33 refrigerated circulator at 10°C using a 0.1 mm path length cell. Spectra for each sample were obtained at a protein concentration of 0.2mg/mL in a buffer contain 20mM tris (pH 7.5), 200 mM NaCl, 1mM TCEP and 1uM Zinc chloride. The background contribution of the buffer alone was subtracted from each protein spectrum. RESULTS 2.1 The Hinge Domain of RbBP5 is Required for Methyltransferase Activity in WRAD and the MLL1 Core Complex Recent studies suggest a sequential mechanism for H3K4 di-methylation by the MLL1 core complex (Gardner et al., 2011; Patel et al., 2009). In this proposed model, the MLL1 SET domain of MLL1 catalyzes monomethylation of histone H3 at active site one. Following this event, the monomethylated peptide is subsequently transferred to a second active site on the WRAD sub-complex that then catalyzes dimethylation of histone H3. WRAD therefore constitutes a previously uncharacterized methyltransferase. Initial studies have shown that not only is RbBP5 essential for the methyltransferase activity within the MLL1 core complex, but is also essential for the newly discovered WRAD activity (Patel et al., 2009; Patel et al., 2011). 39 Nevertheless, the functional role of the RbBP5 protein in these two enzymatic activities has remained unexplored. One approach to better understand the function of RbBP5 is to delineate the role of specific domains within RbBP5 in facilitating H3K4 methylation. Based on sequence analysis RbBP5 contains a predicted N-terminal WD40 repeat Beta-propeller domain, a reported hinge domain (Avdic et al., 2011), which contains several highly conserved residues, and a less conserved C-terminus (Figure 2.2a). In order to study the function RbBP5 and its various domains, RbBP5 constructs corresponding to residues 1-538 (Full-length), 1-402 (β-propeller +Hinge), 1-322 (β-propeller), and 323-402 (Hinge Region) were expressed as 6X his tagged proteins in E.coli, and purified to near homogeneity (Figure 2.2b). The individual constructs were then tested for their ability to catalyze H3K4 methylation in the presence of the other members of the MLL1 core complex (MLL1, WDR5, ASH2L, and DPY30) using an unmodified H3 peptide as a substrate in an in vitro histone methyltransferase activity assay. Given the strong conservation of residues in the hinge region of RbBP5, we hypothesized that this region will be necessary not only for the activity of the MLL1 core complex, but also for activity within the WRAD sub-complex, and could possibly serve as part of a putative catalytic domain when assembled with other members of WRAD. 40 41 FIGURE 2.2. Schematic representation of the human retinoblastoma binding protein five (RbBP5) showing the domain architecture of recombinantly expressed full-length and truncated RbBP5 constructs used in this investigation. A. Full-length human RbBP5 contains 538 amino acid residues with a WD40 repeat domain (residues #14-322) depicted in pink, followed by a predicted globular domain with a stretch of highly conserved residues shown in blue. This predicted globular region has no sequence homology to any know protein. Additionally, RbBP5 has a predicted disordered C-terminus shown in white. B. Coomassie Brilliant Blue-stained SDS-PAGE gel showing the purified RbBP5 full length and truncated constructs. C. Comparison of WRAD catalyzed enzymatic activity on histone H3 peptides, when WRAD is assembled with the wild-type RbBP5 protein and RbBP5 variant constructs. The upper panel shows Coomassie Blue-Stained SDS-PAGE gel, and the lower panel shows [3H] methyl incorporation by fluorography. D. Comparison of the enzymatic activity of the MLL1 core complex when assembled with wild-type RbBP5 or the truncated RbBP5 constructs on unmodified and monomethylated histone H3 peptides. The upper panel shows Coomassie Blue-Stained SDS-PAGE gel, and the lower panel shows [3H]methyl incorporation by fluorography. 42 To test this hypothesis, the ability of each RbBP5 construct to catalyze methylation of unmodified histone H3 was tested in in vitro methyltransferase assays containing either wildtype or various truncated RbBP5 constructs with the remaining members of the WRAD sub complex. As shown in Figure 2.2 c, comparing lanes 1 and 2, when assembled with the remaining members of WRAD the truncated construct RbBP5 (1-402) has similar methyltransferase activity comparable to that of the wild-type WRAD complex (Figure 2.2 c, lanes 1 and 2). However, when the residues constituting the hinge domain are deleted in the RbBP5 (1-322) construct WRAD’s activity could not be observed (Figure 2.2 c, lane 3). Assembly of the RbBP5 fragment containing only residues 323-402 in WRAD shows that these residues alone are not sufficient on their own to restore activity compared to that of the fulllength protein when assembled in the WRAD sub-complex, although a trace amount of activity is observed (Figure 2.2c, lane 4). In sum, the WD40 repeat and hinge domains of (RbBP5 (1402)) constitutes the minimal domain that is sufficient and necessary for the activity of the WRAD sub complex. 2.3 Domains of RbBP5 Required for H3K4 dimethyltransferase Activity within the MLL1 Core Complex Given that the minimal domain of RbBP5 required to reconstitute the activity of WRAD consists of residues 1-402, we anticipated that this fragment of RbBP5 would also be sufficient to recapitulate di-methyltransferase activity within the MLL1 core complex. Therefore, we assembled the RbBP5 constructs we previously generated to test for enzymatic activity within the holo-MLL1 core complex, using both unmodified histone H3 peptide and mono-methylated histone H3 peptide as substrates. Consistent with previous published results, when the MLL1 core complex was assembled with the full-length RbBP5 protein it is capable of methylating an 43 unmodified and a previously mono-methylated peptide on histone H3K4 (Figure 2.2 d, lanes 1 and 2). As expected, the RbBP5 (1-402) construct containing the WD40-repeat region and the hinge region of RbBP5 possesses H3K4 mono- and dimethyltransferase activity similar to that of the wild type MLL1 core complex (Figure 2.2d lanes 3 and 4). However, the shorter construct, lacking the hinge domain exhibited full activity on an unmodified peptide (Figure 2.2d lane 5), but was not able to fully restore activity on a histone H3 peptide previously monomethylated at H3K4. (Figure 2.2d lane 6). Surprisingly, the small construct of RbBP5 containing only the hinge domain that lacked activity with WRAD when assembled in the MLL1 core complex exhibited activity similar to that of wild-type on both unmodified and mono-methylated peptides (Figure 2.2d lane 7 and 8). These results suggest that (residues 323-402) in the hinge region of RbBP5 are not only important for catalytic activity within WRAD, but more specifically contain catalytic residues important for di-methyltransferase activity mediated by the MLL1 core complex. 2.4 Alanine Scanning Mutagenesis Identifies Residues Involved in the Dimethyltransferase Activity of the MLL1 Core Complex Intrigued by the finding that the RbBP5 (323-402) construct was capable of facilitating the di-methyltransferase activity of the MLL1 core complex, we decided to further investigate the role of the hinge domain residues in RbBP5. We hypothesized that putative catalytic residues responsible for the di-methyltransferase activity of the MLL1 core complex probably reside within the 323-402 sequence of RbBP5. A multiple sequence alignment among 19 RbBP5 homologs reveals that this region possesses a 25 amino acid stretch that is highly conserved (Figure 2.3a). To gain insight into why this region displays such a high degree of conservation, we conducted an alanine scanning mutagenesis screen on this stretch of residues. To do so, point 44 mutations were introduced into the hinge region of RbBP5 for each residue from W329-K359 (letters represent the single letter codes for each amino acid). Alanine residues at positions 331 and 333 were mutated to serine residues. Each of the mutant proteins were expressed in E. coli, lysed, and crude extracts were used in assays to compare wild-type and mutant RbBP5 proteins using a H3K4me1 peptide as a substrate. The incorporation of a methyl group from 3[H] SAM into the peptide was monitored and visualized by fluorography. From this mutagenesis screen, we were able to identify several residues that when mutated reduced dimethyltransferase activity to varying degrees within the MLL1 core complex. As controls, reactions were set up with wildtype RbBP5 crude lysate (induced), and a reaction that contained the uninduced wild type RbBP5. To control for expression, crude lysates were probed with an anti-RbBP5 antibody to ensure that any observable decrease in the dimethylation activity of these enzymes assembled within the complex was not a result of the protein not being expressed (Figure 2.3b lower panel). The RbBP5 variant proteins that resulted in reduced di-methyltransferase activity are listed in Table 2.1. 45 46 FIGURE 2.3 ClustalW2 multiple sequence alignment of the predicted globular domain of RbBP5 showing only a subgroup of residues, and high throughput methyltransferase assays showing activity of the RbBP5 protein with mutations in conserved residues identified in its predicted globular region. A. The conserved globular motif of RbBP5 is highlighted in blue. Conserved residues are denoted underneath the alignment by an asterisk, conservative substitutions are denoted by a colon, and semiconservative substitutions are denoted by a period. B. Comparison of the enzymatic activity of the MWRAD complex on mono-methylated histone H3 (1-20) peptide when assembled with mutant RbBP5 proteins. The upper panel shows [3H] methyl incorporation into histone peptides as determined by fluorography. The lower panel shows immunoblotting of RbBP5 crude lysates used in this assay. 