general: Activators - protein-DNA interaction

1. general:Activators - protein-DNA interaction

2. The sequence specific activators: transcription factors • Modular design with a minimum of two functional domains • 1. DBD - DNA-binding domain • 2. TAD - transactivation domain • DBD: several structural motifs  classification into TF-families • TAD - a few different types • Three classical categories • Acidic domains (Gal4p, steroid receptor) • Glutamine-rich domains (Sp1) • Proline- rich domains (CTF/NF1) • Mutational analyses - bulky hydrophobic more important than acidic • Unstructured in free state - 3D in contact with target? • Most TFs more complex • Regulatory domains, ligand binding domains etc DBD N TAD C

3. TF classification based on structure of DBD • Two levels of recognition • 1. Shape recognition • Anhelix fits into the major groove in B-DNA. This is used in most interactions • 2. Chemical recognition • Negatively charged sugar-phosphate chain involved in electrostatic interactions • Hydrogen-bonding is crucial for sequence recognition bHelix-Loop-Helix (Max) Zinc finger Leucine zipper (Gcn4p) p53 DBD NFkB STAT dimer

4. Alternative classification of TFs on the basis of their regulatory role • Classification questions • Is the factor constitutive active or requires a signal for activation? • Does the factor, once synthesized, automatically enter the nucleus to act in transcription? • If the factor requires a signal to become active in transcriptional regulation, what is the nature of that signal? • Classification system • I. Constitutive active nuclear factors • II. Regulatory transcription factors • Developmental TFs • Signal dependent • Steroid receptors • Internal signals • Cell surface receptor controlled • Nuclear • Cytoplasmic

5. Classification - regulatory function Brivanlou and Darnell (2002) Science295, 813 -

6. Sequence specific DNA-binding- essential for activators • TFs create nucleation sites in promoters for activation complexes • Sequence specific DNA-binding crucial role

7. Principles of sequence specific DNA-binding

8. How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogen- bonds Hydrophobic interaction

9. Complementary forms The dimension of anhelix fits the dimensions of the major groove in B-DNA Sidechains point outwards and are ideally positioned to engage in hydrogen bonds

10. Direct reading of DNA-sequenceRecognition of form • The dimension of an a-helix fits the dimensions of the major groove in B-DNA • Most common type of interaction • Usually multiple domains participate in recognition • dimers of same motif • tandem repeated motif • Interaction of two different motifs • recognition: detailed fit of complementary surfaces • Hydration /vann participates • seq specvariation of DNA-structure

11. Example • Steroid receptor

12. Recognition by complementary forms 434 fag repressor

13. DNAs form:B-DNA most common B B-form Major groove Minor groove wide geometry fits a-helix Each basepair with unique H-bonding- pattern Deep and narrow geometry Each basepair binary H-bonding- pattern

14. DNAs form:A-form more used in RNA-binding A A-form Major groove Minor groove Deep and narrow geometry Wide and shallow

15. How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogen- bonds Hydrophobic interaction

16. Next level: chemical recognition - reading of sequence information • Negatively charged sugar-phosphate chain = basis for electrostatic interaction • Equal everywhere - no sequence-recognition • Still a main contributer to the strength of binding

17. Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ - Na+ Na+ - - - - Na+ Na+ - Na+ - Na+ Na+ Na+ Na+ Na+ Counter ions liberated Entropy-driven binding Na+ Electrostatic interactionEntropy-driven binding Na+ Na+ Na+ Na+ Na+ - Na+ Na+ - Na+ - Na+ - Na+ - Na+ - Na+ - Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Negative phosphate chain partially neutralized by a cloud of counter ions

18. How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogen- bonds Hydrophobic interaction

19. D A A Recognition by Hydrogen bonding • Hydrogen-bonding is a key element in sequence specific recognition • 10-20 x in contact surface • Base pairing not exhausted in duplex DNA, free positions point outwards in the major groove

20. Major groove Minor groove AT-base pair Major groove GC-base pair Minor groove Unexploited H-bonding possibilities in the grooves Point outwards in major groove Point outwardsin minor groove

