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This slide series has been used for teaching to students of the Montpellier University in 2004.

This slide series has been used for teaching to students of the Montpellier University in 2004. It explains basic features of the folding of DNA in nucleosomes and chromatin fibers, and discusses the regulatory roles of post-translational histone modificatons

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This slide series has been used for teaching to students of the Montpellier University in 2004.

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  1. This slide series has been used for teaching to students of the Montpellier University in 2004. It explains basic features of the folding of DNA in nucleosomes and chromatin fibers, and discusses the regulatory roles of post-translational histone modificatons The material displayed is inspired from several sources. The literature used is cited within the slides. Moreover, several slides (indicated by the JHW logo) have been copied or modified from Jacob Waterborg, University of Missouri, Kansas City To see his chromatin material in full, see the website: http://sbs.umkc.edu/waterborg/

  2. Compaction by chromosome scaffold / nuclear matrix 10,000 nm DNA compactioninahuman nucleus 11 nm 30nm 1bp (0.3nm) Compaction of DNA by histones JHW

  3. + 2M NaCl histones + 2M NaCl mitotic chromosome histones 1mm chromatid Compaction by chromosome scaffold / nuclear matrix Nuclear - chromosome compaction radial loop chrosomosome model Mitosis DNA loops 10 mm chromatid JHW

  4. H1 HISTONESare highly conserved,small, basic proteins Linker histone H2A H2B helix • Histone acetylation • is a reversible modification • of lysines in the N-termini • of the core histones. • Result: • reduced binding to DNA • destabilization of chromatin Core histones variable H3 H4 conserved N JHW

  5. Core Histones The histone-fold • The basic structure of ALL • core histones is the same: • 1 long hydrophobic alpha-helix, • bordered by • 2 short hydrophobic alpha helices • that form pairs • H2A - H2B and H3 - H4 • which interact. References:Moudrianakis et al. PNAS 88, 10138 (1991); PNAS 90, 10489 (1993); PNAS 92, 11170 (1995) JHW

  6. Histone octamer assembly Histone octamer H3-H4 tetramer H2A-H2B dimer JHW

  7. The Nucleosome as the fundamental chromatin unit Figure, courtesy from Timothy Richmond, ETH Zürich, Switzerland JHW

  8. H4 H3 H2B H2A Nucleosome features • 146-149 bp DNA in a 1.65 turns of a flat, left-handed superhelix • one pseudo twofold axis centered at the “dyad” (reference: 0 helical turns) • one base-pair precisely at the dyad • sharp bends at + 1.5 and + 4-5 turns • Histone-fod domains organize 121 bp of DNA. The DNA is bound at 10 bp intervals through many contacts, including penetration of arginines at all 14 minor grooves facing the protein core • The grooves from neighboring DNA turns line up; forming channels • H3 and H2B N-termini exit one of these channels every 20bp. • The H4 tail establishes contacts with the next core particle. JHW

  9. Histone octamer organizes 145 bp of DNA < 11 nm > • Each core histone dimer • has 6 DNA binding surfaces • that organize 3 DNA turns; • The histone octamer • organizes 145 bp of DNA • in 1 3/4 helical turn of DNA: • 48 nm of DNA packaged in a disc of 6 x 11nm < 6 nm > JHW

  10. Where are the N-termini of the core histones ? Luger, Mader, Richmond, Sargent & Richmond Nature389, 251-260 (1997) Question 1: What is the function of histone N-termini ? Question 2: Are all N-termini functionally equivalent ? JHW

  11. Linker histone H1 H1/H5 globular protein domain On the OUTSIDE of the DNA gyres ? Asymmetric between the DNA gyres ? Zhou et al. Nature 395, 402 (1998) Wolffe et al. Science274, 614 (1996) Linker histone H1 organizes exiting DNA (up to 168 - 200 bp). H1 stabilizes interaction between nucleosomes in compacted chromatin. JHW

  12. 1 mM 5 mM Zhou, Gerchman, Ramakrishnan, Travers, Muyldermans Nature 395, 402 (1998) An, Leuba, van Holde, Zlatanova PNAS 95, 3396 (1998) Chromatosome C Core histone octamer + 1 Linker Histone + 2 full turns of DNA (168 bp) N H1 H3 H3 Linker Histone and histone termini control linker DNA entry/exit of chromatosome in chromatin fiber. JHW

  13. Chromatinfibers 11 nm (beads) 30 nm chromatin fiber highly acetylated core histones (especially H3 and H4) + charged N termini (bind DNA on neigboring nucleosomes) • HIGH level of histone H1 • Reduced level of histone H1 • Gene transcription possible • NO gene transcription JHW

