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University of Tennessee, Knoxville Trace: Tennessee Research and Creative Exchange Masters Theses Graduate School 8-2007 Studies on the Mechanisms of Homolog Pairing and Sister Chromatid Cohesion During Drosophila Male Meiosis Jian Ma University of Tennessee - Knoxville Recommended Citation Ma, Jian, "Studies on the Mechanisms of Homolog Pairing and Sister Chromatid Cohesion During Drosophila Male Meiosis. " Master's Thesis, University of Tennessee, 2007. http://trace.tennessee.edu/utk_gradthes/167 This Thesis is brought to you for free and open access by the Graduate School at Trace: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of Trace: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Jian Ma entitled "Studies on the Mechanisms of Homolog Pairing and Sister Chromatid Cohesion During Drosophila Male Meiosis." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Biochemistry and Cellular and Molecular Biology. Bruce D. McKee, Major Professor We have read this thesis and recommend its acceptance: Hong Guo, Mariano Labrador Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) To the Graduate Council: I am submitting herewith a thesis by Jian Ma entitled: “Studies on the mechanisms of homolog pairing and sister chromatid cohesion during Drosophila male meiosis”. I have examined the final electronic copy of this thesis for form and content and recommend that it can be accepted in partial fulfillment of the requirement for the degree of Master of Science, with a major in Biochemistry, Cellular and Molecular Biology. McKee, Bruce D., Ph.D., Major Professor We have read this thesis and recommend its acceptance: Guo, Hong, Ph.D. Labrador, Mariano, Ph.D. Accepted for the Council: Carolyn R. Hodges, Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Studies on the mechanisms of homolog pairing and sister chromatid cohesion during Drosophila male meiosis. A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville Jian Ma August 2007 ACKNOWLEDGEMENT This thesis could not be finished without the help and support of many people who are gratefully acknowledged here. I am honored to express my deep and sincere gratitude to my major supervisor, Professor McKee, Bruce, Ph. D., Director, Department of Biochemistry, Cellular and Molecular Biology, the University of Tennessee. He has offered me valuable ideas, suggestions and criticisms with his profound knowledge in linguistics and rich research experience. His understanding, encouraging and personal guidance have provided a good basis for the present thesis. I am extremely grateful to Assistant Professor Guo, Hong and Assistant Professor Labrador, Mariano, Department of Biochemistry, Cellular and Molecular Biology, the University of Tennessee. Their ideals and concepts have had a remarkable influence on my thesis. I also wish to thank my lab members: Thomas, Sharon; Matulis, Shannon; Yan, Rihui; Tsai, Jui-He, and Vandiver, Jennifer. Their kind support has been of great value in this study. I owe my loving thanks to my wife Shengli Ding and my son Brian E. Ma. Without their encouragement and understanding it would have been impossible for me to finish this work. ii ABSTRACT Meiosis is a complex process involving one round of DNA replication followed by two rounds of cell divisions. The proper segregation of homologs at meiosis I and sister chromatids during meiosis II is essential for the survival of the offspring. Aberrant chromosome segregation at any stage of meiosis can lead to aneuploidy. Meiotic chromosome segregation without crossing over or chiasmata is a widespread but poorly understand chromosome segregation pathway. In male Drosophila meiosis the absence of recombination in chromosomes makes it easier to identify mutations which influence homologous chromosome pairing and segregation. Modifier of Mdg4 in Meiosis (MNM), a protein encoded by modifier of mdg4, is required for integrity of chromosome territories and stability of achiasmatic bivalents and for normal homolog segregation in male Drosophila meiosis I. MNM localizes to clusters of nucleolar and autosomal foci during meiotic prophase I (PI) and to a novel, compact structure associated with the X-Y bivalent during prometaphase I (PMI) and metaphase I (MI). Stromalin in Meiosis (SNM), a member of the SCC3/STAG cohesion family, is required for homolog pairing in male Drosophila but not for meiotic sister chromatid cohesin. SNM protein co-localizes with MNM to the nucleolus throughout PI and to a prominent focus on the X-Y bivalent during PMI and MI. Mutations of snm and mnm exhibit similar homolog pairing failure during meiosis I. Consequently we used the Yeast Two-Hybrid System to determine whether SNM and MNM can interact with each other. We concluded that MNM can interact with itself and SNM. We also found that SNM interacts with the iii BTB domain of MNM and that the FLYWCH domain in the C-terminus of the MNM protein may play a role in the interaction between MNM and SNM. Sister chromatid cohesion (SCC) is required for proper chromosome segregation during mitosis and meiosis. The protein complex cohesin is a major component of SCC and links sister chromatids together from the time of their replication until their segregation. sisters unbound (sun) is a novel gene required in male and female Drosophila for meiotic SCC. Mutations in sun cause premature sister chromatid segregation (PSCS) and nondisjunction (NDJ) of both homologous and sister chromatids, and also disrupt normal recombination and synapsis in female meiosis. The four chromatids in each bivalent exhibit random segregation at meiosis I. We found that centromeric cohesion is lost in the absence of SUN during mid-prophase (S4). Surprisingly, cytological analysis shows chromosome behavior appears relatively normal during meiosis I. Double mutations sun snm and sun mnm impair the integrity of chromosome territories. In addition we found that SNM, but not MNM, is required for centromere pairing in mid-prophase (S3) and simultaneous loss of SNM and SUN proteins causes PSCS at mid-prophase I (S3), which is earlier than in single mutants in snm or sun. These findings indicated that these two proteins play complementary roles in meiotic cohesion. iv TABLE OF CONTENTS Chapter I – General Introduction 1 I-1 Overview of Meiosis………………………………………………….……..……2 I-2 Meiotic Recombination and Chiasma………………………………………….…4 I-3 Cohesion and Cohesin………………………………………..………..……….…5 I-4 Meiotic Cohesion……………………………………………………….………...12 I-5 Meiosis in Drosophila……………………………………….…………………...16 I-6 Meiotic Cohesion in Drosophila…………………………..…………………….. 21 Chapter II – Evidence for a homolog conjunction complex in Drosophila male meiosis 24 II-1 Introduction……………………………………………………….…………..…25 II-2 Materials and Methods……………………………………………………..……29 II-3 Results and Discussion…………………………………………….…………….36 Chapter III – sun is required for sister chromatid cohesion and sister centromere mono-orientation in male Drosophila meiosis 45 III-1 Introduction…………………………………………………………………….46 III-2 Material and Methods……………..…………………………….…………...…48 III-3 Results…………………………………………………………..………………50 III-4 Discussion…………………………………………………….….……………..59 Chapter IV – Roles of interaction of sun, snm and mnm in meiotic cohesion, centromere pairing and chromosome territory integrity 62 IV-1 Introduction………………………………………………………….…………63 IV-2 Materials and Methods…………………………………..………….….………64 IV-3 Results……………………………………………………………….…………65 IV-4 Discussion…………………………………………………………….……..…73 Chapter V – References 76 Vita 102 v List of Figures Chapter I – General Introduction Figure I-1: Model of Meiosis Figure I-2: Model of chiasmata Figure I-3: Model for ATP hydrolysis-dependent binding of cohesin to DNA Figure I-4: Model for release of sister chromatid cohesion in meiosis Figure I-5: Model for meiotic pairing in male Drosophila 3 6 9 14 20 Chapter II – Evidence for a homolog conjunction complex in Drosophila male meiosis Figure II-1: Structures of the pGAD-C and pGBD-C vectors Figure II-2: Structure of BTB and FLYWCH domain of MNM protein Figure II-3: MNM interact with itself and with SNM Figure II-4: β-gal activity for MNM and SNM interaction Figure II-5: BTB domain interact with SNM Figure II-6: Comparison of β-gal activities 30 32 37 40 42 43 Chapter III – sun, a novel gene, is required for sister chromatid cohesion and sister centromere mono-orientation in male Drosophila meiosis Figure III-1: Molecular characterization of sun Figure III-2: NDJ test of homologs vs sister chromatids for sun mutants Figure III-3: Chromosome morphology during meiosis I in sun mutants Figure III-4: Sister chromatids segregate in meiosis II in sun mutants Figure III-5: sun mutants and wild-type in early-prophase (S1/2) Figure III-6: sun mutants and wild-type in mid-prophase (S3) Figure III-7: sun mutants and wild-type in mid-prophase (S4) Figure III-8: sun mutants and wild-type in mid-prophase (S5) 47 51 52 53 55 56 57 58 Chapter IV – Roles of interaction of sun, snm and mnm in meiotic cohesion, centromere pairing and chromosome territory integrity Figure IV-1: Chromosome morphology in meiosis I in sun snm mutants Table IV-2: Sperms produced by sun mutants & sun snm mutants Figure IV-3: Sister centromere cohesion is lost in sun snm double mutants Figure IV-4: Comparison of > 8 CID spots percentage in prophase I Figure IV-5: Chromosome territories in PI in sun snm & sun mnm mutants vi 66 67 69 70 72 Chapter I - General Introduction 1 I-1 Overview of Meiosis Meiosis, conserved in eukaryotes, is a special cell division which allows for the exchange of genetic material between parental chromosomes to maintain the genetic diversity of offspring. Meiosis comprises a round of DNA replication followed by two successive nuclear divisions, meiosis I and meiosis II (Kleckner, 1996). Meiosis I is a reductional segregation in which homologous chromosomes pair and segregate to opposite poles but sister chromatids stay together. In meiosis II, an equational division which resembles a mitotic division, sister chromatids separate and move to opposite poles. After meiosis a diploid germ cell produces four haploid cells containing half number of chromosomes (Fig.I-1). Meiosis is important for the correct propagation of species in all sexually reproducing organisms and for the diversities of genome and phenotype. Any errors that affect meiosis, such as mutations in homolog pairing or sister chromatid cohesion pathways, can lead to homologous chromosome or sister chromotid nondisjunction (NDJ) and produce aneuploidy, which is the leading cause of genetic illnesses such as Down's syndrome and human spontaneous abortion (Hassold et al., 2001). Considering the significance, it is necessary to uncover the mechanisms of homologous chromosome pairing and disjunction and sister chromatid disjunction. Meiosis I and II are both divided into four phases: prophase, metaphase, anaphase, and telophase. Prophase I (PI) is the first stage of meiosis I, during which several important changes in chromatin architecture should take place. First of all, each individual chromosome condenses and lines up with its homologous 2 ---Brown, 2002 Figure I-1: Model of Meiosis. One member of the pair is red, the other is blue. Image from Brown, 2002. 