21. Unique ”bar code” in major groove Binary ”bar code” in minor groove AT-basepair AT-basepair GC-basepair GC-basepair AT-pair [AD-A] ≠ TA-pair [A-DA] GC-pair [AA-D] ≠ CG-pair [D-AA] D D AT-pair [A-A] = TA-pair [A-A] GC-pair [ADA] = CG-pair [ADA] A A A A D A A A A A ”bar code” in the grooves Unique recognition of a base pair requires TWO hydrogen bonds In the major groove

22. Docked prot side chains exploit the H-bonding possibilities for interaction • Hydrogen-bonding is essential for sequence specific recognition • 10-20 x in contact interphase • Most contacts in major groove • Purines most important • A Zif example

23. Interaction: Protein side chain - DNA bp • Close up • Amino acid sidechains points outwards from the a-helix and are optimally positioned for base-interaction • Still no ”genetic code” in the form of sidechain-base rules • docking of the entire protein

24. Interaction: Protein side chain - DNA bp • Close up • Amino acid sidechains points outwards from the a-helix and are optimally positioned for base-interaction

25. A network of H-bonds • Example: • c-Myb - DNA Protein DNA

26. How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogen- bonds Hydrophobic interaction

27. Hydrophobic contact points Ile

28. Homeodomains

29. The Homeodomain-family: common DBD-structure • Homeotic genes - biology • Regulation of Drosophila development • Striking phenotypes of mutants - bodyparts move • Control genetic developmental program • Homeobox / homeodomain • Conservered DNA-sequence “homeobox” in a large number of genes • Encode a 60 aa “homeodomain” • A stably folded structure that binds DNA • Similarity with prokaryotic helix-turn-helix • 3D-structure determined for several HDs • Drosophila Antennapedia HD (NMR) • Drosophila Engrailed HD-DNA kompleks (crystal) • Yeast MAT2

30. Homeodomain-family: common DBD-structure • Major groove contact via a 3 -helix structure • helix 3 enters major groove (“recognition helix”) • helix 1+2 antiparallel across helix 3 • 16 -helical aa conserved • 9 in hydrophobic core • some in DNA-contact interphase (common docking mechanism?) • Positions important for sequence recognition • N51 invariant: H-binding Adenine, role in positioning • I47 (en, Antp) hydrophobic base contact • Q50 (en), S50 (2) H-bond to Adenine, determining specificity • R53 (en), R54 (2): DNA-contact

31. Engrailed

32. Antennapedia

33. Homeodomain-family: common DBD-structure • Minor groove contacted via N-terminal flexible arm • R3 and R5 in engrailed and R7 in MAT2 contact AT in minor groove • R5 conserved in 97% of HDs • Deletions and mutants impair DNA-binding • ftz HD (∆6aa N-term) 130-fold weaker DNA-binding • MAT2 (R7A) impaired repressor • POU (∆4,5) DNA-binding lost • Loop between helix 1 and 2 determines Ubxversus Antp function • Close to DNA • exposed for protein protein interaction

34. HD-paradox: what determines sequence specificity? • Drosophila Ultrabithorax (Ubx), Antennapedia (Antp), Deformed (Dfd) and Sex combs reduced (Scr): closely similar HD, biological rolle very different • Minor differences in DNA-binding in vitro • TAAT-motif bound by most HD-factors • contrast between promiscuity in vitro and specific effects in vivo • Swaps reveal that surprisingly much of the specificity is determined by the N-terminal arm which contacts the minor groove • Swaps: Antp with Scr-type N-term arm shows Scr-type specificity in vivo • Swaps: Dfd with Ubx-type N-term arm shows Ubx-type specificity in vivo • N-terminal arm more divergent than the rest of HD • R5 and R7 (contacting DNA) are present in both Ubx, Antp, Dfd, and Scr • Other tail aa diverge much more

35. Solutions of the paradox • Conformational effects mediated by N-term arm • Even if the -helical HDs are very similar, a much larger diversity is found in the N-terminal arms that contact the minor groove • Protein-protein interaction with other TFs through the N-terminal arm - enhanced affinity/specificity - the basis of combinatorial control • MAT2 interaction with MCM1 - cooperative interactions • Ultrabithorax- Extradenticle in Drosophila • Hox-Pbx1 in mammals

36. Combinatorial TFs give enhanced specificity • TFs encoded by the the homeotic (Hox) genes govern the choice between alternative developmental pathways along the anterior–posterior axis. • Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbx1 in mammals).

