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Architectural TFs MBV4230 Overview DNA-binding TFs General principles Architectural factors MBV4230 Recognition of response elements Activators vrs Architectural TFs Ordinary activators with sequence specific DNA binding Key recruitment sites for assembly of transcription complexes Architectural transcription factors playing a more structural role in the assembly of transcription complexes MBV4230 Architectural TFs - brief history Transcription activation - focus on more and more dimentions 70-ties: 1-Dimentional understanding 80-ties: 2-Dimentional understanding ≥ RNAPII: TFs binding specific cis-elements required for selective transcription TFs mediate regulatory response Promoters/enhancers: clusters of cis-elements complex regulation - Several buttons have to be pushed simultaneouly Ptashnes simplification - mixed order OK 90-ties: 3-Dimentional understanding Three-dimentional assembly of TFs required for correct biological response MBV4230 3D protein-promoter complexes - factors dedicated architecture some factors has a pure architectural function designated architectural transcription factors They lack a transactivation domain (TAD) Do not function out of their natural context (in contrast to ordinary acitvators) Their function is to confer a specific 3D structure on DNA MBV4230 Classical HMG-proteins non-histone chromatin proteins - original defining criteria high mobility in PAGE soluble in 2-5% TCA small < 30 kDa High content of charged amino acids Extensive post-translational modification abundant: 1 per. 10-15 nucleosomes MBV4230 Classical HMG-proteins Three classes of HMG DNA-binding proteins HMGB HMG-box family HMG-AT-hook family Eks: HMG 1 and HMG 2 Bends DNA substantially Facilitators of nucleoprotein complexes Eks.: HMGI(Y) Antagonizing intrinsic distortions in the conformation of AT-rich DNA HMG-nucleosome binding family HMGA HMGN Eks.: HMG14 and 17 Mediates moderate destabilization of chromatin higher-order structure Not present in yeast or fly HMGB-proteins MBV4230 HMG1 and 2 3 structural domains A and B with high homology (80-90 aa) acidic C-terminal Interaction with DNA (and histones?) Little or no sequence specificty A and B ≈ DNA C-term ≈ histone H1 or unknown function N ++++ A ++++ B DNA ---- C Histon H1? MBV4230 HMG-boxes in architectural proteins One or two HMG-box domains Non-sequence-specific Sequence-specific 30 Asp/Glu 20 Asp/Glu acidic basic MBV4230 First eukaryotic architectural TF: LEF1 (Grosschedl 1992) LEF1: a cell type-specific TF LEF1 contains an HMG-related domain LEF1: a sequence-specific TF that binds CCTTTGAAG found in enhancer of TCR (T cell receptor alpha) LEF1 induces strong bending of DNA - about 130o Induced bending brings nearly TFs in contact MBV4230 LEF1 3D MBV4230 LEF1 3D MBV4230 A whole family of architectural TFs with HMG-domains UBF has repeated HMG-homologous repeats 4-6 ex dimer ≈ 10 HMG-like domains activator of rRNA gener UBF-DNA complex scaffold for SL-1 recruitment Interaction with 180 bp that is packed into a distinct structure DNA-motif in a series of TFs: “HMG-box” designate the DNA-sequence-motif “HMG-domain” designate the protein motif MBV4230 Two subclasses of HMG-domain proteins Proteins with multiple HMG-domains low sequence-specificity Ubiquitous - found in all cell types eks.: HMG1, HMG2, ABF-2, UBF Proteins with single HMG-domain (moderate) sequence-specificity Cell type-specific eks.: LEF-1, SRY, TCF-1, Sox, Mat-1, Ste11, Rox1 MBV4230 HMG1 - Characteristic DNA-binding HMG A domain binds minor groove fails to bend DNA effecively 60o has high affinity for non-canonical DNAstructures such as : cruciform DNA 4-way junctions cisplatin kinked DNA Never found alone HMG B domain binds minor groove bends DNA by over 90o less selectively to distorted DNA Found alone like in HMG-D + MBV4230 NMR-structures Examples HMG1 B-domain LEF-1 SRY Yeast Nhp6p Drosophila HMG-D Common: 3 helix L-form heliks II and III form an angle of about 80o Conserved