  14. Histone modifications 27 Turner (2002). Cell 111, 285-91

  15. 5 16 20 8 12 Ac or Me Ac Ac Ac Ac Ac-S-G-R-G-K-G-G-K-G-L-G-K-G-G-A-K-R-H-R-K-V-L-R-D- + + + + + + + + + + 27 4 18 9 23 14 Ac or Me Me Ac Ac Ac Ac A-R-T-K-Q-T-A-R-K-S-T-G-G-K-A-P-R-K-Q-L-A-T-K-A-A-R-K-S-A-P- + + + + + + + + + - N - O O - - C C C g e - N+ P P - a d b C C C - O O O - - - - - - - N P - - C C N C C e - - - C C C C - O - - O - Acetylation of conserved lysines The N-termini of histones H4 and H3, and their acetylation patterns, are absolutely conserved. H4 N-terminus H3 N-terminus DNA backbone binding Lysine Acetyl-CoA HAT (Histone Acetyl-Transferase) Histone Deacetylase reversible reactions CoA e-N-Acetyl-Lysine no DNA binding JHW

  16. gel type: SDS _ + + + + electrode: + H3.1 135 T T T T T T T T 4 T 3 2 1 0 4 + + 3 + H3.2 2 1 0 + + 135 + H3 T T T T T T T 4 3 H3 2 1 135 0 + + + + + 135 aa H4 4 4 + H4 3 3 H3 T 2 2 T T H4 1 1 H4 0 0 102 102 102 aa _ _ + electrode: Gel Electrophoresis of Histones size + charge + hydrophobicity size + charge Separation by: size AU AUT H3.1 H3.2 H3 H4 H4 Gel scans: alfalfa histones (Waterborg, 90’s) JHW

  17. Example: alga Chlamydomonas reinhardtii Level of histone acetylation: 32 % of H316 % of H4 Average level of acetylation: 2.4 AcLys/H31.6 AcLys/H4 Average AcLys turnover T½: 1.9 0.4 min (H3) 3.5 1.1 min (H4) AcLys turnover T½ (min): 2.8 1.8 1.8 1.6 1.5 Transcriptionally active fraction of genome  20 % 1 2 3 4 5 1 2 3 4 5 #AcLys/histone Fraction of acetylated histone 100 92 104 106 82 39 58 81 100 76 % subject to AcLys turnover:100 12 % of H355 4 % of H4 (67% of multi-acetylated H4) AlmostAll Acetylation is Dynamic WaterborgJ. Biol. Chem. 273, 27602 (1998) JHW

  18. (Ac-Lys) antibody nucleosome ppt DNA loop domain  H A   = -globin genes: domain boundary domain boundary 0 10 30 kb 20 Control: inactive gene DNase I hyper-sensitive site chicken ovalbumin General DNase I sensitivity DNA remaining 0 1 2 DNase I DNase I (U/ml) Acetylation of Chromatin Domains Example: the chicken -globin gene domain • High levels of chromatin acetylation, across complete chromatin domains (DNA loops), induces chromatin changes detected as “general DNase I sensitivity” • Within these chromatin domains, at functional genes or transcription factors, • the chromatin structure is interrupted by small “DNase I hypersensitive sites” Hebbes, Clayton, Thorne, Crane-RobinsonEMBO J. 13, 1823 (1994) JHW

  19. + TR/RXR TH Transcriptional ACTIVATION hormone TH Without Thyroid Hormone TACCCG ACGGTC TACCCG co-activator ADA2 ADA3 p300 CPB HAT co-repressor N-CoR Sin3 GCN5 HAT Histone Deacetylase HAT P/CAF Histone Acetyl Transferases RPD3 HAT HAT TAF 250 II TAF 250 pol.II II TATA TBP TATA TBP Acetylation at Promoters Transcriptional REPRESSION Thyroid Hormone Receptor example of DNA-binding Transcription Factor Adapted from WolffeNature 387, 16 (1997) JHW

  20. transcriptional repressor co-repressor 5 Histone Deacetylase me 3’..pGpCp..5’   RPD3 5 me 5’..pCpGp..3’ MeCP2 Sin3 + + Deacetylation of ChromatinTranscriptional Silencing — X-chromosome Inactivation • 5meC CpG DNA modification is observed in repressed genes and inactivated X chromosomes • 5meC CpG-methylation is maintained after DNA replication by Maintenance Methylase action on hemi-methylated DNA • 5meC binds transcriptional repressor MeCP2 (MethylC-binding Protein-2) • MeCP2 binds Sin3 with RPD3 histone deacetylase CpG methylation Hypo-acetylated repressed chromatin fiber Nan et al.Mol.Cell.Biol. 16, 414 (1996); Cell 88, 1 (1997); Jones et al. Nat.Genet. 19, 187 (1998) JHW