37. a b N-tail in protein-protein interaction- adopt different conformations HD HD Conformation determined by prot prot interaction Mat-a2/Mcm-1

38. The partner may also be a linker histone • Repression of the mouse MyoD gene by the linker histone H1b and the homeodomain protein Msx1. • The first evidence that a linker histone subtype operates in a gene-specific fashion to regulate tissue differentiation

39. It works impressively well • Hox genes

40. POU family

41. POU-family: common DBD-structure • The POU-name : • Pit-1 pituitary specific TF • Oct-1 and Oct-2 lymphoide TFs • Unc86 TF that regulates neuronal development in C.elegans • A bipartite160 aa homeodomain-related DBD • a POU-type HD subdomain (C-terminally located) • et POU-specific subdomain (N-terminally located) • Coupled by a variabel linker (15-30 aa) • POU is a structurally bipartite motif that arose by the fusion of genes encoding two different types of DNA-binding domain.

42. POU: Two independent subdomains • POUHD subdomain • 60 aa closely similar to the classical HD • Only weakly DNA-binding by itself (<HD) • contacts 3´-half site (Oct-1: ATGCAAAT) • docking similar to engrailed. Antp etc • Main contribution to non-specific backbone contacts • POUspec subdomain • 75 aa POU-specific domain • enhances DNA-affinity 1000x • contacts 5´-half site (Oct-1: ATGCAAAT) • contacts opposite side of DNA relative to HD • structure similar to prokaryotic - and 434-repressors • The two-part DNA-binding domain partially encircles the DNA.

43. Flexible DNA-recognition • POU-domains have intrinsic conformational flexibility • and this feature appears to confer functional diversity in DNA-recognition • The subdomains are able to assume a variety of conformations, dependent on the DNA element.

44. A POU prototype: Oct-1 • Ubiquitously expressed Oct-1 (≠ cell type specific Oct-2) • Oct-1 performs many divergent roles in cellular trx regulation • partly owing to its flexibility in DNA binding and ability to associate with multiple and varied co-regulators • Oct-1 activates transcription of genes that are involved in basic cellular processes • Oct-1 activates small nuclear RNA (snRNA) and • S-phase histone H2B gene transcription • cell-specific promoters, particularly in the immune and nervous systems • immunoglobulin (Ig) heavy- and lightchains • Activate target genes by bidning to the “octamer” cis-element ATGCAAAT • Hence the name “Octamer-motif binding protein”

45. Flexibility • On the natural high-affinity Oct-1 octamer (ATGCAAAT) binding site, the two Oct-1 POU-subdomains lie on opposite sides of the DNA • The unstructured linker permits flexible subdomain positioning and hence diversity in Oct-1 sequence recognition.

46. Oct-1: associates with multiple and varied co-regulators • Oct-1 associates with a B-cell specific co-regulator OCA-B (OBF-1). OCA-B stabilizes Oct-1 on DNA and provides a transcriptional activation domain. • B-cell specific activation of immunoglobulin genes - for long a paradox • Depended on octamer cis-elements • B-cell express both ubiquitous Oct-1 and the cell type specific Oct-2  Hypothesis: Oct-2 aktivates IgGs (Wrong!) • oct-2 deficient mouse  normal development of early B-cells and cell lines without Oct-2 produce abundant amounts of Ig • A B-cell specific coactivator mediates Oct-1 transactivation • VP16 - a virus strategy to exploit a host TF

47. Many viruses use Oct-1 to promote infection • When herpes simplex virus (HSV) infects human cells, a virion protein called VP16, forms a trx regulatory complex with Oct-1 and the cell-proliferation factor HCF-1 • VP16 = a strong transactivator, not itself DNA-binding, but becomes associated with DNA through Oct-1 • The specificity of Oct-1 is altered from Octamer-seq to the virus cis-element TAATGARAT • The VP16-induced complex has served as a model for combinatorial mechanisms of trx regulation

48. Pax family

49. Pax family Paired domain

50. Paired domain DBD RED Major groove interaction: Minor groove interaction: Flex? Major groove interaction: PAI