3 chromosome, generating bivalents. Second, the synaptonemal complex (SC), a protein structure consisting of two parallel lateral regions and a central element, forms between two homologous chromosomes to mediate chromosome pairing and recombination (crossing-over) (Cohen et al., 2001). The pachytene substage of PI ends when the SC disappears, and nuclear envelope breakdown marks the start of prometaphase I (PMI). During metaphase I (MI) the condensed homologs are arranged on the metaphase plate. Three important events need to occur prior to MI to ensure that homologs segregate faithfully: a physical linkage between homologous chromosomes has to be established to resist the force from the opposite poles; sister chromatids must be held together beyond meiosis I by a protein complex termed cohesin; sister kinetochores have to attach to microtubules from the same pole to result in a mono-oriented movement (Brian et al,. 2001). Any error within one of these three events will cause homolog NDJ or PSCS in meiosis I. Segregation of homologs to opposite poles initiates at anaphase I (AI) with the release of homolog connection, and formation of two daughter cells at telophase I (TI) concludes meiosis I. Meiosis II has the same four phases as meiosis I and produces four daughter cells with half the number of chromosomes after sister chromatid segregation. I-2 Meiotic Recombination and Chiasma As mentioned above, in the majority of eukaryotic organisms, recombination, or the formation of crossovers is important for proper segregation of homologous chromosomes at MI, as well as providing genetic diversity. Meiotic recombination is a 4 virtually universal feature in most organisms and is initiated via programmed double strands break (DSBs) catalyzed by the topoisomerase-like protein Spo11. Several other proteins including the RAD50/ MRE11/ XRS2 complex also have been proved to involve in the formation of DSBs and in resection of the 5’ strand termini to give molecules with ~ 300 nt 3’ single-stranded tails (Keeney et al., 1997; Bergerat et, al., 1997; Keeney et al., 2001; Neale et al., 2005). The majority of these DSBs are repaired via homologous recombination with the homologous sequence on a homologous chromatids, rather than with the sister chromatids. A fraction of these recombination events proceeds by two long-lived intermediates: single-end invasions (SEIs) and double Holliday junction (dHJs) to result in crossovers and chiasmata, physical connections between homologous chromosomes (Fig. I-2) (Hunter et al., 2001; Allers et al., 2001). The chiasma is an important apparatus for homolog segregation in meiosis I. It holds homologs together after SC is removed and provides the force to resist the pulling power from the opposite poles to prevent the homologous chromosomes from separating prior to AI (Zickler et al., 1999) I-3 Cohesion and Cohesin Sister chromatid cohesion was first named in 1994 (Miyazaki et al., 1994) to refer a physical linkage between two duplicated sister chromatids. It is established from the time of DNA replication in S-phase and holds sister chromatids together through chromosome arms and the centromeres. Sister chromatid cohesion resists the power of microtubules from the opposite poles while aligned at the metaphase plate 5 Figure I-2: Model of chiasmata. Image from Griffiths, 1999 Each line represents a chromatid of a pair of synapsed chromosomes. 6 and is released to allow sister chromatid segregation during anaphase of mitosis or meiosis II. In addition, sister chromatid cohesion has been proved to hold homologs together in meiosis I by stabilizing chiasmata (Lee et al., 2001), and is involved in repair of DNA double-strand breaks during G2 phase (Sjogren 2001). The molecular basis for sister chromatid cohesion is a chromosomal protein complex, called cohesin. Cohesin is a multi-subunit complex containing at least four conserved proteins: SMC1, SMC3, SCC1 (Also known as MCD1 or RAD21) and SCC3 (also known as SA in vertebrate cells) from yeast to human. SMC1 and SMC3 are members of Structural Maintenance of Chromosomes (SMC) proteins, a superfamily that has multiple functions in regulating the structural and functional organization of chromosomes from bacteria to human, such as chromosome and sister chromatid segregation (during mitosis and meiosis), chromosome condensation, chromosome-wide gene regulation and DNA recombinational repair (Losada et al., 2005). All SMC proteins have a conserved characteristic architecture in which they form long coiled-coil arm between a ‘hinge’ domain at one end and an ABC-type ATPase ‘head’ domain at the other by folding back on themselves through an antiparallel coiled-coil interaction. The ‘head domain’ can close and open respectively by the ATP binding and hydrolysis (Hirano et al., 2001; Haering et al., 2002; Arumugam et al., 2003; Hirano, 2006). Cohesin exhibits a tripartite ring structure, within which SMC1 and SMC3 form a V-shaped molecule structure through the association with each other at their hinge domains, and SCC1, which belongs to the kleisin superfamily representing the most conserved SMC-interacting proteins, closes this ring structure by binding to the head domains of 7 SMC1 and SMC3 through its C- and N-terminal domains respectively. The fourth subunit SCC3 binds to the ring through its interaction with SCC1 (Gruber et al., 2003; Schleiffer et al., 2003). Ring-like cohesin is thought to hold sister chromatids together by surrounding and entrapping them, but direct proof is absent. There are several models to explain how cohesin embraces the sister chromatids. One model is that the entry of DNA into cohesin’s ring is based on the ABC-type ATPase ‘head’ domain of SMC proteins (Fig. I-3). Hydrolysis of ATP opens the ring by destroying the interaction of the head domains and allows the DNA to slide inside, and binding of a new ATP molecule then re-closes the ring to embrace the DNA (Arumugam et al., 2003; Weitzer et al., 2003). Recently Gruber et al. proposed another model in 2006 within which the SMC hinge domain is responsible for the entry of DNA into cohesin’s ring. The transient dissociate of the hinge-hinge interface of the SMC1-SMC3 allows the DNA to enter the cohesin ring (Gruber et al., 2006). In yeast cohesion is established during S phase. Cohesin is loaded onto chromosomes with help of the loading complex SCC2 and SCC4 (Ciosk et al., 2000), but the establishment of cohesion in S phase requires several other proteins, such as ECO1/CTF7 (Skibbens et al., 1999; Toth et al., 1999), TFR4 and TRF5 (Wang et al., 2000). Milutinovich proved that the hinge and loop1 regions of SMC1 also play an important role in the binding of cohesin to the specific chromatin sites including cohesion-associated regions (CRAs) and pericentric regions (Milutinovich et al., 2007). In addition, condensin has been found to be required to maintain cohesion at several chromosomal arm sites, but not required at centromere (Lam et al., 2006). 8 ---Uhlmann, 2004 Figure I-3. A model for ATP hydrolysis-dependent binding of cohesin to DNA. The SCC1 binds to the SMC heads to form a ring structure. ATP hydrolysis leads to separation of the SMC heads and let DNA enter the ring structure. When all chromosomes are aligned at metaphase plate, Separase is activated to cleave SCC1 and open the ring; leading to removal of sister chromatid cohesion. 9 Cohesin along the chromosome arms is at lower density (Lengronne et al., 2004; Glynn et al., 2004), but enriched around centromeres. The heterochromatin protein HP1/Swi6 at pericentromeric regions is thought to promote the centromeric enrichment of cohesion, presumably through direct interaction with the cohesin subunit Scc3/SA (Pidoux et al., 2004; Ishiguro et al., 2007). The removal of cohesin is a vital step for the faithful segregation of chromosomes in both mitosis and meiosis. This removal is triggered by proteolytic cleavage of SCC1/RAD21 by Separase at the onset of anaphase in mitosis. Separase is a cysteine protease which is inhibited, for most of the cell cycle, by binding its inhibitor Securin. Once all chromosomes have been bioriented during metaphase, Securin is marked for degradation by a ubiquitin ligase called the Anaphase Promoting Complex or Cyclosome (APC/C). Securin degradation releases Separase allowing it to destroy cohesin and trigger the onset of anaphase (Cohen-Fix et al., 1996; Funabiki et al., 1996b; Ciosk et al., 1998; Uhlmann et al., 1999; Zou et al., 1999). In vertebrate cells, Separase activity is inhibited by not only Securin but also its phosphorylation at the hands of Cdk1 kinase. In these cells, the destruction of Securin and proteolysis of the CDK1 subunit cyclin B simultaneously, both mediated by APC/C, will activate Separase (Stemmann et al., 2001). In contrast to the simultaneous release of cohesion from the chromosome arms and centromeres in budding yeast, in vertebrate cells dissociation of cohesin from chromosomes is carried out in two steps (Waizenegger et al., 2000). The first step is to release cohesin from chromosome arms before metaphase by hyperphosphorylation of 10 SCC3/SA subunits mediated by the Polo-like kinase 1 (PLk1) and Aurora B without SCC1 cleavage. This process is called the ‘prophase pathway’ (Gimenez-Abian et al., 2004; Sumara et al., 2002). Hauf found in 2005 that SA2 is the critical target of Plk1 in this pathway (Hauf et al., 2005). However, centromeric cohesin still persists and is removed at the metaphase-anaphase transition by Separase, which is the second step. The protection of centromeric cohesin from the prophase pathway is accomplished by the centromeric protein Shugoshin (Sgo), an ortholog of the