47 Table 2.1 48 Based on this screen, we decided to take a more quantitative approach to validate the results of this screen. Therefore, RbBP5 proteins containing mutations that resulted in reduced dimethyltransferase activity were expressed and purified as previously described (Patel et al., 2008). To ensure that point mutations were not altering the overall hydrodynamic shape of the proteins, sedimentation velocity analytical ultracentrifugation was conducted on individual proteins. Sedimentation boundaries obtained were analyzed by the continuous sedimentation coefficient distribution (c(s)) method in the program SEDFIT. Fitting of the boundaries demonstrates that these mutant RbBP5 proteins sediment as monodispersed species and with a sedimentation value of 2.4 s* for all proteins (Figure 2.4), similar to that of the wild-type RbBP5 protein (Patel et al., 2009). These results suggest that the mutations introduced in the RbBP5 variants do not significantly alter the overall hydrodynamic shape of RbBP5. Hydrodynamic parameters for all mutant RbBP5 proteins are summarized in Table 2.2. Circular dichroism spectroscopy was also performed on mutant RbBP5 proteins to determine if the point mutations alter the secondary structure fold of RbBP5. Superimposition of CD spectra of all RbBP5 variants suggest that no drastic changes in the secondary structure occurs (Figure 2.5). Once the correct folding of each mutant RbBP5 protein was established, their individual enzymatic contributions to the MLL1 core complex di-methyltransferase activity were assayed utilizing an independent assay. Using a quantitative Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF) methyltransferase assay, the ability for each mutant RbBP5 protein to catalyze mono- and di-methylation when assembled in the presence of other complex members on an unmodified peptide was tested in vitro. 49 FIGURE 2.4. Biophysical characterization of wild-type and mutant RbBP5 proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distributions (c(s)) derived from sedimentation velocity data of individual wild-type and mutant RbBP5 proteins. The experimental sedimentation coefficient (s) are indicated. 50 Table 2.2 Summary of sedimentation coefficients derived from sedimentation velocity analyses of the Retinoblastoma Binding Protein Five (RbBP5) expressed with mutations in various positions or expressed as truncated constructs Protein sa s20,wb f/fo RbBP5 (wild-‐type) 2.43 2.65 1.92 RbBP5(W329A) 2.43 2.65 1.92 RbBP5(W329E) 2.41 2.62 1.94 RbBP5(W329F) 2.43 2.65 1.92 RbBP5(W329R) 2.43 2.65 1.92 RbBP5(W329Y) 2.43 2.65 1.92 RbBP5(S330A) 2.42 2.63 1.93 RbBP5(A331S) 2.44 2.66 1.91 RbBP5(F332A) 2.38 2.59 1.96 RbBP5(A333S) 2.41 2.62 1.94 RbBP5(P334aA) 2.42 2.63 1.93 RbBP5(F336A) 2.42 2.63 1.93 RbBP5(K337A) 2.43 2.65 1.92 RbBP5(E338A) 2.41 2.62 1.94 RbBP5(Y345A) 2.43 2.65 1.92 RbBP5(E346A) 2.41 2.62 1.94 RbBP5(E347A) 2.43 2.65 1.92 RbBP5(S350A) 2.44 2.66 1.91 RbBP5(D358A) 2.44 2.66 1.91 a Experimental sedimentation coefficient determined at 10 °C. b Standard sedimentation coefficient (s20,w) after correcting for water at 20 °C. 51 FIGURE 2.5. Biophysical characterization of wild-type and mutant RbBP5 proteins by circular dichroism. Circular dichroism spectra of wild-type and variant RbBP5 proteins at 0.2 mg/mL. 52 Relative amounts of unmodified, monomethylated, and di-methylated H3K4 methylated species were quantitated over 24 hours. As negative controls, experiments containing only the MLL1 SET domain or the MLL1 core complex lacking the RbBP5 subunit were also performed. Similar to previous results with the MLL1 SET domain, the MLL1 SET domain displayed only mono-methyltransferase activity when using the unmodified histone H3 peptide substrate (Supplementary Figure 5, (S5)). Additionally consistent with earlier findings, absence of the RbBP5 subunit in the MLL1 core complex (MWAD) results in loss of H3K4 dimethylation (Figure 2.6). Unexpectantly, a number of mutants that were identified to have reduced activity in the high-throughput assay when tested for activity in the MALDI-TOF methyltransferase assay displayed enzymatic activity similar to that of the wild-type complex. Discrepancies between these two assays could potentially be attributed to differences in the substrates in the two assays, as well as the use of far less 3H- SAM in the high-throughput assay, as opposed to the MALDITOF based methyltransferase activity assay. Figures showing the enzymatic activity and kinetic behavior of these RbBP5 mutants along with any biophysical characterizations of these proteins are included in the appendices. However, given that the primary interest of this research is focused on identifying residues involved in the stimulation of the MLL1 mediated dimethyltransferase activity, the remaining parts of this chapter will focus on the mutations that displayed decreased dimethyltransferase activity in the high-throughput assay whose decreased activity were validated in separate experiments via the MALDI-TOF based methyltransferase assay. 53 54 FIGURE 2.6. MALDI-TOF mass spectrometry spectra of MWRAD and MWAD complexes. MALDI-TOF mass spectrometry of enzymatic reactions at 30 minutes, 3 hours, 6 hours and 24 hours. MALDI-TOF spectra in the left panel corresponds to the MLL1 core complex enzymatic activity of the complex assembled with full-length wild-type RbBP5. MALDI-TOF spectra in the right panel corresponds to the MLL1 core complex enzymatic activity of the complex assembled in the absence of full-length wild-type RbBP5. 55 Despite these discrepancies, we still were able to identify residues in RbBP5 that when mutated showed reduced dimethyltransferase activity. These residues include W329, F332, F336, and E347. In order to determine how these residues are specifically influencing the kinetic behavior of the MLL1 core complex, we used quantitative MALDI-TOF mass spectrometry to monitor the kinetic progression of H3K4 mono-, di- and tri-methylation of a histone H3 peptide under pre-steady state conditions. The average data obtained based on triplicate experiments were fitted with equations for kinetic models described for multiple irreversible consecutive reactions using the program, Dynafit. Using the following model, we were able to determine pseudo first order rate constants for the first and second methylation events for each individual mutant RbBP5 protein in the MLL1 complex (Figure 2 .7). k1 k2 H3K4 → H3K4me1 → H3K4me2 We were able to fit the MWRAD profiles with or without the addition of RbBP5 variant proteins by using the three equations listed below (Patel et al., 2009). The results of kinetic parameters obtained are summarized in Table 2.3. 1. [A]= [A]0 exp(-k1t) 2. [B]= [A]0 k1 ×[exp(-k1t)− exp(-k2t) k2 - k1 3. [C]= [A]0 {1+1 [k2 exp(-k1t)-k1-k1exp(-k2t)] k1-k2 56 [A]0 is the concentration of the unmodified peptide at time (t) zero. [B] and [C] represent the concentrations of the monomethylated and di-methylated species in single turnover curves, respectively. The terms k1 and k2 represent the pseudo first order rate constants for the conversion of A è B and BèC respectively (Patel et al., 2009; Patel et al., 2011). As shown in Figure 2.7 b, replacing W329 with alanine results in ~5-fold reduction in the k1 rate and a ~25 fold reduction in the k2 rate compared to the MLL1 core complex assembled with the wild-type RbBP5. Replacing, F336 with an alanine results in a ~3-fold reduction in the k1 rate and ~23fold reduction in the k2 rate of the reaction. Significantly, replacement of E347 with alanine results in almost complete abolishment of the k2 rate and a 56-fold reduction in k1. Interestingly, replacement of F332 with alanine in RbBP5 increases the overall k1 rate, but results in a 7-fold decrease in the overall k2 rate. Taken together, these results suggest that the residues, W329, F332, F336, and E347 are critical for the methylation kinetics of the MLL1 core complex. Collectively, these results suggest direct participation by the RbBP5 component in H3K4 dimethylation. 57 FIGURE 2.7. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry. A-E. Comparison of the single turnover progress curves for reactions catalyzed by the MLL1 core complex with wild-type RbBP5 or with the RbBP5 variants; RbBP5(W329A), RbBP5(F332A), RbBP5(F336A), and RbBP5(E347A) from MALDI-TOF mass spectrometry assays. The data for H3K4, H3K4me1, and H3K4me2, species were globally fitted to Equations 1-3 (see “RESULTS, chapter 2) using DynaFIT. Error bars represent ± S.E. from duplicate experiments. F. Normalized activity derived from k1 and k2 rates constants of reactions catalyzed by the MLL1 core complex with RbBP5 wild-type or RbBP5 variants; RbBP5(W329A), RbBP5(F332A), RbBP5(F336A), and RbBP5(E347A). . 58 Table 2.3 Summary of Rate Constants derived from globally fitting experimental data. a Rate constants (± S.E) derived from global fitting the data to equations 1-3 results section of chapter 2. 59 2.5 Alanine substitutions in RbBP5 at positions W329, F332, F366, and E347 disrupt WRAD’s ability to bind MLL1 To determine whether the mutations generated affected the ability of RbBP5 variants to form the WRAD sub-complex and MLL1 core complex, sedimentation velocity analytical ultracentrifugation was used to compare wild-type and variant RbBP5 proteins for their ability to interact with members of the complex. Based on these experiments, we show that compared to the wild-type WRAD complex, sedimentation velocity AUC reveals that the W329A, F332A, F336A, and E347A, proteins are all capable of assembling into the WRAD complex with sedimentation coefficients similar to that of wild type RbBP5 (Figures 2.8-2.10). This suggests that mutations at positions W329A, F332A, F336A (data not shown), and E347A, do not interfere with the mutant proteins’ ability to form WRAD. Varying the concentrations produces slight shifts in the WRAD complexes assembled with the different mutations suggesting that the interactions are less stable within the WRAD complex. This behavior is still consistent with the conclusion that these mutations are not affecting the overall stability of the WRAD complex, as previously published reports observe the same type of behavior over a similar concentration range for wild-type WRAD (Patel et al., 2009; Patel et al., 2011). 