  21. Histone Methylation C C C C C C Histones can be methylated at lysines or arginines. Example: H3 K9 methylation N Lysine C C C g e N+ a d b C C C O S-adenosylmethyionine HMT (Histone Methyl-Transferase) Histone demethylase? N e-N-monomethyl-Lysine N+ C C DNA backbone binding may not be strongly affected, but specific proteins may recognize these modifications C e C C C O S-adenosylmethyionine HMT (Histone Methyl-Transferase) N e-N-dimethyl-Lysine N+ C C C e e S-adenosylmethyionine C C C O HMT (Histone Methyl-Transferase) N N+ C C C e-N-trimethyl-Lysine e e C C C O

  22. Histone methylation : Histone methyltransferases Enhancer of Zeste & K27 Silencing of euchromatic genes Modified from: Lachner and Jenuwein (2002). Current Opin. Cell Biol. 14, 286-98

  23. Tri-; di-; mono-HMTases? S. pombe to man (Tri-met K27) and tri- met K9 Enhancer of zeste N. crassa Multiple roles of H3 K9 methylation Modified from: Lachner and Jenuwein (2002). Current Opin. Cell Biol. 14, 286-98 In contrast to histone acetylation, histone methylation is STABLE. This makes of this mark a candidate for inheritance of chromatin states.

  24. Heterochromatin First discovered in Drosophila in genetic studies of chromosome rearrangements Heterochromatin induces gene silencing Singh, P. B. (1994). Molecular mechanisms of cellular determination: their relation to chromatin structure and parental imprinting. J Cell Sci 107, 2653-2668.

  25. Genes involved in heterochromatin Singh, P. B. (1994). Molecular mechanisms of cellular determination: their relation to chromatin structure and parental imprinting. J Cell Sci 107, 2653-2668.

  26. Heterochromatin formation involves histone H3 lysine 9 methylation by Su(var)3-9 and recruitment of HP-1 Ac Ac Open chromatin H3 K9 K14 HDAC H3 K9 K14 Methylation of K9 (Clr4; Suvar39) Me H3 K9 K14 Recruitment of Swi6 or HP-1 Swi6 Me H3 K9 K14 Recruitment of more Clr4 (Suvar39) molecules by Swi6 (HP-1) Condensed chromatin

  27. Epigenetic regulation of centromere function and RNAi Outer repeats Central domain Outer repeats S. Pombe Centromere dsRNAs Nucleosomes Nucleosomes K9 Me K9 Me Swi6 Swi6 This structure requires proteins that are responsible for the phenomenon of RNA interference -> RNA interference is gene silencing mediated by short RNA molecules produced from double stranded RNA (dsRNA). -> Short RNAs are produced by cleavage of dsRNA by the nuclease called Dicer. Dicer produces short dsRNA called siRNA duplexes. Then, a complex of proteins called RISC unwinds the duplex and produces siRNA. siRNA hybridize to the target mRNA which is degraded. …But RNAi also acts on chromatin -> Centromeres produce dsRNA, and Dicer as well as the RISC are required for centromere silencing. Transposons induce gene silencing in a similar manner. Mechanism for coupling of RNAi and chromatin regulation? Model: K9 methylation, Swi 6 recruitment! Nascent RNA Ago (RISC), Clr4 Dcr complex si RNA See also: Grewal and Moazed (2003). Science 301, 798-802

  28. Opposing functions of H3 K9 versus K4 methylation Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98

  29. A functional link between histone H3 methylation and DNA cytosine methylation in Arabidopsis thaliana HP1? Modified from: Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98

  30. Multiple biological phenomena involving histone methylation E(z) H3-K27 methylation and and H3-K27 methylation Modified from: Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98

  31. Summary and perspectives Chromatin is packaged in a hierarchy of structures. Each of these levels of packaging has regulatory roles in the genome The level of packaging we know best, also thanks to formidable tools & technology development in the recent years, is the nucleosome. In particular, post-translational histone modifications play key roles in regulation of genome function, and the combinatorial power of these modifications is only beginning to be unraveled. Future challenges will be to decrypt the detailed meaning of these modifications, and to understand the roles of the higher order levels of chromatin and chromosome folding

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