MEI-S332 protein from Drosophila melanogaster (Watanabe, 2005; Lee et al., 2005). Sgo was found in 2004 by genetic screening in budding and fission yeast as a protector of centromere cohesion in mitosis and meiosis. So far, budding yeast, worm and Drosophila possess only a single orthologue, Sgo1 and MEI-S332, respectively, that is expressed in mitotic as well as meiotic cells. Fission yeast and humans have two Sgo proteins. Researchers found that human Sgo1, hSgo1, localizes to centromere in mitosis in the presence of Bub1, a spindle checkpoint protein, to prevent the premature centromeric cohesin dissociation, and disappears during anaphase. Three recent papers bring new light on the molecular mechanism of this protection: phosphatase 2A (PP2A), a major partner of Sgo in human and yeast cells, is involved in centromeric cohesin protection. PP2A colocalizes with Sgo at the centromere by binding its PP2A-B’ subunit to Sgo’s N-terminal region from prophase to anaphase in the presence of Bub1 and counteracts the phosphorylation of cohesion subunits by PLK1. In addition, Tang et al. found in 2006 that although PP2A is required for the centromeric localization of hSgo1 and for counteracting the phosphorylation of hSgo1 11 by Polo kinase, which would otherwise result in removal of hSgo1 from chromosome, hSgo1 may also have a PP2A-independent function on the protection of centromeric cohesin because the precocious sister chromatid separation phenotype of PP2A-deficient human cell, but not that of hSgo1-deficient cells, can be rescued by the depletion of Polo kinases. hSgo2 is another human Sgo protein. Kitajima concluded that hSgo2 has a role in PP2A recruitment to human mitotic chromosomes (Kitajima et al., 2006; Riedel et al., 2006; Tang et al., 2006). Another interesting study showed that neither PLK1 depletion nor Aurora B inhibition suppresses PSCS in the absence of hSgo1, indicating that additional proteins could contribute to the prophase dissociation pathway (McGuinness et al., 2005). In addition, S. cerevisiae Sgo1 plays a role in the bi-polar attachment of kinetochores by activating the spindle checkpoint, which indicates a molecular link between sister chromatid cohesion and tension-sensing at the kinetochore-microtubule interface (Indjeian et al., 2005). I-4 Meiotic Cohesion Sister chromatid cohesion is also required for both meiotic divisions, but differs in several ways from its role in mitosis. In meiosis I, cohesion is involved in at least three unique functions. First, cohesion along chromosome arms promotes recombination between homologous chromosome and the formation of synaptonemal complexes (SCs) (Revenkova et al., 2004). Second, cohesion close to crossovers stabilizes chiasmata and is required for the disjunction of homologues in meiosis I (Klein et al., 1999; Petronczki et al., 2003). Third, cohesion functions in 12 mono-orientation of sister centromeres. In meiosis II, cohesion has similar roles to mitosis, including providing tension to promote sister chromatids alignment and holding sister centromeres together until anaphase II (AII). The most important change in the cohesin complex between meiosis and mitosis is that Scc1/Rad21 in mitosis is replaced largely by a meiotic-specific paralog Rec8 (Watanabe et al., 1999; Stoop-Myer et al., 1999; Kitajima et al., 2003). This Rec8 replacement may promote DNA recombination, synaptonemal complex formation, monopolar attachment and persistent centromeric cohesion until to AII. However, in budding yeast, most of these meiosis-specific properties can be substituted by Scc1/Rad21 except for the maintenance of sister centromere cohesion until AII (Toth et al., 2000; Yokobayashi et al., 2003; Xu et al., 2005). In addition, other meiosis-specific cohesin subunits have been found in various organisms. For example, in S. pombe, PSC3, which is the SCC3 subunit, is replaced in some meiotic cohesin complexes by a meiosis-specific counterpart, Rec11. Budding yeast Spo13, a novel protein, does not localize to the centromere, but is crucial for retention of centromeric cohesin in meiosis and when ectopically expressed in mitosis it can inhibit release of cohesin (Lee et al., 2002; Shonn et al., 2002). In budding yeast, sister chromatid cohesion is disrupted in two-step process in meiosis by the cleavage of Rec8 on chromosome arms at AI and at centromeres at AII by Separase, the same enzyme that cleaves the mitotic cohesin Scc1/Rad21 (Fig. I-4) (Buonomo et al., 2000). First, Rec8 along the chromosome arm is cut, resulting in disassociation of cohesin from arms. In addition, condensin and Cdc5, a Polo-like 13 --- JESSBERGER, 2005 Figure I-4. A model shows that SCC in meiosis is released in two steps. At meiosis I, the homologs move toward opposite poles, while the sister chromatids of each homolog remain connected because of centromeric cohesin. In meiosis II, centromeric cohesin is removed, resulting in sister chromatid segregation. 14 kinase, also can facilitate the removal of cohesin from chromosome arms before AI (Yu et al., 2005). However sister chromatid cohesion is preserved at centromeres throughout AI until MII due to the protection of centromeric Rec8 by the proteins Shugoshin (Sgo1) and PP2A phosphatase which shield centromeric cohesin from Polo kinase-dependent removal by dephosphorylating Rec8, as in mitotic animal cells (Kitajima et al., 2006; Riedel et al., 2006). Another protein involved in the protection of centromeric cohesion is Bub1 which is involved in protection of centromeric cohesin by recruiting Sgo to centric localization (Tang et al., 2004; Hamant et al., 2005; Kiburz et al., 2005; Kitajima et al., 2005). Mutations in each of the above genes cause PSCS during meiosis I. Second, centromeric Rec8 is cleaved by Separase at the onset of AII, resulting in sister chromatids segregating into each daughter cell. During meiosis I sister chromatids of a homolog develop potentially two independent kinetochores. However each sister kinetochore must attach to microtubules emanating from same spindle pole (monopolar attachment), instead of opposite poles, so that sister chromatids can move to the same pole at meiosis I. Although the mechanism of sister kinetochore mono-orientation is poorly understood, some research showed that centromeric cohesin complexes contribute to this monopolar attachment by holding sister kinetochores together. One line of evidence is that in fission yeast, mutations in the meiosis-specific cohesin subunit Rec8 result in bipolar attachment of sister kinetochores in meiosis I (Watanabe 1999; Yokobayashi et al., 2003; Yamamoto et al., 2003; Hauf et al., 2004). In addition, Monopolin in S. cerevisiae and Moa1 in S. pombe have been proved to be required for 15 mono-orientation of sister chromatids in meiosis I and Moa1 can interact with Rec8 (Toth et al. 2000; Yokobayashi et al. 2004). However, in both organisms Rec8 cohesin complex also play a role in the monopolar attachment of sister kinetocores, and loss of Rec8 function causes a random chromatid orientation (S. cerevisiae), or an equational orientation (S. pombe) at meiosis I (Klein et al., 1999; Watanabe et al., 1999). I-5 Meiosis in Drosophila Meiosis in Drosophila as a model has been investigated for several decades because of its several advantages. The first is that scientists can visualize the meiotic divisions in both male and female meiosis. The second is that there are a valuable collection mutations affecting meiosis. The third is Drosophila has a short life cycle and only four paired bivalents. The fourth is that the mechanisms of male and female Drosophila meiosis systems are different. Female Drosophila has both chiasmate and achiasmate meiotic mechanisms. The three large chromosomes have chiasmata to stabilize the interhomolog connections during meiosis and mutations affecting recombination can cause aberrant homolog segregation. For instance, mutations in homologous recombination genes including rad51/spindle-A (spnA), spindle-B (spnB), spindle-D (spnD) and okra have been found to affect meiotic recombination in females, and cause a reduction in recombination and increase of NDJ (Ghabrial et al., 1998; Morris et al., 1999, Yoo et al., 2004). However, these three large chromosomes can also segregate correctly using an achiasmate pathway termed distributive 16 segregation if exchange is suppressed by heterozygosity for multiple inversions. The tiny fourth chromosomes which never recombine always segregate via the distributive pathway (Orr-Weaver 1995). Male Drosophila has a unique meiotic system in which recombination does not occur and SC and chiasmata are not detectable. Nevertheless, these non-exchange homologs can pair and segregate faithfully during meiosis I. Mutations in genes affecting the exchange segregation pathway in females, such as the Spo11 homolog mei-W68 or spnA, spnB, spnD and okr, do not affect male meiosis. Also, mutations in genes required for the non-exchange distributive pathway such as nod (no distributive disjunction) and ncd (nonclaret disjunctional) have no effect on male meiosis (Knowles et al., 1991; Orr-Weaver 1995). Thus genes neither affecting recombination nor distributive segregation in female can explain the mechanism of male meiosis. In addition, mutations in the SC gene c(3)G (crossover suppressor on 3 of Gowen) abolish both SC formation and meiotic recombination in female Drosophila but does not alter the male homologs segregation pattern (Walker et al., 2000; McKim et al., 2002; McKee, 2004). These data strongly support that male meiosis in Drosophila lacks crossovers and involves a different meiotic segregation system. However, it is unclear how homologs pair in male Drosophila and what kind of apparatus provides the power to hold homologs together and balance the forces from opposite poles. Cooper demonstrated in 1964 that the X and Y chromosome pair at specific sites, thread-like structures of heterochromatin known as collochores near the nucleolus organizers (NORs), where the repeated genes for the 18s and 28s are located (Cooper 17 et al., 1964). Mckee and coworkers proved a 240 bp repeated sequence within the intergenic spacer of the ribosomal DNA (rDNA) can mediate disjunction of the X and Y chromosomes and the efficiency of segregation is dependent on the copy number of the intergenic spacer. But the autosomes do not contain any rDNA, which means they likely have multiple pairing sites accounting for their segregation (McKee et