60 FIGURE 2.8. Biophysical characterization of wild-type WRAD and WR(W329A)AD proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the WRAD sub-complex assembled with stoichiometric amounts of wild-type WDR5, RbBP5, Ash2L, and DPY30 (upper panel) and the WRAD sub-complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(W329A) , Ash2L, and DPY30 (lower panel) . The experimental sedimentation coefficient (s) are indicated. 61 FIGURE 2.9. Biophysical characterization of wild-type WRAD and WR(F332A)AD proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the WRAD sub-complex assembled with stoichiometric amounts of WDR5, RbBP5, Ash2L, and DPY30 (upper panel) and the WRAD sub-complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(F332A), Ash2L, and DPY30 (lower panel). The experimental sedimentation coefficient (s) are indicated. 62 FIGURE 2.10. Biophysical characterization of wild-type WRAD and WR(E347A)AD proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the WRAD sub-complex assembled with stoichiometric amounts of WDR5, RbBP5, Ash2L, and DPY30 (upper panel) and the WRAD sub-complex assembled with stoichiometric amounts of WDR5, RbBP5(E347A) , Ash2L, and DPY30 (lower panel). The experimental sedimentation coefficient (s) are indicated. 63 We next added stoichiometric amounts of MLL1 to the individual WRAD complexes, and tested for their ability to interact with the MLL1 SET domain (Figure 2.11-2.14). Sedimentation velocity analytical ultracentrifugation reveals that the addition of MLL1 to the WR(W329A)AD sub complex does not shift the sedimentation coefficient of WRAD indicating that this complex is unable to bind the MLL1 SET domain (Figure 2.11). Similar results are obtained when MLL1 is added in a stoichiometric amount to the WR(F332A)AD, and WR(E347A)AD complexes (Figure 2.12 and 2.14). A slight difference in behavior is observed when MLL1 is added to the WR(F336A)AD complex (Figure 2.13). At a higher concentration, the complex sediments with an s- value intermediate between wild-type WRAD and the wildtype MLL1 core complex (Figure 2.13). Dilution of this mixture, resulted in the sedimentation peak shifting to a lower s value, suggesting that the interaction between the MLL1 and WR(F336A)AD dissociates on a relatively rapid time scale when compared to the time scale of sedimentation (Figure 2.13). In addition, to test if there are some weak interactions that exist between WR(W329A)AD and the MLL1 SET domain, the MLL1 protein was added in excess to WRAD. Titration of excess MLL1 reveals no shift in the WR(W329A)AD sedimentation profile consistent with the hypothesis that MLL1 does not interact with WRAD (Figure 2.15.). As a whole, these results suggests that although the interaction is much weaker for the WR(F336A)AD complex between the MLL1 SET domain, the WR(F336A)AD still can form some contacts with the MLL1 SET domain. In summary, these results provide evidence that the residues W329, F332, F336, and E347 are essential for interaction between MLL1 and WRAD and are indispensable for H3K4 dimethyltransferase activity of the MLL1 core complex. 64 FIGURE 2.11. Biophysical characterization of wild-type MWRAD and MWR(W329A)AD proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5, Ash2L, and DPY30 (upper panel) and the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(W329A) , Ash2L, and DPY30 (lower panel). The experimental sedimentation coefficient (s) are indicated. 65 FIGURE 2.12. Biophysical characterization of wild-type MWRAD and MWR(F332A)AD proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5, Ash2L, and DPY30 (upper panel) and the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(F332A) , Ash2L, and DPY30 (lower panel). The experimental sedimentation coefficient (s) are indicated. 66 FIGURE 2.13. Biophysical characterization of wild-type MWRAD and MWR(F336A)AD proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5, Ash2L, and DPY30 (upper panel) and the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(F336A), Ash2L, and DPY30 (lower panel). The experimental sedimentation coefficient (s) are indicated. 67 FIGURE 2.14. Biophysical characterization of wild-type MWRAD and MWR(E347A)AD proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5, Ash2L, and DPY30 (upper panel) and the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(E347A), Ash2L, and DPY30 (lower panel). The experimental sedimentation coefficient (s) are indicated. 68 FIGURE 2.15. Biophysical characterization of wild-type MWR(W329A)AD protein by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with excess MLL13745, and stoichiometric amounts of WDR5, RbBP5(W329A), Ash2L, and DPY30.The experimental sedimentation coefficient (s) are indicated. 69 The results presented emphasize a more intricate role of MLL1 SET domain methyltransferase activity than previously thought. Notably, three out of the four residues identified in the high-throughput screen important for interaction with MLL1 were aromatic residues. Intrigued by this finding, we wondered if the loss of activity observed by replacing the aromatic residues with an alanine replacement could be rescued with another aromatic residue. Therefore, we replaced W329 with both phenylalanine and tyrosine. We also replaced the conserved tryptophan residue with an arginine, an amino acid residue with some aromatic properties, to see what effect these residues would have on the enzymatic activity and the assembly of the MLL1 core complex. We hypothesized that replacement of tryptophan at position 329 with aromatic residues would restore dimethyltransferase activity, and restore the ability of the RbBP5 protein to incorporate back into the MLL1 core complex. Therefore, we expressed and purified these proteins as previously described. CD spectra of these variant proteins show that the mutations do not introduce secondary structure alterations (Figure 2.16). Methylation kinetics were monitored under presteady state conditions using quantitative MALDI-TOF mass spectrometry. 70 FIGURE 2.16. Biophysical characterization of wild-type and mutant RbBP5 proteins by circular dichroism. Circular dichroism spectra of RbBP5 wild-type and mutant RbBP5 proteins at 0.2 mg/mL A. Superimposition of circular dichroism spectra of RbBP5 wild-type and mutant RbBP5 proteins RbBP5(W329A), RbBP5(W329Y), and RbBP5(W329F). B. Superimposition of circular dichroism spectra of RbBP5 wild-type and mutant protein RbBP5(W329R). 71 When the enzymatic activity assay was conducted, we observe partial restoration of dimethylation for the W329F and the W329Y mutations when assembled in the complex (Figure 2.17). For the W329F RbBP5 protein, we observe a slight increase in the k1 rate, and dimethyltransferase activity restoration; however, the overall catalyzed reaction k2 rate is slower than the k2 rate of wild-type. Similarly, when W329 is substituted with tyrosine, partial restoration of the enzymatic activity of the overall complex is observed. Substitution of the conserved tryptophan instead with arginine (W329R), a residue with some aromatic properties but with a positive charge, however, is unable to restore the enzymatic activity of the MLL1 core complex. After identifying the methylation kinetics, when wanted to know if the (W329F) RbBP5, the (W329Y) RbBP5, and the (W329R) RbBP5 proteins were capable of assembling into the MLL1 core complex. Sedimentation velocity analytical ultracentrifugation of the (W329F) RbBP5 shows that this protein is capable of forming complex; however, addition of this mutation results in a different overall hydrodynamic shape compared to wild-type (Figure 2.18). Similarly, sedimentation velocity analytical ultracentrifugation reveals that the W329Y variant RbBP5 is capable of forming the complex but with a different hydrodynamic shape. Lastly, substitution of the conserved tryptophan with arginine (W329R) prevents assembly in the MLL1 core complex (Figure 2.19). Taken together, these results suggest that the aromatic properties of the conserved tryptophan residue at position 329 of RbBP5 are required for a direct interaction with MLL1 within the MLL1 core complex. 72 FIGURE 2.17. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry. A-E. Comparison of the single turnover progress curves for reactions catalyzed by the MLL1 core complex with wild-type RbBP5 or with the RbBP5 variants; RbBP5(W329A), RbBP5(W329F), RbBP5(W329F) and RbBP5(W329R), from MALDI-TOF mass spectrometry assays. The data for H3K4, H3K4me1, and H3K4me2, species were globally fitted to Equations 1-3 (see “RESULTS,” Chapter 2) using DynaFIT. Error bars represent ± S.E. from duplicate experiments. . 73 Table 2.4 Summary of Rate Constants derived from globally fitting experimental data. a Rate constants (± S.E) derived from global fitting the data to equations 1-3 results section of chapter 2. 74 FIGURE 2.18. Biophysical characterization of wild-type MWRAD, MWR(W329F)AD, and MWR(W329Y)AD by analytical ultracentrifugation. A. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5, Ash2L, and DPY30. B. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(W329F), Ash2L, and DPY30. C. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(W329Y), Ash2L, and DPY30. The experimental sedimentation coefficient (s) are indicated. 75 FIGURE 2.19. Biophysical characterization of wild-type MWRAD, and MWR(W329R)AD, by analytical ultracentrifugation. A. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5, Ash2L, and DPY30. B. Diffusion-free sedimentation coefficient distribution (c(s)) derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with stoichiometric amounts of MLL13745, WDR5, RbBP5(W329R), Ash2L, and DPY30. The experimental sedimentation coefficient (s) are indicated. 