Architectural TFs

1. Architectural TFs

2. Overview DNA-binding TFs General principles Architectural factors

3. Recognition of response elementsActivators 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

4. Architectural TFs - brief history • Transcription activation - focus on more and more dimentions • 70-ties: 1-Dimentional understanding • ≥ RNAPII: TFs binding specific cis-elements required for selective transcription • TFs mediate regulatory response • 80-ties: 2-Dimentional understanding • 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

5. 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

6. 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 • abundant: 1 per. 10-15 nucleosomes

7. Classical HMG-proteins • Three classes of HMG DNA-binding proteins • HMG-box family • Eks: HMG 1 and HMG 2 • Bends DNA substantially • Facilitators of nucleoprotein complexes • HMG-AT-hook family • Eks.: HMGI(Y) • Antagonizing intrinsic distortions in the conformation of AT-rich DNA • HMG-nucleosome binding family • Eks.: HMG14 and 17 • Mediates moderate destabilization of chromatin higher-order structure • Not present in yeast or fly HMGB HMGA HMGN

8. HMGB-proteins

9. HMG1 and 2 • 3 structural domains • A and B with high homology (80-90 aa) • acidic C-terminal • Interaction with DNA (and histones?) • A and B ≈ DNA • C-term ≈ histone H1 or unknown function + + + + + + + + - - - - A B N C Histon H1? DNA

10. HMG-boxes in architectural proteins • One or two HMG-box domains 30 Asp/Glu acidic basic

11. 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 • LEF1 induces strong bending of DNA - about 130o • Induced bending brings nearly TFs in contact

12. LEF1 3D

13. LEF1 3D

14. 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

15. 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-a1, Ste11, Rox1

16. Characteristic DNA-binding • binds minor groove • induce bending of DNA • has high affinity for non-canonical DNA-structures such as : • cruciform DNA • 4-way junctions • cisplatin  kinked DNA +

17. 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 concave side interact with DNA

18. Similar structures of HMG domains

19. 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”)

20. 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) • Inserted side chain unstacks two basepairs • side chain as stacking-partner • Full (ETS1) • side chain penetrates into the helix • side chain (Trp) as new stacking-partner • Result: helix axis direction altered

21. 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

22. 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 • TCRa and Interferon b

23. 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

24. The charge neutralization model

25. 2. subgruppe: HMGA .. First described by Søren Laland, an almost forgotten discovery

26. HMGA - proteins with AT-hook • The mammalian HMGI/Y (HMGA) proteins participate in a wide variety of cellular processes • including regulation of gene trx and induction of neoplastic transformation and promotion of metastatic progression. • All members have multiple copies of a DNA-binding motif called the `AT hook' • 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.

27. 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.

28. 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.

29. 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: T53 and T78 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 Lys65 by CBP and at Lys71 by PCAF • …as well as methylated and poly-ADP ribosylated • Hypothesis: Modifications may alter DNA-binding specificity?

30. 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.

31. Maniatis: HMGI(Y) contributes to formation of enhanceosomes • virus-inducible enhancer in the IFN- gene (human interferon ) • 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”)

32. Recentverision

33. 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.

34. 3. subgruppe: HMGN

35. 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. • Binding of HMGN proteins to nucleosomes decreases the compactness of chromatin, and facilitates trx

36. 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 • The CHUD domain interacts with the amino terminus of histone H3. • May also affect H1 binding • Incorporation of HMGN proteins into chromatin is believed to reduce the compactness of the chromatin fiber.