al., 1992; McKee et al., 1993; McKee, 2004). Currently, only a few genes have been identified to affect homolog segregation and cause meiosis I NDJ in male Drosophila including the male-specific genes, teflon (tef) and mei-s8, both of which localize to the 2nd chromosome. The teflon gene is required for disjunction of all autosomes, but not for sex chromosomes (Tomkiel et al., 2001). Thomas et al., 2005 identified two novel genes on the 3rd chromosome: stromalin in meiosis (snm) and modifier of mdg4 in meiosis (mnm). These two genes are required for segregation of all homolog pairs in male Drosophila meiosis I, but are dispensable for female meiosis and for sister chromatid segregation. Mutations in snm and mnm disrupt homolog conjunction and result in high frequencies of homolog NDJ in male Drosophila, but premeiotic and meiotic homolog pairing is normal. Using antibodies against SNM and MNM, MNM and SNM proteins were found in the nucleolus during PI and localize co-dependently to the X-Y bivalent pairing site during late PI through MI and disappear at AI. SNM is independent of MNM but MNM depends upon SNM with respect to their nucleolar localization. Using an MNM-GFP construct that fully rescues mnm mutations, MNM was also found to localize to multiple autosomal foci throughout meiosis I and this autosomal binding was found to depend on the teflon gene (Tomkiel et al., 2001, 18 Thomas et al., 2005). Recently, a Venus-tagged SNM protein that fully rescues snm mutations has been found to localize to autosomal foci as well as to the nucleolus and X-Y bivalent (S Thomas. personal communication). Another distinguishing character of meiosis in male Drosophila is meiotic prophase I (PI), a growth phase during which the primary spermatocytes undergo an approximate 25-fold increase in volume, lasts approximately 90 hours and is divided in substages S1-S6 depending on size of the nucleus and chromatin architecture (Cenci et al., 1994). In stages S1 and S2 (early-G2), the nucleus lies in an eccentric position within the cytoplasm and the chromatin positions at the center of the nucleus as a compact mass. Homologous chromosomes are tightly paired at this stage via pairing sites present in the euchromatin (Vazquez et al., 2002). When spermatocytes enter the late S2b or early S3, the chromatin mass subdivides into three main territories, presumably corresponding to the three major bivalents: XY, 2nd, and 3rd (Vazquez et al., 2002). Interestingly, euchromatic pairing is dissolved at the time (S2b/S3) that are established. These homologous chromatin masses remain at the inner nuclear envelope from mid-prophase I (S3-S4) until the onset of PMI. Then the chromosomes condense very rapidly which marks the onset of MI (Fig. I-5). Following two cell divisions, and a series of dramatic morphological changes, the resulting spermatids contains the half of the original eight chromosomes (Fig. I-5) (Cenci et al., 1994). 19 ---Vazquez et al., 2002 Figure I-5. A model for meiotic pairing in male Drosophila (A) A summary of spermatocyte development. Shading indicates chromatin. The formation of chromosome territories marks the beginning of stage S3 in mid-G2. (B) A model for meiotic chromosome pairing in male Drosophila. Forming the distinct territories represents the beginning of stage S3. 20 I-6 Meiotic Cohesion in Drosophila Drosophila has been a good model for genetic research for many years, but the mechanism and functions of meiotic cohesion are still an enigma because of no mutations in cohesin genes. The Drosophila genome contains single SMC1 and SMC3 genes and two members each of the SCC1 (RAD21 and C(2)M) and SCC3/SA (SA and SNM) families. C(2)M), although it exhibits weak similarity to SCC1 and REC8, is a synaptonemal complex component and functions only in synapsis and recombination during PI in females, not in centromeric cohesion in either sex (Manheim et al., 2003; Heidmann et al., 2004; Anderson et al., 2005). SNM identified by Thomas et al. in 2005 as a new meiosis-specific SCC3/SA paralog works exclusively in homolog conjunction in meiosis I in male Drosophila but is not required for sister chromatid cohesion in males or for any aspect of female meiosis (Thomas et al., 2005; Soltani-Bejnood et al., 2007). In addition, a Rec8 subunit has not been identified in Drosophila (Heidmann et al., 2004) MEI-S332 and ORD are two meiotic cohesion proteins found in Drosophila so far. Mei-S332 is the founding member of the Shugoshin family that protects centromeric cohesion. Mei-S332 localizes to centromeres in both male and female meiosis from PMI to MII, and dissociates concomitantly with segregation of sister chromatids, which is consistent with its role in maintaining sister chromatid cohesion (Kerrebrock et al., 1995; Moore et al., 1998). Several papers showed that mutations in the mei-S332 gene cause premature separation of the sister chromatids in AI, resulting in random sister chromatid segregation in meiosis II (Davis 1971; Goldstein et al., 21 1980; Kerrebrock et al., 1992; Tang et al., 1998). These findings implied that Mei-S332 functions in centromeric cohesion protection at AI, but the detailed mechanism is unclear. Mei-S332 is also expressed in mitotic cells and localizes to the centromeres from prometaphase to anaphase, however, unlike the mitotic shugoshins, it is not required for mitotic cohesion, nor for centromere retention of Rad21 (Katis et al., 2004; Kitajima et al., 2004; Lee et al., 2005). In addition, the centromeric localization of Mei-S332 in mitosis and meiosis is directly regulated by the chromosomal passenger complex, INCENP and Aurora B, and does not depend on the cohesin complex (Lee et al., 2004; Resnick et al., 2006). Recently, Clarke et al. (2005) showed that Mei-S332 activity is inhibited by POLO kinase and its removal from the centromeric region is regulated by phosphorylation by POLO kinase (Clarke et al., 2005; Lake et al., 2005). ORD is a meiotic cohesion protein with no homologs in other organisms (Bickel et al., 1996). ORD associates with the meiotic chromosomes during early PI in male meiosis, but localizes to centromeres after chromosome condensation in PI and persists until centromeric cohesin is released during AII (Balicky et al., 2002). ORD functions in sister centromere orientation in meiosis I as well as in maintaining sister chromatid cohesion in meiosis (Miyazaki et al., 1992; Bickel et al., 1997). ORD also localizes to synaptonemal complexes in PI in females and is required for normal recombination levels and for homologue bias during meiotic recombination. Mutations in ord can cause premature desynapsis and reduced recombination. In addition, Webber found that ORD is required for chiasmata maintenance in 22 Drosophila oocytes (Bickel et al., 1997; Bickel et al., 2002; Webber et al., 2004). 23 Chapter II – Evidence for a homolog conjunction complex in Drosophila male meiosis 24 II-1 Introduction Stromalin in Meiosis (SNM) SNM is a protein which contains 973 amino acids and shares homology with yeast SCC3, S. pombe REC11 and the vertebrate SA/STAG proteins, which are components of cohesin and essential for sister chromatid cohesion (Prieto et al., 2001; Kitajima et al., 2003). SNM is a meiosis-specific protein, but it does not function in sister chromatid cohesion and is not orthologous to these proteins. It is a paralog of Drosophila SA protein, which is thought to function in sister chromatid cohesion. Other meiosis-specific SCC3/SA paralogs, such as S. Pombe Rec11 and vertebrate STAG, have been described, but each of these proteins is more similar to its mitotic paralog than to the other meiosis-specific paralogs, suggesting independent origins for the meiotic paralogs (Thomas et al., 2005) Modifier of Mdg4 in Meiosis (MNM) MNM is a BTB-domain protein encoded by the mod (mdg4) locus, a complex locus which encodes over 30 different proteins by alternative splicing and functions in Drosophila in several processes, including chromatin boundary formation, nuclear architecture, position effect variegation (PEV), apoptosis, regulation of homeotic genes and early development, meiotic pairing of chromosomes and neurogenesis. These Mod(mdg4) protein isoforms share a common N-terminal region of 402 amino acids containing the BTB/POZ domain, a widely conserved protein-protein interaction motif which is responsible for the formation of multimeric complexes or interaction with other proteins. These isoforms vary in their C-terminal ends which contain from 25 28 to 208 amino acids. Most of the C-termini encode a Cys2His2–embedded FLYWCH domain named by the six conserved hydrophobic amino acids (FLYWCH) (Dorn et al., 1993; Gerasimova et al., 1995; Mackay J.P., et al., 1998; Gorczyca et al., 1999; Buchner et al., 2000; Dorn et al.,, 2003, Thomas et al., 2005). In addition to Drosophila, the FLYWCH motif is also present in three predicted mammalian proteins and three additional C. elegans proteins of unknown function. The function of the FLYWCH domain has not been proved, but Beaster-Jones found it is required for the DNA binding and in vivo function of C. elegans PEB-1 (Beaster-Jones et al., 2004). In addition, two Mod (Mdg4) isoforms, Mod(mdg4)-67.2 and Mod(mdg4)-56.3/DOOM/MNM, indicate a role for this domain in protein-protein interactions. Mod(mdg4)-67.2 interacts with the DNA binding protein Su(Hw) via its specific C-terminus containing FLYWCH (Gause et al., 2001; Ghosh et al., 2001). For the interaction between Mod(mdg4)-56.3/DOOM/MNM and the inhibitor of apoptosis protein of Baculovirus OpIAP, the FLYWCH domain has been proved to be necessary and sufficient (Harvey et al., 1997; Dorn et al., 2003). The BTB domain (also known as the POZ domain (Poxvirus zinc finger)) in the N-terminus of protein MNM was first identified by Koonin in 1992 as a conserved sequence motif in genes of DNA virus and named from the Drosophila transcription factors Bric-a-brac, Tramtrack, and Broad Complex which contains a similar sequence at their N terminus. (Koonin et al., 1992; Godt et al., 1993; Zollman et al.,1994). The BTB domain is a versatile protein-protein interaction motif and known for its ability of dimerization, oligomerization and interaction with a number of other 26 BTB proteins or non-BTB proteins using its unique tri-dimensional fold with a large interaction surface formed by approximately 95 core amino acids (Albagli et al., 1995; Mazur et al., 2005). This unique character provides the BTB-containing proteins