76 2.6 Confirmation that Valine Residues 375 and 377 are Critical in the RbBP5-WDR5 Interaction Previous reports have suggested that residues Valine 375 and Valine 377 of RbBP5 confer its binding to WDR5 (Odho et al., 2010). Taking a more stringent biophysical and kinetic approach, I have been able to further characterize the WDR5-RbBP5 interaction. Given that Valine 375 and Valine 377 of RbBP5 were identified as major residues recognized by WDR5 to establish an interaction, I decided to mutate both Valine 375 and Valine 377 to alanine residues. All plasmids were sequenced and verified to ensure that they contained the correct mutation and no additional unwanted mutations. These proteins were recombinantly expressed in E.coli, and purified as described in the experiment procedures section. Sedimentation velocity analytical ultracentrifugation experiments were performed demonstrating that mutating Valine 375 and Valine 377 to alanine residues did not distort the overall hydrodynamic shape of the mutant proteins. As shown in Figure 2.20, sedimentation velocity analytical ultracentrifugation of the isolated RbBP5 variants containing alanine substitutions at positions 375 and 377 reveal monodispersed proteins in solution with an experimental sedimentation coefficient of 2.4 s like that of the wild-type protein (Figure 2.20). Circular dichroism spectroscopy was also performed to establish that the mutant proteins also displayed unaltered overall secondary structures. As show in Figure 2.20b there are no major conformation changes in CD spectra of the mutant RbBP5 proteins when compared to the wild-type protein. After confirmation that these mutations did not disrupt the overall structure of RbBP5, I wanted to determine how these proteins influence the enzymatic behavior of the MLL1 core complex when assembled in the presence of other interacting proteins. To do so, MALDI-TOF methyltransferase assays were conducted and rate constants by globally fitting single turnover kinetic curves measured by the MALDI-TOF mass spectrometry were determined (Figure 77 2.21a). As shown in Figure 2.21 substituting Valine 375 with alanine results in ~2-fold reduction in the k1 rate and a ~4-fold reduction in the k2 rate. Replacing, Valine 377 with an alanine results in a small overall reduction in the k1 rate and an ~ 3- fold overall reduction in the k2 rate. These results suggests that although Valine 375 and Valine 377 may establish important contacts with WDR5, these are not the only contributions to this interaction, and the ability of the RbBP5Ash2L-DPY30 sub complex to interact with the MLL-WDR5, is not entirely abolished. The most likely scenario is that the M-W sub complex is in rapid exchange with the RbBP5-Ash2LDPY30 sub complex and the activity observed is reminiscent of these transient interactions. To determine whether the Valine mutations in RbBP5 disrupt its interactions with WDR5, sedimentation velocity analytical ultracentrifugation was performed of stoichiometric amounts of WDR5-RbBP5 binary complexes (Figure 2.22) Results suggest that mutations of Valines 375 and 377 do not completely abolish the protein’s ability to interact with WDR5. Taking an alternative approach I have been able to validate the previous findings on the WDR5-RbBP5 interaction. All results here are consistent with previous findings. 78 FIGURE 2.20. Biophysical characterization of RbBP5 wild-type and RbBP5 RbBP5 (V377A) proteins. (V375A) and A. Diffusion-free sedimentation coefficient distributions (c(s)) derived from sedimentation velocity data of individual wild-type and mutant RbBP5 proteins. The experimental sedimentation coefficient (s) are indicated. B. Circular dichroism spectra of wild-type and mutant RbBP5 proteins at 0.2 mg/mL 79 FIGURE 2.21. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry. A-C. Comparison of the single turnover progress curves for reactions catalyzed by the MLL1 core complex with wild-type RbBP5 or with the RbBP5 variants; RbBP5(V375A), and RbBP5(V377A) from MALDI-TOF mass spectrometry assays. The data for H3K4, H3K4me1, and H3K4me2, species were globally fitted to Equations 1-3 (see “RESULTS,” Chapter 2) using DynaFIT. Error bars represent ± S.E. from duplicate experiments. D. Normalized activity derived from k1 and k2 rates constants of reactions catalyzed by the MLL1 core complex with RbBP5 wild-type or RbBP5 variants; RbBP5(V375A), and RbBP5(V377A). E. Summary of rate constants derived from globally fitting experimental data. 80 FIGURE 2.22. Biophysical characterization of WDR5-RbBP5 RbBP5(V377A) interactions. (V375A) and WDR5- A. Diffusion-free sedimentation coefficient distributions (c(s)) derived from sedimentation velocity analytical ultracentrifugation data of binary complexes at a concentration of 5uM WDR5-RbBP5(V375A) and B. 5uM WDR5-RbBP5(V377A). 81 DISCUSSION: Histone H3K4 methylation is a prevalent mark that positively regulates transcriptional activation. The SET1 family of enzymes, which include MLL1-MLL4 and SET1A and SET1B carry out the bulk of H3K4 di- and trimethylation in vivo. As noted above, studies on the representative member of this family, MLL1, has provided several insights on mechanistic determinants underlying the regulation of multiple lysine methylation. In common with the vast majority of lysine methyltransferases, MLL1 contains an evolutionarily conserved SET domain required for its enzymatic activity. However, unlike most other lysine methyltransferase, the MLL1/SET1 class of enzymes can only catalyze multiple methylation in the context of a multisubunit core configuration of proteins, which includes WDR5, RbBP5, Ash2L, and DPY30. These proteins can exist independent of MLL1 and form a sub -complex called WRAD. WRAD binds in close proximity to the catalytic SET domain of MLL1 and together forms what we call the MLL1 core complex. Currently, there are two proposed models that have emerged detailing the mechanism behind multiple methylation catalyzed by the MLL1 core complex. The first model “the one active site model” posits that mono-, di-, and tri-methylation of H3K4 can be attributed solely to the methyltransferase activity of the SET domain alone, and that the WRAD module merely functions as an allosteric regulator of the MLL1 catalytic SET domain (Couture et al., 2008; Dillon et al., 2005; Southall et al., 2009). However, it has been more recently demonstrated that an isolated MLL1 SET domain is predominantly a mono-methyltransferase (Patel et al., 2009). This is consistent with the hypothesis that the presence of a conserved tyrosine in the active site of SET domain enzymes constrains them to be monomethyltransferases. Unexpectantly, it has also been shown that WRAD forms an 82 independent sub-complex that possesses an intrinsic histone methyltransferase activity of its own in the absence of the MLL1 SET domain (Patel et al., 2011). Furthermore, when WRAD is assembled within the MLL1 core complex it is capable of catalyzing H3K4 di-methylation. These studies have thereby lead to a new model by which the degree of methylation of histone H3 at lysine 4 is regulated in a sequential fashion catalyzed by a multi-subunit complex that contains two distinct active sites for the addition of each methyl group. In agreement with this model, additional studies reveal that the loss of the WDR5, RbBP5, or Ash2L subunit of WRAD results in the loss of di- methylation in vitro, without significant changes in H3K4 monomethylation. Taken together, these studies provide evidence that suggests members of the conserved core configuration of proteins in the WRAD complex possess essential protein structural features underlying the shared enzymatic activities of the MLL1 core complex. Although, there has been a wealth of reports on the assembly and function of the MLL1 core complex’s enzymatic activity, there is a lack of information that exists on the functions and contributions of individual conserved components and their detailed characterizations. In this thesis, I sought to thoroughly characterize the role of the RbBP5 hinge domain in facilitating the di-methyltransferase activity in the MLL1 core complex. RbBP5 is a member of the Rb pathway, and the absence of RbBP5 in the MLL1 core complex results in no observable dimethylation in vitro (Patel et al., 2011). To begin to explore the role of this protein in the MLL1 core complex di-methyltransferase activity, a series of RbBP5 constructs were engineered to identify the minimal domain required for activity. A small fragment of RbBP5 consisting of residues (323-402) was able to restore di-methylation near to wild-type levels. Further sequence alignments reveal that this fragment contains an absolutely conserved stretch of amino acid residues. High-throughput scanning alanine mutagenesis of this stretch identified a number of 83 residues that when mutated result in decreased levels of dimethylation relative to that of wildtype. Further characterization of residues identified in this screen, reveal that residues W329, F332, F336, and E347 are all required for di-methylation within the MLL1 core complex, as mutations in any of these residues result in significantly reduced di-methyltransferase activity of the complex. Surprisingly, when any of the mutations were assembled within the MLL1 complex, they diminish WRAD’s ability to directly interact with the MLL1 SET domain. Although, previous studies have implicated interaction surfaces between RbBP5 and the MLL1 SET domain, to date there have been no studies that have clearly demonstrated potential surfaces of RbBP5 involved in this interaction. Therefore, through extensive biochemical and biophysical characterization of the RbBP5 hinge domain, we have been able to uncover a novel, previously unidentified MLL1 interaction motif in this B-propeller protein that preferentially recognizes the SET domain of MLL1. In light of our finding that three out of the four residues identified responsible for contacts with MLL are aromatic, we propose that these residues may create a hydrophobic interface with consensus hydrophobic residues on the surface of MLL1. Mutation of conserved amino acids residues in the MLL1 SET domain will facilitate the identification surfaces in MLL1 that confer its binding to RbBP5. Preferential binding of RbBP5 to MLL1 through this motif, may alter the conformation of the MLL1 SET domain, therefore