aromatic aa in kink Basic / hydrophobic concave side interact with minor groove in DNA MBV4230 Similar structures of HMG domains intercalation partial intercalation MBV4230 Minor groove binding, intercalation and bending Objective: shorten the distance between cis-elements facilitating interaction between bound factors DNA <500bp relatively stiff induced bending required Mechanism for induced bending of DNA Protein scaffold HMG B-domain: L-shaped protein TBP: sadle Minor groove binding DNA-binding face = hydrophobic surface that conforms to a wide, shallow minor groove 4 residues inserted deep into the minor groove Full or partial intercalation (“kile”) MBV4230 Two points of intercalation, X and Y Basic tail Binds Major groove X only X and Y Y only X = major kink and intercalation site , Y=second kink due to partial intercalation MBV4230 Intercalation in protein-induced DNA-bending Partial intercalation in the DNA helix of a protein side chain introduces a kink in the DNA enhancing the bend Large hydrophobic residues (N-term helix I) partially intercalates between two base pairs The A-box HMG domain has only an Ala in the X position not large enough to intercalate, Intercalation linked to bending also seen in other factors Partial (TBP) Full (ETS1) Inserted side chain unstacks two basepairs side chain as stacking-partner side chain penetrates into the helix side chain (Trp) as new stacking-partner Result: helix axis direction altered MBV4230 Cooperation with TFs A major role of non-seq.spec. architectural factors is to facilitate formation of complex nucleoprotein assemblies Need interaction with sequence specific TF to be directed to precise locations An introduced bend could facilitate binding of one factor, and this could subsequently assist a second factor The seq.spec. architectural factors is known to participate in the formation of complex nucleoprotein assemblies like enhanceosomes TCR and Interferon b MBV4230 Are all TFs architectural? A large number of publications “TFx bends DNA” positive reports “TFx bends DNA” negative reports “TFx does not bend DNA” All TFs that bind on one side of DNA will induce bending due to one-sided neutralization of charge Degree of bending will depend on ionic condition Uncertain if biologically relevant The term “Architectural TFs“ should be reserved for factors with a particularly developed bending mechanism MBV4230 The charge neutralization model - - - - - - - - - - -- -- - - + - - - ++ --+ + + -+ + + + --+ + 2 --3 + -1 - - --Sp1 Asymmetrical charge neutralization Bending + - -+-+- - - -+ + + - - - - -+-+ + + - - -- + - -- -+ + +- - - - - ++- -+ + + ++ - - - + -+ + --- + + 2 --+ 3 1 Sp1 Bending effect of charge neutralization reduced in the pr esence of multvalent cations 2. subgruppe: HMGA .. First described by Søren Laland, an almost forgotten discovery MBV4230 HMGA - proteins with AT-hook The mammalian HMGI/Y (HMGA) proteins participate in a wide variety of cellular processes All members have multiple copies of a DNA-binding motif called the `AT hook' including regulation of gene trx and induction of neoplastic transformation and promotion of metastatic progression (early in embyonic life – less in most adult cells). that binds to the narrow minor groove of stretches of AT-rich sequence. The proteins have little secondary structure in solution but assume distinct conformations when bound to DNA or other proteins Their flexibility allows the HMGI/Y proteins to induce both structural changes in chromatin substrates and the formation of stereospecific complexes called `enhanceosomes'. Reciprocal conformational changes occur in both the HMGI/Y proteins themselves and in their interacting substrates. MBV4230 Members 4 known members Alternatively splicing gives rise to two isoform proteins, HMGA1a (HMGI) and HMGA1b (HMGY). These two are identical in sequence except for a deletion of 11 residues between the the first and second AT hook in the latter. Alternative splicing also produces HMGA1c. The related HMGA2 (HMGI-C) protein is coded for by a separate gene. Conserved Homologues of the mammalian HMGA proteins have been found in yeast, insects, plants and birds, as well as in all mammalian species examined. MBV4230 HMGA - AT-hook binding to DNA Each HMGA protein possesses 3 similar, but independent, AT hooks which have an invariant peptide core motif of Arg-Gly-Arg-Pro (”palindromic” consensus PRGRP) flanked on either side by other conserved positively charged residues. The HMGA proteins bind, via the AT hooks, to the minor groove of stretches of AT-rich DNA but recognize substrate structure, rather than nucleotide sequence. MBV4230 HMGA proteins heavily modified The HMGA proteins are among the most highly phosphorylated proteins in the mammalian nucleus. Cell cycle-dependent phosphorylation pga cdc2 activity in the G2/M phase of the cycle. Sites: T52 and T77 situated at the N-terminal ends of the 2. and 3. AT-hook. Phosphorylation significantly reduces (>20-fold) DNA binding. HMGA proteins are the downstream targets of a number of signal transduction pathways that lead to phosphorylation. HMGA proteins are also acetylated at K64 and at K70 …as well as methylated and poly-ADP ribosylated ? Hypothesis: Modifications may alter DNA-binding specificity? MBV4230 Architectural effects Architectural effects Binding of full-length HMGA proteins can bend, straighten, unwind and induce loop formation in linear DNA molecules in vitro. Multiple contact points with DNA may alter conformation of DNA A single AT-hook preferentially binds to stretches of 4-6 bp of AT-rich sequence, and partially neutralizes the negatively charged backbone phosphates on only one face of the DNA helix. The number and spacing of AT-rich binding sites in DNA influences the conformation of bound DNA and the biological effects elicited. HMGA may also induce conformational change in proteins HMGA forms protein-protein interactions with other transcription factors, which alters the 3D structure of the factors resulting in enhanced DNA binding and transcriptional activation. MBV4230 Maniatis: HMGI(Y) contributes to formation of enhanceosomes virus-inducible enhancer in the IFN-b gene (human interferon b) cis-elements for NF-kB, IRF-1, ATF-2-c-Jun Synthetic (multiple cis-elements) enhancer ≠ natural Too high basal transcription Less induction Responds to several stimuli, while natural enhancer only responds to virus Biological function depends of HMGI(Y) as architectural component HMG I(Y) First described by Lund and Laland binds AT-rich DNA in minor groove (“AT-hook”) MBV4230 Recent verision IFN-b MBV4230 Other functions of HMGA proteins HMGA and cancer HMGI/Y proteins are also involved in a diverse range of other cellular processes including pathologic processes such as neoplastic transformation and metastatic progression. Chromosomal translocations in a long 3.intron Intron 3 of the HMGA2 genes is extremely long (>25 kb in human and >60 kb in mouse) and separates the three exons that contain the AT hook motifs from the remainds of the 3´untranslated tail region of the gene. Translocation within the exceptionally long third intron are commonly observed in benign mesenchymal tumors. 3. subgruppe: HMGN MBV4230 HMGN proteins Three functional domains of the HMGN proteins: a bipartite nuclear localization signal (NLS), a nucleosomal binding domain (NBD) and a chromatin-unfolding domain (CHUD). The CHUD domain has a net negative charge. Extensive post-translational modifications: Ac by p300, P of Ser Binding of HMGN proteins to nucleosomes decreases the compactness of chromatin, and facilitates trx MBV4230 HMGN: architectural elements reducing compactness of chromatin Model of the binding of HMGN proteins to chromatin HMGNs interact with both the DNA and the histone component of the nucleosome CHUD domain interacts with H3 histone tail and H2B Either HMGN1 or HMGN2 homodimers May also affect H1 binding Incorporation of HMGN proteins into chromatin is believed to reduce the compactness of the chromatin fiber.