with a variety of functional roles in transcription repression (Melnick et al., 2000; Ahmad et al., 2003), cytoskeleton regulation (Ziegelbauer et al., 2001; Kang et al., 2004), tetramerization and gating of ion channels (Kreusch 1998; Minor et al., 2000; ), protein ubiquitination /degradation (Pintard et al., 2003; Furukawa et al., 2004; Wilkins et al., 2004) and neurite outgrowth (Kim et al., 2005). The sequence of BTB domains among BTB-proteins have been proved to be variable, though there are a dozen highly conserved hydrophobic residues, and to form four known structural classes with different solvent exposed surfaces, which is responsible for the different oligomerization or protein-protein interaction states involving different surface-exposed residues (Stogios et al., 2005). For instance, the T1 domains in ion channel T1 proteins consist only of the core BTB fold without any amino- or carboxy-terminal extensions and have a tendency of tetramerization, but Skp1 proteins contain the core BTB fold with two additional carboxyl-terminal helices which provide another binding site (Kreusch et al., 1998; Bai et al., 1996). The other two families are the BTB-zinc finger (BTB-ZF) family and ElonginC proteins. The former can self-associate and dimerize because of an amino-terminal extension in BTB domain, however the latter lacks the last alpha-helix of BTB domain which affects its protein-protein interaction state (Ahmad et al., 1998; Botuyan et al., 2001). Currently, BTB-protein families show more than two dozen different domains are 27 associated with one single copy of BTB domain, of which five are much more frequent than others including MATH, Kelch, NPH3, Ion transport and Zinc finger (ZF). The largest group is BTB-ZF proteins including the newly identified protein MNM (Stogios et al., 2005; Perez-Torrado et al., 2006). Two findings attract us to explore the interaction between proteins MNM and SNM. One is their similar functions. Present evidence indicates that MNM and SNM have similar functions in homolog conjunction. Mutations in the mnm and snm genes result in homolog NDJ in meiosis I in male Drosophila. The second factor is their appearance and localization. Both of them appear at the onset of PI and co-localize to the XY homologous pairing site at MI, then disappear suddenly at AI (Thomas et al., 2005). Are they co-partners or different components of s homologous conjunction complex in male Drosophila? 28 II-2 Materials and Methods E.coli strains, Yeast Strains and Plasmids The Chemically Competent E. coli { F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu) 7697 galU galK rpsL (StrR) endA1 nupG} was purchased from Invitrogen company and transformation of E.coli were performed according to its protocol: cells on LB agar plates or liquid with 100 µg/ml ampicillin were incubated at 37 °C over night in the incubator (REVCO) or with shaking at 220 r.p.m. respectively. Yeast strain PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4 gal80 LYS2::GAL1–HIS3 GAL2–ADE2 met2::GAL7–lacZ; James et al., 1996) was a gift from Dr. Guan. PJ69-4A contains three separate reporter genes under the independent control of three different GAL4 promoters respectively (GAL1-HIS3, GAL2- ADE2 and GAL -lacZ) and provides a high level of sensitivity with respect to detecting weak interactions, coupled with a low background of false positives (James et al., 1996). The plasmids, pGAD-C1 and pGBD-C1 (Fig II-1), were gifts also from Dr. Guan and their structures are shown in the follow figure. The vector pGAD-C has a selective gene leu2 to ensure that yeast colony containing the corresponding plasmid or plasmid construct can grow up on plates minus leucine. The vector pGBD-C has a selective gene trp1 to select the yeast colony which contains the plasmid or plasmid construct on plates minus tryptophan. Plasmid Construction and DNA Sequencing The mnm cDNA was PCR amplified using the sense primer with an EcoRI site (5’-GATCGAATTCATGGCGGACGACGAG-3’) and the anti-sense primer with a 29 Figure II-1. The structures of the pGAD-C and pGBD-C vectors. Stippled regions indicate the ADHl promoter (P) and transcription termination (T) elements. GAL4 AD (activation domain) in pGAD-C encodes amino acids (768-881). GAL4 BD (DNA binding domain) in pGBD-C encodes amino acids (1-147). Restriction sites following by GAL4 AD or GAL4 BD are shown on each map including EcoRI, SmaI, BamHI, ClaI, SalI, PstI and BglII. Both vectors contain the amp gene to allow the E.coli selection on the LB plate added with ampicillin. 30 BamHI site (5´-GATCGGATCCCTACAAATGGTTGTGC-3´). The gene snm cDNA was amplified using the forward primer with a BamHI site (5’-CAGCTTGGATCCATGAGTGATATATCTTTTGATG-3’) and the reverse primer with a SalI site (5´-GTAGCGTCGACCATCCTGTAAGTTGTATCCTTC -3´). Full-length snm and mnm cDNA templates were described in Thomas et al., 2005 and were kindly provided by Dr. S. Thomas. The btb cDNA was amplified by the sense primer with an EcoRI site (5’-GATCGAATTCATGGCGGACGACGAG-3’) and the anti-sense primer with a BamHI site (5´-GATCGGATCCCTACAAATGGTTGTGC-3´).This btb product contains 127 amino acids of the N-terminus of MNM protein (Fig II-2). The mnm PCR product with both vectors were digested with both EcoRI and BamHI (Invitrogen) and were ligated separately by T4 DNA ligase (Invitrogen) in according with the provided protocols to construct pGAD-MNM and pGBD-MNM, simply AD-MNM (AM) and BD-MNM (BM). The same process was performed on the snm and btb PCR products to constitute AD-SNM (AS), BD-SNM (BS), AD-BTB (AT) and BD-BTB (BT). After confirmation by DNA sequencing, these constructs were transformed into E.coli (FmcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 ara 139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG) provided by Invitrogen. Colony PCR and Plasmid Extraction The optimized colony PCR reaction mixture contained 1X PCR amplification buffer [20 mM (NH4)2SO4, 72.5 mM Tris/HCl, 0.1% Tween 20, pH 9·0], 2.5 mM 31 Figure II-2. Structure of BTB domain and FLYWCH domain of MNM protein. 32 MgCl2, 200 µM each deoxynucleotide triphosphate, 2.5 µM each primer, 1.25 U Supertherm DNA polymerase (LPI) in 50 µl PCR reaction mixture. A final concentration of 100 µg / ml of acetylated BSA (New England BioLabs), 3% dimethyl sulfoxide (DMSO) (Sigma) and 1 M betaine (Sigma) as PCR additives were also added to the reaction mixture. Colonies approximately 1 mm in diameter were picked up with a sterilized toothpick and directly transferred to the PCR tube as DNA templates. The thermal cycle program, run on a GeneAmp PCR system 9700 (Perkin Elmer) consisted of one cycle of 94 °C for 10 min, 51 °C for 2 min, 72 °C for 2 min, and 35 cycles of 94 °C for 20 s, 57 °C for 45 s (decreased by 1 s per cycle), 72 °C for 1 min, and then incubation at 72 °C for 5 min, and a final incubation at 4 °C. (Sheu et al., 2000). After amplification, a PCR product was subjected to gel electrophoresis analysis and DNA sequence analysis to select the plasmid that contains recombinants of the vector and the protein of interest without any mutation. Yeast Two-Hybrid Assay and β-galactosidase Assay Two-hybrid assays were performed using yeast strain PJ694A and plasmids provided by Dr. Guan. For growth assays, plasmids were transformed into yeast strain PJ694A by the lithium acetate method described in the yeast protocols handbook (Clontech). Bait and target fusion proteins were produced constitutively under the control of the ADH1 promoter. Co-transformants were plated on selective media minus tryptophan and leucine. After incubation at 30°C for 3 days, plates were replicated on selective media lacking tryptophan, leucine and histidine or selective 33 media lacking tryptophan, leucine and adenine (selective plates for the reporter gene), and growth on both plates was compared. Appearance of transformants on the selective plates indicates a positive interaction. Single colonies were subsequently streaked out on selective plates to obtain the plates shown in the following figures. β-Galactosidase activity was determined for liquid cultures grown in yeast-peptone-dextrose medium by using the ONPG (O-nitrophenyl-b-D-galactopyranoside) assay. One yeast colony from each section on the media without leucine and tryptophan was picked and shook (230 rpm) overnight in 5ml YPD liquid at 30℃. Cells were suspended in breakage buffer (0.1 M Tris pH 8.0, 1 mM DTT, 20% glycerol ) containing 1 mM PMSF (phenylmethane sulfonyl fluoride, Sigma). Glass beads were added to disrupt the cells and the suspensions were cleared by centrifugation after disrupting cells with glass beads. Protein concentration was measured, and 300 ug of protein were used to perform the β-gal activity assays in the Z buffer containing 200 ul of ONPG (ortho nitrophenyl galactoside, Sigma) 4mg/ml. The final volume of each reaction was 500 ul. Finally, the reactions were incubated at 32℃ until the solution became faint yellow and were stopped by adding 0.5 ml of 1 M sodium carbonate. The OD420 of each sample was measured and the β-gal units were calculated. Statistics Analysis All data were evaluated for normality of distribution and equality of variance prior to statistical analysis. Outcomes were shown as means ± SD and were evaluated 34 for statistical significance by one-way analysis of variance (ANOVA) and t test to compare group means using SPSS 14.0 (SPSS Inc, Chicago, IL). 