enhancing catalysis. We envision an intriguing scenario whereby the RbBP5 component binds to the MLL1 SET domain, so that these aromatic residues may directly participate in catalysis, by facilitating the optimal alignment of the lysine substrate and the co-factor SAM (Figure 2.23). It has been well- established from the most well-studied SET domains, that the formation of the substrate lysine binding channel involves the inclusion of several aromatic residues which insert themselves between histone H3 and s-adensoyl methionine to achieve a favorable physio- 84 chemical environment for the transfer of a methyl group from SAM to the amine group of lysine four on H3. Strikingly, the recent crystal structure report of the MLL1 SET domain reveals that the SET-I subdomain is not in an optimal alignment to form this archetypal binding site and lacks the highly conserved aromatic residues. Therefore, it is quite possible that conserved aromatic residues in RbBP5 elicit binding with MLL1 to achieve this key structural characteristic. It is important to note that one residue, E347 identified in this thesis was neither hydrophobic nor aromatic that was also shown to be important for binding MLL1, it’s role in the catalysis remains to be explored. In our model, we also propose that in addition to binding to MLL1 to form the hydrophobic channel that accommodates and connects the substrate and cofactor, this channel also links the first active site to the second active site in WRAD for transfer of the peptide substrate to WRAD. In this scenario, RbBP5 when in complex with other members of WRAD, binds the MLL1 SET domain enhancing catalysis by first proper alignment of substrate and cofactor and increasing the overall rate of mono-methylation, by forming the hydrophobic channel, while simultaneously facilitating the transfer of the peptide to the second active site in WRAD. In WRAD RbBP5 is needed for making important contacts between the WDR5 and Ash2L subunits to mediate MLL1 core complex dimethyltransferase activity by aiding in formation of the second active site in WRAD. The results presented in this thesis suggests this mode of catalysis, however solving the high-resolution crystal structure of the MLL1 core complex with bound peptide will prove imperative in determining if this mechanism of catalysis is at play. 85 FIGURE 2.23. Model for multiple methylation by the MLL1 core complex. In this model, the MLL1 SET (c-terminal) domain catalyzes the first methylation event monomethylation of histone H3. This rate of reaction is increased by making contacts with RbBP5 and the formation of a hydrophobic channel. The hydrophobic channel created by RbBP5 allows for optimal alignment of the substrate and a subsequent more efficient transfer of the substrate to the second active site in WRAD which catalyzes di-methylation of histone H3. 86 Chapter Three: Crystallization Trials and Probing the Structure of RbBP5 By Limited Proteolysis Identifies Two Previously Uncharacterized Stable Subdomains 87 INTRODUCTION: The RbBP5 gene, a member of the Rb pathway, encodes a protein of 538 amino acid residues. The only know conserved domain of this protein based on homology predictions is the WD40 repeat domain (Figure 3.1). Known structures of WD40 repeat proteins shown that this motif generally adopts a beta-propeller architecture often consisting of seven blades. Several reports have established RbBP5 as a component of H3K4 methyltransferase complexes. Histone H3 lysine four methylation, is an epigenetic mark commonly associated with transcriptionally active states of chromatin. Additional studies have shown that disruption or deletion of RbBP5 elicits decreased H3K4 methylation in vitro and in vivo. Despite its seemingly critical role in H3K4 methyltransferases, the function of RbBP5 is not well defined. Knowledge of a protein’s three-dimensional structure is often critical for understanding function. Therefore, the second part of the work in this thesis was focused on the determination of a high-resolution structure of RbBP5 by X-ray crystallography. Although, there was a report documenting the 3-dimensitional structure of WDR5 in complex with a short synthetic RbBP5 peptide (Odho et al., 2010), there is currently no three dimensional structure of full-length RbBP5. The general approach I took in obtaining well diffracting crystals of RbBP5 can be summarized in the following steps, 1) rapid sparse matrix screening of crystallization conditions by the hanging drop vapor diffusion of the RbBP5 protein, followed by optimization of promising conditions and finally collection of data at synchrotrons. The structure would have been determined by molecular replacement. 88 FIGURE 3.1. Schematic representation of the human retinoblastoma binding protein five (RbBP5) showing the domain architecture of recombinantly expressed full-length RbBP5. Full-length RbBP5 contains 538 amino acid residues with a WD40 repeat domain based on homology modeling. 89 Perhaps, the most challenging part of this goal of my project was the crystallization and structure determination of RbBP5 itself. Unfortunately, no suitable crystals were obtained by this first approach taken on the full- length human RbBP5 protein. Therefore, I subjected the full -length protein to limited proteolysis treatment to identify a stable construct that was amendable to crystallization. From the limited proteolysis, I was able to obtain and identify two stable subdomains of the RbBP5 protein. These minimal domains were re-cloned, purified, and subjected to more sparse matrix screening crystallization trials. Although no crystals emerged from early crystallization trials, I was able to biophysically characterization the intrinsic properties of the two stable domains identified. The experiment procedures and results are presented below. EXPERIMENTAL PROCEDURES Protein expression and purification: The RbBP5 gene was purchased from open biosystems and subcloned into a pHis parallel vector which encodes a tobacco etch virus (TEV) cleavage Nterminal 6x-His fusion. Versions of full-length human RbBP5 (1-538) and RbBP5 constructs were overexpressed in Escherichia coli (Rosetta II, Novagen), by growing cells containing a given plasmid at 37°C in Terrific Broth medium containing 50ug/mL carbenicillin. The temperature was then lowered to 15 °C and cells were induced for 16–18 h with 750uM isopropyl-1-thio-D-galactopyranoside (IPTG). Cells were harvested, re-suspended in lysis buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 3 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, and 1 EDTA-free protease inhibitor tablet (RocheApplied Science)), lysed with a microfluidizer cell disrupter, and clarified by centrifugation. Supernatants containing the RbBP5 His-tagged proteins were purified by nickel affinity chromatography 90 (HisTrap column, GE Healthcare). For the first step of purification, the crude lysate was passed through a HisTrap column (GE healthcare) containing nickel beads. The bound 6x-His-RbBP5 was eluted from the column using a linear gradient of elution buffer containing 500mM imidazole. The peak fractions that contained RbBP5 were collected, pooled, and then dialyzed against column buffer that contains 50 mM Tris pH 7.4, 300mM NaCl, 30 mM Imidazole, and 3mM DTT at 4°C to remove the excess imidazole and to cleave the 6X-His tag in the presence of TEV (Tobacco Etch Virus) protease. The dialysis buffer was changed three times. For the second step of purification, the dialyzed protein was passed through the His-Trap column and the flow through fractions that contained the untagged version of RbBP5 were pooled and combined. As a final step of purification, the protein was passed through a gel filtration column (Superdex 200TM GE Healthcare) pre-equilibrated with the sample buffer containing 20mM Tris (pH 7.5), 300mM NaCl, 1mM Tris (2-carboxyethyl) phosphine (TCEP), and 1µM zinc chloride. All other proteins used in methyltransferase assays were purified as previously described (Patel et al., 2008). Mutagenesis: Premature stop codons were introduced into human RbBP5 clones using the QuickChange site directed mutagenesis kit (Stratagene) to create truncated RbBP5 constructs. Plasmids were sequenced to verify the presence of the intended mutations and the absence of additional mutations. Integrated DNA Technologies synthesized oligonucleotide primers used in the mutagenesis. Limited Proteolysis Experiments: Fresh Trypsin Stock was prepared at 0.2ng/uL. For maximum of activity sequencing grade modified trypsin was resuspended in trypsin resuspension buffer and the trypsin enzyme mixture was allowed to warm at room temperature for 15 minutes. 100uL reactions with trypsin at a final concentration of 0.01ng/uL and a final protein concentration of 91 0.5mg/mL were prepared in 1.5mL micro centrifuge tubes. The ingredients were mixed gently by flicking. Limited proteolysis of the protein was carried out in a 37 °C water bath. The reactions were quenched at various time points by the addition of PMSF. SDS-PAGE followed by coomassie staining was used to visualize any stable subdomains identified in the reaction mixture at various times points and their approximate molecular weights. Experiments were repeated in triplicate sets to confirm appropriate conditions for the limited proteolysis of the protein. Reaction mixtures with stable subdomain masses were confirmed using ESI Mass Spectrometry (Yale School of Medicine, W.M. Keck Biotechnology Resource Laboratory). Sequences were confirmed using N-terminal sequencing (Johns Hopkins Institute for Nano biotechnology, Proteometrics Synthesis and Sequencing Facility.) MALDI-TOF mass spectrometry methyltransferase assay: Mass spectrometry assays were conducted as mentioned earlier. 7 µM of enzyme was incubated with 250 µM s-adenosylmethionine and 10 µM histone H3 peptide (1-20) at 15 °C in 50mM Tris pH 9.0, 200mM NaCl, 3 mM DTT, and 5% glycerol. The reactions were quenched at various time points by the addition of trifluoroacetic acid to 0.5%. The quenched samples were diluted 1:4 with α-cyano-4hydroxycinnamic acid. MALDI TOF mass spectrometry was performed on a Bruker AutoFlex mass spectrometer (State University of New York, ESF) operated in reflectron mode. Final spectra were averaged from 100 shots/position at 10 different positions. Analytical Ultracentrifugation: Analytical ultracentrifugation experiments were carried out using a Beckman Coulter ProteomeLabTM XL-A analytical ultracentrifuge equipped with absorbance optics and a 4-hole An-50 Ti analytical rotor. Sedimentation velocity experiments were carried out at 10°C and 50,000 rpm (200,000 x g) using 3 mm two sector charcoal-filled Epon centerpieces with quartz windows. For each sample, 300 scans were collected with the time 92 interval between scans set to zero. Protein samples in 20mM Tris pH 7.5, 300mM NaCl, 1mM TCEP and 1µM ZnCl2 were run at various concentrations as described in results. Sedimentation boundaries were analyzed by the continuous distribution (c(s)) method using the program SEDFIT. The program SEDNTERP version 1.09 was used to correct the experimental S-value (s*) to standard conditions at 20°C in water (S20,w) and to calculate the partial specific volume of each protein. Circular Dichroism Spectroscopy: CD spectra were collected on an AVIV Model 420 ds spectropolarimeter equipped with a Neslab CFT-33 refrigerated circulator at 10°C using a 0.1 mm path length cell. Spectra for each were obtained at a protein concentration of 0.2mg/mL in a buffer contain 20mM TRIS (pH 7.5), 200 mM NaCl, 1mM TCEP and 1uM Zinc Chloride. The background contribution of the buffer alone was subtracted from each protein spectrum. RESULTS: Through limited proteolysis of the RbBP5 protein (Figure 3.2), two stable subdomains were identified and confirmed through the use of N-terminal sequencing, electrospray ionization mass spectrometry, and an in silico digestion of predicted cleavage sites in full-length RbBP5. These subdomains correspond to residues #1-495 and #1-337 in RbBP5. To characterize the hydrodynamic properties of these constructs in isolation sedimentation velocity analytical ultracentrifugation was performed. Sedimentation velocity profiles were fitted to an array of Lamn equations solutions with a set of experimental parameters to determine the diffusion free sedimentation coefficient distributions (c(s)). As show in Figure 3.3, RbBP5 (#1-495) sediments as a monodispersed protein with a sedimentation coefficient like that of wild-type. (Figure 3.3) CD spectra of this protein reveals that the deletion of the C-terminal residues does not induce any 93 drastic conformational changes in this protein (Figure 3.4). In contrast sedimentation velocity analytical ultracentrifugation of the RbBP5 (#1-337) proteins reveals that this proteins sediments as a monodispersed protein as well with a c(s) value of 2.16 (Figure 3.5). CD spectra of this protein reveals, that the deletion of its C-terminus alters in overall secondary structural conformation (Figure 3.6). To determine the methylation kinetics of these two protein constructs, enzymatic assays were set up under pre-steady state conditions, and methylation of an unmodified histone H3 peptide was monitored using MALDI-TOF mass spectrometry. As shown in Figure 3.7, the overall k1 and k2 rates of reactions catalyzed by MWR(#1-495)AD are much slower compared to that of the wild-type, although similar levels of dimethylation are observed after 24 hours. With respect to MWR(#1-337)AD, a loss of di-methylation is readily observed with a slow monomethylation rate. This could possibly be attributed to less stability of the complex when the constructs are assembled with the other components. 94 1 2 3 4 5 6 7 8 FIGURE 3.2. Limited proteolysis of full-length RbBP5. Full-length RbBP5 was incubated with trypsin. Aliquots were quenched at various time points and SDS-PAGE followed by Coomassie Staining was performed. Lane one shows the standard. Lane two shows the full length RbBP5 protein in the absence of trypsin at a concentration of 0.5mg/mL. Lanes 3-8 shows the full-length protein after being incubated with 0.01ng/uL of trypsin for various amounts of time. Aliquots at each time point were quenched by the addition of PMSF. 95 FIGURE 3.3. Biophysical characterization of wild-type RbBP5 (#1-495) proteins by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distributions (c(s)) derived from sedimentation velocity data of the RbBP5 (#1-495) construct. The experimental sedimentation coefficient (s) are indicated. 96 FIGURE 3.4. Biophysical characterization of RbBP5 wild-type and RbBP5 (#1-495) proteins by circular dichroism. Circular dichroism spectra of full-length wild-type and variant RbBP5 proteins at 0.2 mg/mL 97 FIGURE 3.5. Biophysical characterization of RbBP5 (#1-337) by analytical ultracentrifugation. Diffusion-free sedimentation coefficient distributions (c(s)) derived from sedimentation velocity data of the RbBP5 (#1-337) construct. The experimental sedimentation coefficient (s) are indicated. 98 FIGURE 3.6. Biophysical characterization of RbBP5 (#1-337) by circular dichroism. Circular dichroism spectra of wild-type and the RbBP5 (#1-337) proteins at 0.2 mg/mL. 99 FIGURE 3.7. Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry. A-C. Comparison of the of single turnover progress curves for reactions catalyzed by the MLL1 core complex with a, wild-type RbBP5, b, RbBP5(#1-495) and c, RbBP5(#1-337) from MALDITOF mass spectrometry assays. The data for H3K4, H3K4me1, and H3K4me2, species were globally fitted to Equations 1-3 (see “RESULTS,” Chapter 2) using DynaFIT. Error bars represent ± S.E. from duplicate experiments. 100 Table 3.1 Summary of rate constants derived from globally fitting experimental data. a Rate constants (± S.E) derived from global fitting the data to equations 1-3 results section of chapter 2. 101 DISCUSSION: The RbBP5 gene encodes a nuclear protein which belongs to a family of WD-repeat proteins. The encoded protein binds directly to the retinoblastoma protein, which regulates cell proliferation. Despite RbBP5’s critical role in mammalian methyltransferase complexes relatively little is know about this protein’s involvement in this activity and currently there is no high resolution structure of the protein available. One goal of this thesis was to attempt to solve the three dimensional structure of RbBP5. After extensive sparse matrix screening on full-length RbBP5, suitable crystals were not obtained. Therefore, I chose to take an alternative approach utilizing limited proteolysis of fulllength RbBP5 in attempt to identify and obtain a stable truncated protein that may potentially be more suitable for crystallization. Given that proteins generally have a modular structure consisting of several distinctive domains tethered by flexible regions, I hypothesized that these flexible regions could potentially be creating a source of heterogeneity preventing crystallization with full-length RbBP5. One general aspect of protein crystallization that is universally recognized is that rigid, stable proteins are much more likely to crystallize than proteins that are internally flexible or have dynamic surfaces. Thus, deleting or re- moving the flexible regions at the termini or within a protein may help in the crystallization by minimizing any interfering effects from the micro heterogeneity. Taking a limited proteolysis approach, I was able to identify two stable subdomains in RbBP5 consisting of residues #1-495 and residues #1-337. These domains were cloned and characterized. These results presented in the aforementioned further confirm domain mapping experiments performed by Anamika Patel. Consistent with previous findings, deleting the end most c-termini as in the construct containing residues #1-495, 102 does not significantly effect the overall enzymatic activity of the complex, although it does results in a rate fold reduction. Deletion of residues containing the hinge domain of RbBP5 result in a complete lost of di-methyltransferase activity within the MLL1 core complex. When the RbBP5 constructs were put into sparse matrix screening crystallization trials, no crystals emerged from early screening trials. However, the identification of these two stable sub-domains provide a strong starting point for more crystal screening trials with the potential of identifying suitable crystals for the high resolution x-ray crystallography structure determination of stable domains in RbBP5. 103 OVERALL CONCLUSIONS AND FUTURE PROSPECTIVES: Understanding the molecular means by which enzymes that introduce variation into histones in the form of covalent posttranslational modifications is anticipated to lead to a deeper understanding of how eukaryotic transcription is controlled in the context of chromatin. Such knowledge would be valuable in understanding how the perturbation of this enzymatic machinery can cause developmental defects and diseases. In turn these studies could lead to the development of targeted therapeutics for human diseases associated with the misregulation of chromatin modifications. The work in this thesis focuses on understanding the molecular mechanisms underlying one particular modification H3K4 methylation. H3K4 methylation is a prominent mark that is required for the inheritance of transcriptionally active states of chromatin. In humans, this mark is predominantly catalyzed by the Mixed Lineage Leukemia (MLL) family of enzymes. The MLL proteins contain evolutionarily conserved SET domains and associate with a core configuration of proteins including WDR5, RbBP5, Ash2L, and DPY30. The work in this thesis has focused on the beta propeller protein RbBP5 a critical component of the MLL1 core complex. Through a careful biochemical dissection of the functional role of RbBP5, the work in this thesis has shown that in the absence of RbBP5, the MLL1 core complex loses di-methyltransferase activity. Additionally, the studies in this thesis have identified several residues that when mutated result in the loss of dimethyltransferase activity. Surprisingly, the same residues appear to be important for interaction with the MLL1 SET domain subunit within the context of the MLL1 core complex. This finding constitutes a 104 previously unidentified SET domain interaction motif located in the hinge region of RbBP5. Based on the data in these studies we propose a model whereby RbBP5 plays a unique role in creating a structural platform where in the presence of WDR5, RbBP5 mediates interaction with the MLL1 c-terminal SET domain for optimal catalytic activity and to bridge the other complex components namely, Ash2L and DPY30, to form the second catalytic site. For future studies it will be interesting to identify the specific sites of interaction in the MLL1 SET domain required for its interaction with RbBP5. Additionally, defining from a structural perspective where the interaction surfaces between the MLL1 SET domain and RbBP5 lie would be crucial in identifying RbBP5’s contribution to the MLL1 SET domain mediated catalysis and substrate interactions. Such future studies could lead to an essential understanding in how the regulation of H3K4 methylation by the MLL1 core complex is integrated in eukaryotic transcriptional programs, how these programs are misregulated, and to better design and development of drugs that target this misregulation in patients with certain forms of leukemia. 