35 II-3 Results and Discussion 1. MNM can interact with SNM and with itself The finding that two proteins MNM and SNM co-localize to the X-Y bivalents from late G2 through PMI and MI and disappear simultaneously at Ana I, and that mutations in both mnm and snm show high similar meiotic phenotype, suggests a hypothesis that these two proteins interact directly with each other. We used the yeast two-hybrid system to test this hypothesis under in vivo conditions. We cloned the full-length coding sequence of MNM and SNM, and that of the BTB domain of MNM, into both the pGAD-C1 (yeast Gal4 activation domain) and pGBD-C1 (yeast Gal4 DNA binding domain) yeast two-hybrid vectors. Here BTB-only clones served as a positive control because the BTB domain is a versatile protein-interacting motif and the BTB domain of Mod(mdg4) has been proved to interact directly and strongly with itself (Albagli et al., 1995; Mazur et al., 2005). These clones were then co-transformed into PJ69-4A yeast cells which contain three independent reporter genes (GAL1-HIS3, GAL2-ADE2 and GAL7-lacZ). If two proteins can interact with each other, the reconstituted intact Gal4, a transcription regulating protein, has the ability to activate transcription of these reporter genes, and allow growth of colonies. As expected, the colonies carrying BTB-only AD+DB combination can grow on the media minus adenine (Fig.II-3-A) and media minus histidine (Fig.II-3-B), indicating that the BTB domain can interact with itself and can be used as a positive control. Also, the colonies carrying MNM-only AD+DB combination also can grow on the media minus adenine (Fig. II-3-A) and the media 36 Figure II-3. MNM and SNM interact directly and weakly with each other. Growth of yeast strain PJ69-4A expressing different proteins BTB, MNM, and /or SNM pairs on non-selective (left) or selective (right) media for the reporter gene HIS3 (A) and ADE (B) used in yeast two-hybrid system. The numbers on the plate indicate the following: 1, yeast expressing AD-BTB+BD-BTB; 2, yeast expressing AD-MNM+BD-MNM; 3, yeast expressing AD-SNM+BD-MNM; 4, yeast expressing AD-MNM+BD-SNM; 5, yeast expressing AD-MNM+BD; 6, yeast expressing AD+BD-MNM The yeast colonies carrying both BTB-only AD+DB combination and MNM-only AD+DB combination also can grow on the media minus histidine and the media minus adenine, indicating that MNM can interact with itself. 37 2 2 3 3 1 1 4 4 6 6 5 5 Figure II-3-A 2 2 3 3 1 1 4 4 6 6 5 Figure II-3-B 38 5 without histidine (Fig.II-3-B), indicating that MNM, as a BTB-containing protein, can interact with itself. Yeast co-transformed with SNM-BD and MNM-AD or with SNM-AD and MNM-BD grew well on the media without histidine (Fig. II-3-B) but poorly on the media without adenine (Fig.II-3-A). HIS3 is not ideal reporter because of its leaky expression to yield high false positive frequency, but it is still useful for the effective detection of weak interactions (James et al., 1996). Notably, both SMNM-MNM combinations (section 3 and 4) exhibited much better growth on hisplates than did either MNM-BD +AD (section 5) and MNM-AD + BD (section6), indicating that the growth in the SNM-MNM combination is not due only to leaky HIS3 expression. Thus, these data suggest that MNM-SNM interact with each other. The SNM-MNM interaction was further confirmed in liquid culture assays of β-galactosidase activity, which is the third reporter gene of the system. Yeast co-transformed with MNM-only AD+DB combination, SNM-BD and MNM-AD , or SNM-AD and MNM-BD produced similar levels of β-gal, which are significantly higher than when expressing either MNM-BD or MNM-AD alone (p<0.05) (Fig.II-4). Although this expression is lower than theβ-gal units produced by the BTB-BTB interaction, we still can draw the conclusions that MNM protein interacts with itself, and that MNM and SNM proteins can interact with each other. In addition, yeast which expresses AD+BD-MNM was also found to grow up on the media without histidine. We think that it is the consequence of the leaky expression of histidine because its β-gal unit is much lower than the other positive combinations and similar to the other negative control which contains AD-MNM+BD. 39 β - gal Uni t 250 * 200 150 100 ** ** 50 0 AT+BT AM+BM ** AM+BS AS+BM *** *** AM+BD AD+BM Figure II-4. β-galactosidase activity, expressed as Milller units, in extracts of yeast strains carrying combinations of BTB, MNM, and /or SNM protein pairs. From left to right: 1, yeast carrying AD-BTB+BD-BTB; 2, yeast carrying AD-MNM+BD-MNM; 3, yeast carrying AD-MNM+BD-SNM; 4, yeast carrying AD-SNM+BD-MNM; 5, yeast carrying AD-MNM+BD; 6, yeast carrying AD+BD-MNM * Value is significantly different from ** value by One-way ANOVA (p<0.05) * Value is significantly different from *** value by One-way ANOVA (p<0.05) ** Value is significantly different from *** value by One-way ANOVA (p<0.05) 40 2. What part of MNM is responsible for the interaction between MNM and SNM? BTB domains or other BTB-ZF proteins have been shown to interact with other proteins. Is the MNM BTB domain also involved in the interaction between MNM and SNM proteins? In support of this, we found that yeast co-transformed with SNM-BD and BTB-AD or with SNM-AD and BTB-BD grew vigorously on the media without histidine, suggesting that the BTB domain alone interacts with SNM protein (Fig.II-5). However, theβ-gal units produced by yeast co-transformed with SNM and BTB were much lower than those produced by yeast expressing SNM and MNM (Fig. II-6). This implies that another part of MNM protein is involved in the interaction between MNM and SNM. 3. Unanswered questions and future experiments Although BTB-only interacted with SNM to activate both HIS3 and lacZ reporter genes, full-length MNM interacted more strongly with SNM than did BTB-only, at least in the β-gal assay, suggesting that an additional domain of MNM outside the BTB domain may contribute to the SNM-MNM interaction. Previous research proved that the FLYWCH domains in the C-termini of proteins Mod (mdg4)-67.2 and Mod (mdg4)-56.3/DOOM/MNM have roles in specific protein-protein interactions. They can interact independently with Su(Hw) and OpIAP respectively (Gause et al., 2001; Ghosh et al., 2001). Therefore, the FLYWCH domain of MNM is a good candidate for the second SNM-MNM interaction domain. An experiment is in progress to determine 41 3 2 2 3 4 4 1 1 5 5 7 7 6 6 Figure II-5. Growth of yeast strain PJ69-4A expressing different proteins BTB, MNM, and /or SNM pairs on non-selective (left) or selective (right) media for the reporter gene HIS3 used in yeast two-hybrid system. The numbers on the plated denote the following: 1, yeast expressing AD-BTB+BD-BTB; 2, yeast expressing AD-BTB+BD-MNM; 3, yeast expressing AD-MNM+BD-BTB; 4, yeast expressing AD-BTB+BD-SNM; 5, yeast expressing AD-SNM+BD-BTB; 6, yeast expressing AD-BTB+BD; 7, yeast expressing AD +BD-BTB. 42 90 80 70 60 50 40 30 20 10 0 66. 85 49. 55 MNM & SNM BTB & SNM 19. 425 9. 725 1 2 Figure II-6. β-galactosidase activity, expressed as Milller units. Comparison of β-gal units produced by the yeast colony containing MNM and SNM proteins and by the yeast colony carrying BTB domain and SNM protein. 1: β-gal units produced by AM+BS (blue) and AT+BS (red) 2: β-gal units produced by AS+BM (blue) and AS+BT (red) 43 the interaction between the FLYWCH domain of MNM and SNM by using the isolated C-terminus of MNM. If the isolated FLYWCH domain is able to interact with SNM, a useful follow-up experiment would be to determine whether this interaction depends on the C2H2 motif because a single amino acid substitution in the C2H2 motif that changes it to CYCH was found to completely abolish MNM meiotic function and disrupt its location to the nucleolus and to chromosomes (Thomas et al.,2005; Soltani-Bejnood et al., 2007). Thus, it would be informative to introduce the CYCH mutation into both full length MNM and FLYWCH only MNM to determine whether the C2H2 motif is required for the interaction with SNM. In all of the experiments reported above, the Drosophila proteins were fused at their N-termini to the C-terminus of either Gal4-BD or Gal4-AD. Mazur found in 2005 that when GAL4 activation domain (AD) is fused with the N-termini of the BTB domain or of a BTB-containing protein, its ability to induce transcription is disturbed, resulting in difficulty in detecting the interaction between proteins using the Yeast Two-Hybrid Assay (Mazur et al., 2005). So, an alternative experiment in the future is to switch the BTB and MNM protein from the C-termini of AD to the N-termini of AD to measure the interaction strength between BTB and SNM or between MNM and SNM. 44 Chapter III – sun is required for sister chromatid cohesion and sister centromere mono-orientation in male Drosophila meiosis 45 III – 1 Introduction Recently, S. Thomas and B. McKee have identified a new Drosophila gene, sisters unbound (sun) which was discovered in a genetic screen for EMS-induced mutations that disrupt paternal transmission of the small 4th chromosome (Wakimoto B.T. et al., 2004), and mapped to the 68D3 region on the left arm of 3rd chromosome. All four sun alleles have mutations in the predicted coding sequence of CG32088, a gene predicted to consist of 9 exons and to encode a protein 760 amino acids in length (Fig III-1). sun is required for centromeric cohesion. Mutations in sun cause abnormal meiosis in both male and female Drosophila including high homologous and sister chromatid NDJ, resulting in aneuploid sperm. sun mutations also affect recombination and synapsis in females meiosis. My study focused on the role of sun in centromeric cohesion. How and when do sun mutations affect cohesion and sister chromatid segregation in meiosis in male Drosophila? During which phase is centromeric cohesion first lost, PI or MI? The proteins SNM and SUN interact in sister chromatid cohesion in male Drosophila. 46 CG32088/sun Z3-5839 Del. Stop 5’ 1 3’ 2 3 4 6 5 7 8 Z3-4085 Z3-1550 Z3-1956 G170R Missense Q557 Stop W839R Stop 9 Figure III-1. Molecular characterization of CG32088/sun. All alleles are shown above and below the gene model. Grey shading represents the exons. 47 III- 2 Material and Methods Fly stocks, Special Chromosomes and Drosophila Culture Methods. The sun mutations in this study were from the Zuker-3 (Z3) collection of more than 6000 lines with EMS-mutagenized third chromosomes which was produced in a screen for paternal 4th chromosome loss (Koundakjian et al., 2004; Wakimoto et al., 2004), and were provided kindly by B. Wakimoto. All flies were maintained at 23°C. Compound chromosomes and markers are described in Flybase. CID-GFP stocks were furnished kindly by S. Henikoff. Unless otherwise noted, tested males were crossed singly to two or three females in shell vials. Crosses were incubated at 23◦C on cornmeal-molasses-yeast-agar medium. Parents were removed from the vial on day 10 and progeny were counted between day 13 and day 21. Sex Chromosome Nondisjunction Test in males Sex chromosome nondisjunction was measured in crosses of trans-heterozygous males for two sun alleles (sunZ3-5839/sunZ3-1956) having a marked Y chromosome to the females carrying the compound-X chromosomes, C(1)RM. These female can produce only nullo-X and diplo-X chromosomes eggs equally. The nullo-X eggs generate viable progeny when fertilized by either XX or XY sperm, the sperm classes that are diagnostic for sister chromatid and homolog NDJ, respectively, and the diplo-X eggs generate viable progeny when fertilized by nullo-XY (O) sperm. The cross can also yield progeny from XXY, XYY and XXYY sperm, but such progeny were recovered only very rarely and were neglected in this analysis. 