105 APPENDIX SUPLEMMENTARY FIGURE 1: Determination of rate constants from single turnover progress curves measured by MALDI-TOF mass spectrometry of reactions catalyzed by the MLL1 core complex with RbBP5 wild-type or RbBP5 variants. A-L. Comparison of the single turnover progress curves for reactions catalyzed by the MLL1 core complex with wildtype RbBP5 or with the RbBP5 variants from MALDI-TOF mass spectrometry assays. The data for H3K4, H3K4me1, and H3K4me2, species were globally fitted to Equations 1-3 (see “RESULTS,” Chapter 2) using DynaFIT. Error bars represent ± S.E. from duplicate experiments. 106 S.2$ Protein Complex MWR (wild-type)AD MWR(W329A)AD MWR(W329E)AD MWR(W329F)AD MWR(W329R)AD MWR(W329Y)AD MWR(S330A)AD MWR(A331S)AD MWR(F332A)AD MWR(A333S)AD MWR(P334A)AD MWR(F336A)AD MWR(K337A)AD MWR(E338A)AD MWR(Y345A)AD MWR(E346A)AD MWR(E347A)AD MWR(S350A)AD MWR(D358A)AD MWR(V375A)AD MWR(V377A)AD K1 1.8 0.40 1.37 2.11 1.17 1.66 1.08 0.83 2.05 1.21 1 0.576569 2.56 2.5 0.84 1.8 0.032 1.74 0.64 0.85 1.43 k2 0.42 0.017 4.0e-007 0.075 0.0052 0.25 0.57 0.27 0.063 0.44 0.26 0.0186185 0.36 0.58 N.D. 0.27 0.013 0.24 0.12 0.095 0.17 k3 0.0024 N.D. N.D. N.D. N.D. N.D 0.0011 N.D N.D. 0.004 N.D. 0.0518556 0.00086 0.0054 N.D. 0.0027 N.D. N.D. N.D. N.D. N.D. SUPPLEMENTARY FIGURE 2: Table Summary of Rate Constants derived from globally fitting experimental data of reactions catalyzed by the MLL1 core complex with RbBP5 wild-type or RbBP5 variants. 107 S.3$ A. 140 k1 Normalized&Activity 120 100 80 60 40 20 W3 WT W329A W329E W3 29F W329R S3329Y A3 0A F3331S A3 2A P3 33S 3 F334A K3 6A 3 E337A Y348A E345A E346A S3 7A D350A V358A V375A 77 A 0 RbBP5&Variant B. 140 k2 Normalized Activity 120 100 80 60 40 20 W3 WT 2 W3 9A W329E W3 29F W329R 2 S33 9Y A3 0A 3 F33 1S A3 2A 3 P3 3S 3 F334A K3 6A 3 E337A Y348A E345A E346A S357A D3 0A 5 V3 8A 7 V3 5A 77 A 0 RbBP5&Variant SUPPLEMENTARY FIGURE 3: Normalized activity derived from k1 and k2 rates constants of reactions catalyzed by the MLL1 core complex with RbBP5 wild-type or RbBP5 variants. 108 S.4$ A. 140 k1 k2 Normalized&Activity 120 100 80 60 40 20 W3 WT W329A W329E W3 29F W329R S3329Y A3 0A F3331S A3 2A P3 33S 3 F334A K3 6A 3 E337A Y348A E345A E346A S3 7A D350A V358A V375A 77 A 0 RbBP5&Variant SUPPLEMENTARYY FIGURE 4: A. Normalized activity derived from k1 and k2 rates constants of reactions catalyzed by the MLL1 core complex with RbBP5 wild-type or RbBP5 variants. 109 SUPPLEMENTARY FIGURES S5-S25. The following pages are raw spectra obtained from MADLI-TOF mass spectrometry methyltransferase assays. The left column represents the wildtype (MWRAD) control and the right column represents MWRAD assembled with mutant RbBP5 proteins. Times points are indicated on each panel. 110 S5. Un2modified## Mono2## 10000 1400 MLL1$ 1200 Di2## MWRAD$ 30#minutes# 30#minutes# 8000 6000 Intensity Intensity 1000 800 600 Un2modified## 4000 400 2000 200 0 0 2650 2660 2670 2680 2690 2650 2700 5000 2660 2670 2680 2690 2700 18000 3#hours# 3#hours# 16000 4000 14000 Intensity Intensity 12000 3000 2000 10000 8000 6000 4000 1000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 6#hours# 1400 2670 2680 2690 2700 6#hours# 14000 1200 12000 1000 10000 Intensity Intensity 2660 16000 1600 800 8000 600 6000 400 4000 200 2000 0 0 2650 2660 2670 2680 2690 2650 2700 2660 2670 2680 2690 2700 8000 4000 24#hours# 24#hours# 6000 Intensity Intensity 3000 2000 4000 2000 1000 0 0 2650 2660 2670 2680 2690 2700 2650 Mass (m/z) 2660 2670 Mass (m/z) 111 2680 2690 2700 S6. 10000 3000 MWRAD& MWR(W329A)AD& 30#minutes# 30#minutes# 2500 8000 Intensity Intensity 2000 6000 4000 1500 1000 2000 500 0 0 2650 2660 2670 2680 2690 2700 2650 18000 2660 2670 2680 2690 2700 700 3#hours# 16000 3#hours# 600 14000 500 10000 Intensity Intensity 12000 8000 400 300 6000 200 4000 100 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 2660 2670 2680 2690 2700 1200 6#hours# 14000 6#hours# 1000 12000 800 Intensity Intensity 10000 8000 600 6000 400 4000 200 2000 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 600 8000 24#hours# 24#hours# 500 6000 Intensity Intensity 400 4000 300 200 2000 100 0 0 2650 2660 2670 2680 2690 2700 Mass (m/z) 2650 2660 2670 Mass (m/z) 112 2680 2690 2700 S7. 10000 5000 30#minutes# 8000 4000 6000 3000 Intensity Intensity MWRAD& 4000 2000 2000 0 2650 2660 2670 2680 2690 2700 2650 18000 2660 2670 2680 2690 2700 2000 3#hours# 16000 3#hours# 1800 1600 14000 1400 Intensity 12000 Intensity 30#minutes# 1000 0 10000 8000 6000 1200 1000 800 600 4000 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 16000 2660 2670 2680 2690 2700 5000 6#hours# 14000 6#hours# 4000 12000 Intensity 10000 Intensity MWR(W329E)AD& 8000 6000 3000 2000 4000 1000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 8000 2660 2670 2680 2690 2700 1400 24#hours# 24#hours# 1200 6000 Intensity Intensity 1000 4000 800 600 400 2000 200 0 0 2650 2660 2670 2680 2690 2650 2700 2660 2670 Mass (m/z) Mass (m/z) 113 2680 2690 2700 S8. 3000 10000 MWRAD& MWR(W329F)AD& 30#minutes# 30#minutes# 2500 8000 Intensity Intensity 2000 6000 4000 1500 1000 2000 500 0 0 2650 2660 2670 2680 2690 2650 2700 18000 2660 2670 2680 2690 2700 7000 3#hours# 16000 3#hours# 6000 14000 5000 Intensity Intensity 12000 10000 8000 4000 3000 6000 2000 4000 1000 2000 0 0 2650 2660 2670 2680 2690 2650 2700 2660 2670 2680 2690 2700 16000 5000 6#hours# 14000 6#hours# 4000 12000 Intensity Intensity 10000 8000 6000 3000 2000 4000 1000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 8000 2660 2670 2680 2690 2700 1400 24#hours# 24#hours# 1200 6000 Intensity Intensity 1000 4000 800 600 400 2000 200 0 2650 2660 2670 2680 2690 2700 0 2650 Mass (m/z) 2660 2670 Mass (m/z) 114 2680 2690 2700 S9. 12000 10000 MWRAD& 30#minutes# MWR(W329R)AD& 30#minutes# 10000 8000 Intensity Intensity 8000 6000 4000 6000 4000 2000 2000 0 0 2650 2660 2670 2680 2690 2650 2700 18000 2660 2670 2680 2690 2700 16000 3#hours# 16000 3#hours# 14000 14000 12000 10000 Intensity Intensity 12000 10000 8000 8000 6000 6000 4000 4000 2000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 16000 2660 2670 2680 2690 2700 14000 6#hours# 14000 6#hours# 12000 12000 10000 Intensity Intensity 10000 8000 6000 8000 6000 4000 4000 2000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 8000 2660 2670 2680 2690 2700 6000 24#hours# 24#hours# 5000 6000 Intensity Intensity 4000 4000 3000 2000 2000 1000 0 0 2650 2660 2670 2680 2690 2650 2700 2660 2670 Mass (m/z) Mass (m/z) 115 2680 2690 2700 S10. 10000 6000 MWRAD& 30#minutes# MWR(W329Y)AD& 30#minutes# 5000 8000 Intensity Intensity 4000 6000 4000 3000 2000 2000 1000 0 0 2650 2660 2670 2680 2690 2700 2650 18000 2660 2670 2680 2690 2700 7000 3#hours# 16000 3#hours# 6000 14000 5000 Intensity Intensity 12000 10000 8000 4000 3000 6000 2000 4000 1000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 2660 2670 2680 2690 2700 7000 6#hours# 14000 6#hours# 6000 12000 5000 Intensity Intensity 10000 8000 6000 3000 2000 4000 1000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 8000 2660 2670 2680 2690 2700 7000 24#hours# 24#hours# 6000 6000 5000 Intensity Intensity 4000 4000 4000 3000 2000 2000 1000 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 Mass (m/z) Mass (m/z) 116 2690 2700 S11. 10000 8000 MWRAD& MWR(S330A)AD& 30#minutes# 30#minutes# 8000 6000 Intensity Intensity 6000 4000 4000 2000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 18000 2660 2670 2680 2690 2700 7000 3#hours# 16000 3#hours# 6000 14000 5000 Intensity Intensity 12000 10000 8000 4000 3000 6000 2000 4000 1000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 2660 2670 2680 2690 2700 1000 6#hours# 6#hours# 14000 800 12000 Intensity Intensity 10000 8000 6000 600 400 4000 200 2000 0 0 2650 2660 2670 2680 2690 2700 2650 8000 2660 2670 2680 2690 2700 3000 24#hours# 24#hours# 2500 6000 Intensity Intensity 2000 4000 1500 1000 2000 500 0 0 2650 2660 2670 2680 2690 2700 2650 Mass (m/z) 117 2660 2670 2680 2690 2700 S12. 10000 2500 8000 2000 6000 1500 4000 30#minutes# 1000 2000 500 0 0 2650 2660 2670 2680 2690 2700 2650 18000 2660 2670 2680 2690 2700 2710 3000 3#hours# 3#hours# 16000 2500 14000 12000 2000 Intensity Intensity MWR(A331S)AD& 30#minutes# Intensity Intensity MWRAD& 10000 8000 1500 6000 1000 4000 500 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 2660 2670 2680 2690 2700 5000 6#hours# 6#hours# 14000 4000 12000 Intensity Intensity 10000 8000 6000 3000 2000 4000 1000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 8000 2660 2670 2680 2690 2700 700 24#hours# 24#hours# 600 6000 Intensity Intensity 500 4000 400 300 200 2000 100 0 0 2650 2660 2670 2680 2690 2700 2650 Mass (m/z) 118 2660 2670 2680 2690 2700 S13. 