48 Testis Immunostaining and FISH For anti-α-tubulin/DAPI experiments, testes were fixed according to Cenci et al. (1994) and stained according to protocol 5.6 (Bonaccorsi et al., 2000). FITC-conjugated monoclonal anti-α-tubulin (Sigma) was diluted 1:150. (Thomas et al., 2005). CID-GFP Detection in Spermatocytes For CID-GFP experiments, testes were dissected from young adults in testes buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF), gently squashed and frozen in liquid nitrogen. Slides were then incubated with 1 mg/ml 4'-6-Diamidino-2-phenylindole (DAPI) at room temperature for 10 minutes and washed with 1XBBS twice for five minutes. Microscopy and Image Processing All images were collected using an Axioplan (ZEISS) microscope equipped with an HBO 100-W mercury lamp and high-resolution CCD camera (Roper). Image data were collected and merged using Metamorph Software (Universal Imaging Corporation). For CID signals, all images were taken as Z-series by using a 100× oil-immersion objective, and were deconvolved by using Metamorph Software to obtain maximum projections. 49 III- 3 Results 1. NDJ of homologous and sister chromatids in sun males To measure whether sun mutations disrupt segregation of the sex chromosomes in Drosophila male meiosis, sun males (sunZ3-5839/sunZ3-1956) carrying a dominantly marked Y chromosome were crossed to C(1)RM females which carry the attached-X chromosome and can generate either nullo-X or diplo-X eggs at equal ratio. After the cross, we confirmed that mutations in sun cause both homolog and sister chromatid NDJ, resulting in XX and XY sperm classes (Fig. III-2). Assuming that YY sperm are produced at frequencies comparable to those of XX sperm, the ratio of sister chromatid to homolog NDJ (S/H) is 0.70, which indicates that the four sex chromatids separate prematurely during meiosis I and segregate approximately randomly through both divisions. 2. Sister centromere cohesion is lost at mid-prophase in sun spermatocytes Surprisingly, in DAPI-stained preparations we found that chromosome morphology in meiosis I in sun mutants is similar to that in wild-type. Bivalents were nearly always intact during PMI and MI and segregated evenly to opposite poles (Fig. III-3), although it was often seen that sister chromatids segregate unevenly in MII (Fig.III-4). One possible explanation here is that SNM & MNM hold bivalents together and prevents the complete separation of homologous and sister chromatids in sun mutants in meiosis I. In order to assess the effects of sun mutations on centromeric cohesion and to determine in which stage centromeric cohesion is lost in 50 Test of homol og vs si st er chr omat i d NDJ f or sun mut ant s 300 250 244 200 200 176 150 99 100 35 50 0 X Figure III-2. Y XX XY 0 NDJ test of homologs vs sister chromatids for sun mutants. The X-coordinate represents different possible sperm classes and the Y-coordinate is the number of F1 male Drosophila. The S/H ratio {(XX+YY)/XY} here was 0.70 and the overall NDJ frequency was 0.44. 51 PMI MI AI TI Wild Type sun Figure III-3. Chromosome morphology is normal during meiosis I in sun spermatocytes (sunZ3-5839/sunZ3-1956), compared to wild-type. Bivalents were nearly always intact during PMI and MI and segregated evenly to opposite poles in AI and TI. Red-DAPI, Green-α-tubulin. 52 PMII MII AII TII Wild Type sun Figure III-4. Sister chromatids segregate unevenly in meiosis II in sun spermatocytes (sunZ3-5839/sunZ3-1956). Red-DAPI, Green-α-tubulin. 53 meiosis I, we generated trans-heterozygous sun males (sunZ3-5839/sunZ3-1956) which express a CID-GFP fusion protein. CID (Centromere identifier) is the Drosophila homolog of the CENP-A centromere-specific H3-like proteins and localizes exclusively to the centromeres (Ahmad et al., 2001; Blower et al., 2001). In our study we used CID to diagnose the sister centromere’s cohesion status in meiosis in wild-type or sun spermatocytes. In wild-type spermatocytes, sister centromeres are tightly held together throughout meiosis I because of centromeric cohesion, so there are never more than eight separate CID spots, corresponding to the eight chromosomes in a diploid cell. As expected, sun mutants exhibited too many CID spots in mid-prophase (S4) stage (Fig. III-7). Most bivalents in sun spermatocytes exhibited 3 or 4 spots instead of the normal 2 spots and the total spot number per nucleus was more than 8, indicating a defect in centromeric cohesion. Similar results were obtained with mature spermatocytes in stages S5 (Fig. III-8). However, during early-(S1/2) and mid-prophase (S3) the numbers of CID foci in sun mutants were similar to wild-type (Fig. III-5 and III-6) (Cenci et al., 1994). We concludethat in sun mutants centromere cohesion becomes lost after resolution of chromosome territories in mid-prophase (S4). These observations strongly suggest that the aberrant meiosis I segregation patterns apparent in the cross data result from premature loss of centromeric cohesion, allowing sister chromatids to segregate nearly randomly. 54 DNA CID MERGE S1/2 Wild Type sun Figure III-5. Less than 4 CID-GFP spots are frequent in both sun (sunZ3-5839/sunZ3-1956) and wild-type spermatocytes in early-prophase I (S1/2). Each circle represents one cell. Green – CID-GFP; Red – DAPI. 55 DNA CID MERGE S3 Wild Type sun Figure III-6. In mid-prophase I (S3) both sun (sunZ3-5839/sunZ3-1956) and wild-type spermatocytes showed less than 4 CID-GFP spots. Each circle represents one cell. Green – CID-GFP; Red – DAPI. 56 DNA CID MERGE Wild Type sun Figure III-7. Centromeric cohesion is lost at stages S4 in sun spermatocytes (sunZ3-5839/sunZ3-1956). Green – CID-GFP; Red – DAPI. 57 S4 DNA CID MERGE S5 Wild Type sun Figure III-8. Centromeric cohesin is absent during stages S5 in sun spermatocytes (sunZ3-5839/sunZ3-1956). Green – CID-GFP; Red – DAPI. It is more clear that the sun spermatocyte exhibits more than 8 CID-GFP spots in late-prophase I. 58 III-4 Discussion sun is required for centromere cohesion Our data showed that SUN is required for sister centromere cohesion in male Drosophila meiosis. Mutations in sun cause loss of sister centromere cohesion as early as mid-prophase (S4), although bivalents remain intact through meiosis I. Homologous and sister chromatids segregate approximately randomly and yield aneuploid sperm. The recovery of sperm containing both X and Y chromosomes indicates that mono-orientation of sister centromeres during meiosis I is disrupted in sun mutations. However it is not clear whether this disruption is a consequence of loss of sister centromere cohesion or represents another function for SUN. There is evidence that cohesion between sister centromeres is required for their “mono-orientation” to the same pole on the meiosis I spindle, although some new proteins also have been proved to be required for sister centromere mono-orientation, such as Monopolin in S. cerevisiae and Moa1 in S. pombe (Toth et al., 2000; Yokobashi et al., 2004). For example, mutations in the meiosis-specific cohesin subunit Rec8 result in bipolar attachment of sister kinetochores in meiosis I (Watanabe 1999; Yokobayashi et al., 2003; Yamamoto et al., 2003; Hauf et al., 2004). So, it is important to determine the real function of SUN in mono-orientation of sister centromeres. Is SUN a component of cohesin complex? Although SUN is required for sister centromere cohesion, we do not know 59 whether SUN is a component of cohesin complex or it works as the protector for sister centromere cohesion. In S. cerevisiae and S. pombe, meiotic cohesion functions are fulfilled by cohesin complexes that include meiosis-specific subunits such as REC8, which replaces the mitotic kleisin subunit RAD21. Rec8 conserved among most eukaryotes, but no true REC8 homolog has been identified in Drosophila, (Petronczki et al., 2003; Heidmann et al., 2004; Losada et al., 2005). In addition, no mutations in cohesin genes have been available in Drosophila. Thus the role of cohesin in meiotic cohesion is unclear. The Drosophila ORD is a meiotic cohesion protein with no homologs in other organisms. Protein ORD localizes to centromeres after chromosome condensation in PI and is required in meiosis for both arm and centromere cohesion (Bickel et al. 1997; Balicky et al. 2002). The phenotypes of sun mutations are similar to those of ord mutations in Drosophila. Both sun and ord mutations cause premature loss of centromere cohesion during meiosis I, leading to NDJ of both homologous and sister chromatids (Miyazaki et al. 1992; Bickel et al. 1997). So, it is interesting to uncover the relationship between sun and ord. Does SUN also localize to centromeres? Do they work together as partners, or is SUN a protector of ORD? In addition, proteins Shugoshin (Sgo1) and PP2A phosphatase have been proved to work together to shield centromeric cohesin in yeast (Kitajima et al., 2006; Riedel et al., 2006). As the founding member of the Shugoshin family, Drosophila MEI-S332 is also a meiotic cohesion protein and protects centromeric cohesion. Mutations in the mei-S332 cause premature separation of the sister chromatids in 60 meiosis II. It is another direction for us to discover the real function of SUN in centromere cohesion and the relationship between SUN and MEI-S332. 61 Chapter IV – Roles of interaction of sun, snm and mnm in meiotic cohesion, centromere pairing and chromosome territory integrity 62 IV-1 Introduction Crossovers are important for homologous chromosomes segregation during meiosis I. However, in several organisms, such as male Drosophila, it has been shown that nonexchange pair of chromosome also can be segregated properly, which demonstrates that some unknown mechanisms, other than exchange, are involved in the nonexchange segregation. Studies in Drosophila females and S. cerevisiae have shown that pairing at the centromeric region is required for correct homologous chromosome segregation when chiasmata are absent. In S.cerevisiae centromere regions undergo meiotic pairing which is sequencing-independent, and this pairing orients the kinetochore of the nonexchange partners (Karpen et al., 1996; Kemp et al., 2004). Another finding also using S. cerevisiae showed that initially centromeric interactions occur mainly between non-homologous chromosome centromeres, and then switch to homologous centromeres prior to zygotene. These centromeric interactions are dependent on a component of the SC, Zip1, and the transition from non-homologous to homologous centromeres pairing is dependent on Spo11, the endonuclease required for DSB formation during meiosis (Tsubouchi et al., 2005). Nsl1p, a new protein important for chromosome segregation, has also been found to play a role in transient centromere pairing in S.cerevisiae (Nekrasov et al., 2002). However in male Drosophila, it is not clear about homologous centromeres pairing. In our research, we found that SNM protein play a roles in homologous centromeres pairing in the stage S3 in PI, and loss of SNM causes homologous centromere unpaired. 63 IV-2 Materials and Methods All materials and methods in this chapter are similar to that in the chapter III. In this chapter trans-heterozygous males for two snm alleles (snmZ3-2138/snmZ3-0317) and for two mnm alleles (mnmZ3-5578/mnmZ3-3401) were used. 64 IV-3 Results 1. The homolog conjunction protein SNM prevent complete separation of homologous and sister chromatids in sun mutants To test the possibility that the homolog conjunction proteins can hold sister chromatids together in the absence of SUN, males doubly mutant for sun snm (sunZ3-5839 sunZ3-1956 / snmZ3-2138 snmZ3-0317) were generated and their chromosomes were examined by DAPI staining. SNM is a homolog conjunction protein in male Drosophila and plays a role in holding homologous together from PI to AI. Loss of SNM will cause homolog NDJ in meiosis I (Thomas et al., 2005). As shown in Figure IV-1, the double mutants displayed a much more severe phenotype during meiosis I than single sun mutants, or single snm mutants, and fully separated sister chromatids can be seen during PMI and MI. In addition, the theoretical S/H (sister/homolog) NDJ ratio of random 2x2 segregation at meiosis I followed by random segregation at meiosis II should be 0.5, whereas the observed ratio in single sun mutants is 0.70, which implies that the homolog conjunction protein promotes reductional (XX:YY) segregations at meiosis I. To test this possibility, sun and snm males carrying BSYy+ were crossed with C(1)RM/O females (Table IV-2). The S/H ratio was reduced to 0.53, close to 0.50, confirming that sister chromatids segregate completely randomly in double mutants males. So we concluded that the homolog conjunction protein SNM prevents complete separation of homologous and sister chromatids in sun mutants. Although SNM alone does not suffice to maintain sister chromatid mono-orientation, it does 65 Wild type sun snm sun & snm PMI MI Figure IV-1. Sister chromatids segregate completely during PMI and MI in sun snm double mutants (sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317) Red-DAPI, Green-α-tubulin 66 mutants X Y XX YY XY O 244 200 35 35 99 176 sun 30.9% 25.3% 4.4% 4.4% 12.5% 22.3% 691 546 57 57 217 953 sunsnm 27.4% 21.6% 2.3% 2.3% 8.6% %NDJ S/H 44% 0.70 51% 0.53 37.8% Table IV-2. The percentage comparison of different sperm classes produced by single sun (sunZ3-5839/sunZ3-1956) mutants and sun snm (sunZ3-5839 sunZ3-1956 / snmZ3-2138 snmZ3-0317) double mutants males. The S/H ratio {(XX+YY)/XY} in single sun mutants was 0.70, and in sun snm double mutants was 0.53 which is close to 0.50, confirming that sister chromatids segregate completely randomly in double mutants males. 67 promote a non-random excess of reductional segregation. 2. SNM, but not MNM, is required for the pairing of homologous centromeres during mid-prophase (S3) Vazquez et al. reported in 2002 that homologous centromeres in wild type males can pair transiently during stage S3, but suddenly lose pairing by stage S4 (Vazquez et al., 2002). To determine whether the homolog conjunction complex plays a role in the centromeric pairing, both snm and mnm males expressing CID-GFP were generated. In wild-type males and mnm males, each newly formed chromosome territory shows a single CID-GFP spot in S3 (no more than 4 CID-GFP spots can be seen in one nucleus), and eight CID spots from stage S4 through MI (data not shown). However, in snm males, more than 99% of S3 nuclei exhibited five or more CID-GFP spots, and most exhibited 6-8 spots (Fig.IV-3). Thus, we concluded that SNM, but not MNM, is required for the pairing of homologous centromeres in the stage S3, and this pairing is transient and is lost between stages S3 and S4. Remarkably, sister centromeres separated earlier in PI in sun snm double mutants than in either single mutants. In sun snm males there were more than 8 CID-GFP spots in 30% of S3 nuclei, 95% of S4, and 100 % of S5/6. However, in single sun males, S3 nuclei exhibited normal frequencies CID-GFP spots. After that 42% of S4 and 90% of S5/6 nuclei showed more than 8 CID-GFP spots. This indicated that SNM can maintain cohesion between sister centromeres in the absence of SUN in the stage S3 (Fig. IV-4). 68 CID DNA MERGE Wild Type sun snm snm & sun Figure IV-3. Sister centromere cohesion is lost in sun snm double mutants (sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317) in S3 stage, which is earlier than sun single mutants, indicating that SNM can maintain cohesion between sister centromeres in the absence of SUN in mid-prophase (S3). Red-DAPI, Green-CID-GFP 69 CID spots in prophase I > 8 CID spots percentage 120% 100% 95% 100% 90% 80% WT snm sun snm sun 60% 42% 40% 30% 20% 0% 0% S1/ 2 0% S3/ 4 S4 0% 0% S5/ 6 Prophase I Figure IV-4. A comparison of > 8 CID spots percentage in different stages in prophase among wild-type, single sun (sunZ3-5839/sunZ3-1956) mutants, single snm (snmZ3-2138/snmZ3-0317) mutants, and sun snm (sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317) double mutants. 70 3. Mutations in sun snm and sun mnm impair the integrity of chromosome territories Beginning at stage S3 (mid-G2) in wild type males the chromatin masses form three main territories, presumably corresponding to the three major bivalents: XY, 2nd, and 3rd chromosomes (Vazquez et al., 2002), and remain separate until the onset of MI. In sun mutants, although centromeric cohesion is impaired and sister centromeres separate prematurely, chromosome morphology and behavior during PI appear normal. Three separate DAPI-stained masses, corresponding to the three major bivalents, were regularly seen. In single snm mutants territory formation at stage S2b/S3 appeared normal but in stage S4-S6, approximate 50% of snm spermatocytes exhibited unusual diffuse territory (Thomas et al., 2005). Surprisingly, sun snm and sun mnm double mutants severely disrupted the integrity of chromosome territories. Nearly all territories during late PI were subdivided into smaller subterritories and exhibited more than 8 chromatin clumps, each of which, we think, corresponds to a single chromatid from a large chromosome (Fig. IV-5). We conclude that the integrity of chromosome territories is dependent solely and jointly on proteins that regulate homolog pairing and sister chromatid cohesion. 71 Wild Type mnm & sun snm & sun Figure IV-5: The integrity of chromosome territories in late PI are impaired in mutations in sun snm (sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317) and sun mnm double mutants (sunZ3-5839/sunZ3-1956 mnmZ3-5578/mnmZ3-3401). Red-DAPI, Green-α-tubulin. 72 IV-4 Discussion 1. What does SNM do in centromere pairing and sister chromatid cohesion? What is the role of homologous centromeres pairing in meiosis? One explanation here is that homologous centromeres pairing are prerequisite to homologous chromosomes pair and proper segregation during meiosis I. However, little is known about centromere pairing. Tsubouchi et al., proposed in 2005 that in S. cerevisiae homologous chromosomes pair dependent on the interaction switch from non-homologous centromeres to homologous centromeres. In S. cerevisiae, Zip1 and Spo11 are two proteins proved to be required for centromeric interactions and the transition from non-homologous to homologous centromeres pairing respectively (Tsubouchi et al., 2005). Nsl1p is another protein with a role in transient centromeres pairing in S.cerevisiae (Nekrasov et al., 2002). However in Drosophila, the mechanism of homologous centromeres pairing is not clear and no proteins have been found. In our research, we first found that homologous conjunction protein SNM, but not MNM, is involved in homologous centromeres pairing in the stage S3 in PI. In snm mutations homologous centromeres are unpaired and two CID spots can be seen in each territory, compared to wild-type, in which centromeres transiently pair from S3 to S4 stage and each territory contains one spot, indicating that centromere pairing is disrupted at the absence of SNM. As a homolog of SCC3 / REC11 and SA/STAG proteins, which are components of cohesin (Prieto et al., 2001; Kitajima et al., 2003), SNM was previously found to have no role in sister chromatid cohesion (Thomas et 73 al., 2005), however it still provides a rule for us to explore the relationship between cohesin complex and homologous centromeres pairing. In addition, Kemp et al. in 2004 provided evidence that in budding yeast centromeres pairing is required for an achiasmate segregation in meiosis (Kemp et al., 2004). Here we show that SNM is required for centromeres pairing, which suggests there may be a conserved pathway in achiasmate segregation. Mutations in sun and snm interact to cause sister chromatid separation in mid-prophase (S3), which is earlier than single sun mutants. Taken together with the different morphology of chromosomes in single sun mutants and sun and snm double mutants, we suggest SNM has another function in addition to homolog conjunction and plays a role in sister centromere cohesion and in meiotic centromere cohesion. 2. How cohesion and pairing proteins affect the integrity of chromosome territories? Chromosome territories correspond to confined chromosomes in discrete regions within nuclei. Their distinct properties have been extensively discussed and are important for gene regulation and genome stability in health and disease (Meaburn et al., 2007). However, little is known about the forces that determine how chromosomes are organized. In Drosophila three large bivalents are organized into three separated territories throughout PI. Vazquez et al., proposed in 2002 that the territories are defined by connections between the chromosomes and components of the nuclear envelope (Vazquez et al., 2002). 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