10000 5000 30#minutes# 8000 4000 6000 3000 Intensity Intensity MWRAD& 4000 2000 MWR(F332A)AD& 30#minutes# 2000 1000 0 0 2650 2660 2670 2680 2690 2700 2650 18000 2660 2670 2680 2690 2700 4000 3#hours# 16000 3#hours# 14000 3000 10000 Intensity Intensity 12000 8000 2000 6000 1000 4000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 2660 2670 2680 2690 2700 4000 6#hours# 14000 6#hours# 12000 3000 Intensity Intensity 10000 8000 6000 4000 2000 1000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 8000 2660 2670 2680 2690 2700 1400 24#hours# 24#hours# 1200 6000 Intensity Intensity 1000 4000 800 600 2000 400 200 0 2650 2660 2670 2680 2690 2700 2650 Mass (m/z) 119 2660 2670 2680 2690 2700 S14. 10000 30000 MWRAD& MWR(A333S)AD& 30#minutes# 30#minutes# 25000 8000 Intensity Intensity 20000 6000 4000 15000 10000 2000 5000 0 0 2650 2660 2670 2680 2690 2700 2650 18000 2670 2680 3#hours# 16000 2690 2700 3#hours# 2500 14000 12000 2000 Intensity Intensity 2660 3000 10000 8000 6000 1500 1000 4000 500 2000 0 0 2650 2660 2670 2680 2690 2700 2650 16000 2660 2670 2680 2690 2700 6000 6#hours# 14000 6#hours# 5000 12000 4000 Intensity Intensity 10000 8000 6000 3000 2000 4000 1000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 8000 2660 2670 2680 2690 2700 12000 24#hours# 24#hours# 10000 6000 Intensity Intensity 8000 4000 6000 4000 2000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 Mass (m/z) 120 2660 2670 2680 2690 2700 S15. 4000 10000 MWRAD& 30#minutes# MWR(P334A)AD& 30#minutes# 8000 6000 Intensity Intensity 3000 4000 2000 1000 2000 0 0 2650 2660 2670 2680 2690 2650 2700 3#hours# 16000 2670 2680 2690 2700 3#hours# 1600 14000 1400 12000 1200 Intensity Intensity 2660 1800 18000 10000 8000 1000 800 6000 600 4000 400 2000 200 0 0 2650 2660 2670 2680 2690 2650 2700 16000 2660 2670 2680 2690 2700 2000 6#hours# 14000 6#hours# 1800 1600 12000 1400 Intensity Intensity 10000 8000 6000 1200 1000 800 600 4000 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 1800 8000 1600 24#hours# 24#hours# 1400 6000 Intensity Intensity 1200 4000 1000 800 600 2000 400 200 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 Mass (m/z) Mass (m/z) 121 2680 2690 2700 S16. 10000 18000 MWRAD& 8000 MWR(F336A)AD& 16000 30#minutes# 30#minutes# 14000 Intensity Intensity 12000 6000 4000 10000 8000 6000 4000 2000 2000 0 0 2650 2660 2670 2680 2690 2650 2700 18000 16000 3#hours# 2670 2680 2690 2700 3#hours# 18000 14000 16000 12000 14000 10000 12000 Intensity Intensity 2660 20000 8000 6000 10000 8000 6000 4000 4000 2000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 14000 6#hours# 2680 2690 2700 6#hours# 12000 10000 10000 Intensity Intensity 2670 14000 12000 8000 6000 8000 6000 4000 4000 2000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 8000 2660 2670 2680 2690 2700 5000 24#hours# 24#hours# 4000 Intensity 6000 Intensity 2660 16000 4000 3000 2000 2000 1000 0 2650 2660 2670 2680 2690 2700 0 2650 Mass (m/z) 2660 2670 Mass (m/z) 122 2680 2690 2700 S17. 10000 5000 MWRAD& MWR(K337A)AD& 30#minutes# 30#minutes# 4000 6000 3000 Intensity Intensity 8000 4000 2000 2000 1000 0 2650 2660 2670 2680 2690 0 2700 2650 18000 3#hours# 16000 2660 2670 2680 2000 2690 2700 3#hours# 14000 1500 10000 Intensity Intensity 12000 8000 1000 6000 4000 500 2000 0 2650 2660 2670 2680 2690 0 2700 2650 2660 2670 2680 2690 2700 3000 16000 6#hours# 14000 6#hours# 2500 12000 2000 Intensity Intensity 10000 8000 6000 1500 1000 4000 500 2000 0 0 2650 2660 2670 2680 2690 2650 2700 8000 2660 2670 2690 2700 1000 24#hours# 24#hours# 800 Intensity 6000 Intensity 2680 4000 600 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 Mass (m/z) Mass (m/z) 123 2680 2690 2700 S18. 10000 MWR(E338A)AD& 1000 800 6000 Intensity Intensity 8000 1200 30#minutes# MWRAD& 4000 600 400 2000 200 0 2650 2660 2670 2680 2690 0 2700 18000 3#hours# 16000 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 500 14000 400 10000 Intensity Intensity 12000 8000 6000 300 200 4000 100 2000 0 2650 2660 2670 2680 2690 0 2700 16000 1800 6#hours# 14000 1600 1400 12000 1200 Intensity Intensity 10000 8000 6000 1000 800 600 4000 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 8000 1400 24#hours# 1200 6000 Intensity Intensity 1000 4000 800 600 2000 400 0 200 2650 2660 2670 2680 2690 2700 Mass (m/z) Mass (m/z) 124 S19. 4000 10000 MWRAD& 30#minutes# MWR(Y345A)AD& 8000 6000 Intensity Intensity 3000 4000 2000 1000 2000 0 0 2650 2660 2670 2680 2690 2650 2700 18000 2660 2670 2680 2690 2700 4000 3#hours# 16000 14000 3000 Intensity Intensity 12000 10000 8000 2000 6000 1000 4000 2000 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 2500 16000 6#hours# 14000 2000 12000 Intensity Intensity 10000 8000 6000 4000 1500 1000 500 2000 0 0 2650 2660 2670 2680 2690 2700 8000 1400 24#hours# 1200 6000 Intensity Intensity 1000 4000 800 600 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 Mass (m/z) 2660 2670 Mass (m/z) 125 2680 2690 2700 S20. 10000 3500 MWRAD& 30#minutes# MWR(E346A)AD& 3000 8000 Intensity Intensity 2500 6000 4000 2000 1500 1000 2000 500 0 2650 2660 2670 2680 2690 0 2700 2650 18000 3#hours# 16000 2660 2670 2680 2690 2700 3500 3000 14000 2500 10000 Intensity Intensity 12000 8000 2000 1500 6000 1000 4000 500 2000 0 2650 2660 2670 2680 2690 0 2700 16000 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 4000 6#hours# 14000 12000 3000 Intensity Intensity 10000 8000 6000 4000 2000 1000 2000 0 2650 2660 2670 2680 2690 0 2700 8000 2000 24#hours# 1800 1600 6000 Intensity Intensity 1400 4000 1200 1000 800 600 2000 400 200 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 Mass (m/z) Mass (m/z) 126 2680 2690 2700 S21. 10000 3500 MWRAD& 30#minutes# MWR(E347A)AD& 30#minutes# 3000 8000 Intensity Intensity 2500 6000 4000 2000 1500 1000 2000 500 0 0 2650 2660 2670 2680 2690 2700 2650 18000 3#hours# 16000 2660 2670 3500 2680 2690 2700 3#hours# 3000 14000 2500 Intensity Intensity 12000 10000 8000 2000 1500 6000 1000 4000 500 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 2660 2670 2680 2690 2700 2000 6#hours# 14000 6#hours# 1800 1600 12000 1400 Intensity Intensity 10000 8000 6000 1200 1000 800 600 4000 400 2000 200 0 2650 2660 2670 2680 2690 0 2700 2650 8000 2660 2670 2680 2690 2700 1200 24#hours# 24#hours# 1000 6000 Intensity Intensity 800 4000 600 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 Mass (m/z) Mass (m/z) 127 2680 2690 2700 S22. 10000 1600 MWRAD& 30#minutes# 1400 MWR(S350A)AD& 30#minutes# 8000 1000 6000 Intensity Intensity 1200 4000 800 600 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 18000 2650 2660 2670 2680 2690 2700 1400 3#hours# 16000 3#hours# 1200 14000 1000 Intensity Intensity 12000 10000 8000 800 600 6000 400 4000 200 2000 0 0 2650 2660 2670 2680 2690 2700 16000 6#hours# 14000 2660 2670 2680 2690 2700 6#hours# 1600 1400 12000 1200 Intensity 10000 Intensity 2650 1800 8000 6000 1000 800 600 4000 400 2000 200 0 2650 2660 2670 2680 2690 0 2700 2650 2660 2670 2680 2690 2700 600 8000 24#hours# 24#hours# 500 6000 Intensity Intensity 400 4000 300 200 2000 100 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 Mass (m/z) Mass (m/z) 128 2680 2690 2700 S23. 10000 1800 MWRAD+ 30#minutes# 1600 8000 MWR(D358A)AD+ 30#minutes# 1400 Intensity Intensity 1200 6000 4000 1000 800 600 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 18000 3#hours# 16000 2660 2670 2680 3000 2690 2700 3#hours# 2500 14000 2000 Intensity Intensity 12000 10000 8000 6000 1500 1000 4000 500 2000 0 0 2650 2660 2670 2680 2690 2700 2650 16000 2660 2670 2680 2690 2700 1400 6#hours# 14000 6#hours# 1200 12000 1000 Intensity Intensity 10000 8000 6000 600 400 4000 200 2000 0 0 2650 2660 2670 2680 2690 2700 2650 8000 2660 2670 2680 2690 2700 1600 24#hours# 24#hours# 1400 6000 1200 1000 Intensity Intensity 800 4000 800 600 2000 400 200 0 2650 2660 2670 2680 2690 2700 0 2650 Mass (m/z) 2660 2670 Mass (m/z) 129 2680 2690 2700 S24. 10000 20000 MWRAD& 30#minutes# MWR(V375A)AD& 18000 8000 30#minutes# 16000 12000 Intensity Intensity 14000 6000 4000 10000 8000 6000 2000 4000 2000 0 2650 2660 2670 2680 2690 0 2700 2650 18000 3#hours# 16000 2680 2690 2700 3#hours# 1600 1400 Intensity 12000 Intensity 2670 1800 14000 10000 8000 6000 1200 1000 800 600 4000 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 16000 2660 2670 2680 2690 2700 2500 6#hours# 14000 6#hours# 2000 12000 Intensity 10000 Intensity 2660 2000 8000 6000 1500 1000 4000 500 2000 0 0 2650 2660 2670 2680 2690 2700 2650 2660 2670 2680 2690 2700 1000 8000 24#hours# 24#hours# 800 Intensity Intensity 6000 4000 600 400 2000 200 0 0 2650 2660 2670 2680 2690 2700 2650 Mass (m/z) 2660 2670 Mass (m/z) 130 2680 2690 2700 S25. 10000 8000 7000 30#minutes# MWRAD& MWR(V377A)AD& 30#minutes# 6000 Intensity Intensity 5000 6000 4000 4000 3000 2000 2000 1000 0 0 2650 2660 2670 2680 2690 2700 2650 18000 2670 2680 3#hours# 16000 2690 2700 3#hours# 2500 14000 12000 2000 10000 Intensity Intensity 2660 3000 8000 6000 1500 1000 4000 500 2000 0 2650 2660 2670 2680 2690 0 2700 2650 16000 2660 2670 2680 2690 2700 1400 6#hours# 14000 6#hours# 1200 12000 1000 Intensity Intensity 10000 8000 6000 600 400 4000 200 2000 0 2650 2660 2670 2680 2690 0 2700 2650 8000 2660 2670 2680 2690 2700 1600 24#hours# 24#hours# 1400 6000 1200 1000 Intensity Intensity 800 4000 800 600 2000 400 200 0 2650 2660 2670 2680 2690 2700 0 2650 Mass (m/z) 2660 2670 Mass (m/z) 131 2680 2690 2700 References Allison, S.P. 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(1995). Altered Hox expression and segmental identity in Mll-‐mutant mice. Nature 378, 505-‐508. 137 BIOGRAPHICAL DATA NAME Melody Sanders PLACE OF BIRTH Detroit, Michigan HOMETOWN Atlanta, Georgia HIGH SCHOOL Dekalb School of the Arts, Atlanta, Georgia Major: Instrumental Music (Alto Saxophone) Minor: Visual Arts Attended: August 2002- May 2003 August 2003- May 2004 Open Campus Highschool, Atlanta, Georgia Attended: August 2004-May 2005 Graduated May 2005 COLLEGE Syracuse University, Syracuse, New York Attended: August 2005-December 2005, August 2007-May 2009 Awards: Bachelor of Science, 2009 POST-GRADUATE Syracuse University, Syracuse, New York Department of Biology Masters of Science, 2013 Awards and Honors: Syracuse Univesity Science Technology and Engineering (STEM) Fellowship 138