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DNA compaction in a human nucleus

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1 DNA compaction in a human nucleus
Chromatin 9-98 10,000 nm DNA compaction in a human nucleus 11 nm 30nm 1bp (0.3nm) Compaction of DNA by histones Compaction by chromosome scaffold / nuclear matrix JHW 3-99

2 HISTONES are highly conserved, small, basic proteins
Chromatin 9-98 H1 HISTONES are 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 3-99

3 Core Histones The histone-fold The basic structure of ALL
Chromatin 9-98 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, (1991); PNAS 90, (1993); PNAS 92, (1995) JHW 3-99

4 Histone octamer assembly
Chromatin 9-98 Histone octamer assembly Histone octamer H3-H4 tetramer H2A-H2B dimer JHW 3-99

5 The Nucleosome as the fundamental chromatin unit
9-98 The Nucleosome as the fundamental chromatin unit JHW 3-99

6 Chromatin 9-98 Nucleosome features 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 and turns Histone-fold 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. H2B H4 H3 H2A JHW 3-99

7 Chromatosome H1 H3 H3 Linker Histone and histone termini
Chromatin 9-98 Chromatosome C Core histone octamer + 1 Linker Histone + 2 full turns of DNA (168 bp) 5 mM 1 mM N H1 H3 H3 Linker Histone and histone termini control linker DNA entry/exit of chromatosome in chromatin fiber. Zhou, Gerchman, Ramakrishnan, Travers, Muyldermans Nature 395, 402 (1998) An, Leuba, van Holde, Zlatanova PNAS 95, 3396 (1998) JHW 3-99

8 Chromatin fibers 11 nm (beads) 30 nm chromatin fiber
9-98 Chromatin fibers 11 nm (beads) 30 nm chromatin fiber + charged N termini (bind DNA on neigboring nucleosomes) highly acetylated core histones (especially H3 and H4) HIGH level of histone H1 Reduced level of histone H1 NO gene transcription Gene transcription possible JHW 3-99

9 Alternative chromatin fiber models
9-98 Alternative chromatin fiber models Zigzag fiber

10

11 Histone modifications
Chromatin Histone modifications 9-98 27 Cell (2002) 111,

12 Acetylation of conserved lysines
Chromatin 9-98 Acetylation of conserved lysines The N-termini of histones H4 and H3, and their acetylation patterns, are absolutely conserved. 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- + Ac or Me Ac 5 8 12 16 20 H4 N-terminus 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- Me Ac or Me Ac + 4 9 14 18 23 27 H3 N-terminus O N C N+ a b g d e P - - DNA backbone binding - - - Lysine - - Acetyl-CoA - - HAT (Histone Acetyl-Transferase) - reversible reactions Histone Deacetylase - - - CoA - O N C e P - e-N-Acetyl-Lysine no DNA binding - - - - - - - - JHW 3-99

13 Chromatin 9-98 Acetylation of Chromatin Domains: analysis by the technique of Chromatin Immunoprecipitation (ChIP) Example: the chicken -globin gene domain domain boundary  H A  10 20 30 kb DNase I hyper-sensitive site  = -globin genes: (Ac-Lys) antibody nucleosome ppt DNA loop domain General DNase I sensitivity Control: inactive gene chicken ovalbumin (U/ml) DNA remaining 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-Robinson EMBO J. 13, 1823 (1994) JHW 3-99

14 Acetylation at Promoters leads to transcriptional activation
Chromatin 9-98 Acetylation at Promoters leads to transcriptional activation TH Thyroid Hormone Receptor example of DNA-binding Transcription Factor + Transcriptional ACTIVATION hormone TATA HAT P/CAF p300 CPB TACCCG TH HAT GCN5 ACGGTC ADA2 ADA3 co-activator Histone Acetyl Transferases HAT TAF 250 II pol.II Hyper-acetylated decondensed Chromatin fiber TBP JHW 3-99

15 Deacetylation at Promoters leads to Transcriptional repression
Chromatin 9-98 Deacetylation at Promoters leads to Transcriptional repression Transcriptional REPRESSION Without Thyroid Hormone TATA TACCCG N-CoR Sin3 RPD3 co-repressor Histone Deacetylase HAT X TAF 250 II Hypo-acetylated condensed Chromatin fiber TBP JHW 3-99

16 Acétylation vs déacetylation des histones
Chromatin 9-98 Acétylation vs déacetylation des histones Chromatine décondensée: Etat transcriptionnel actif Acétylation des histones Chromatine condensée: Etat transcriptionnel réprimé Déacetylation des histones

17 Chromatin Histone Methylation 9-98 Histones can be methylated at lysines or arginines. Example: H3 K4 methylation N Lysine C C g C e N+ a b d O C C C S-adenosylmethyionine HMT (Histone Methyl-Transferase) Histone demethylase DNA backbone binding may not be strongly affected, but specific proteins may recognize these modifications N C e-N-monomethyl-Lysine C C C N+ e O C C C S-adenosylmethyionine HMT (Histone Methyl-Transferase) Histone demethylase C N e-N-dimethyl-Lysine N+ Histone demethylases found for K4, K9, K27 and K36 C C C e e S-adenosylmethyionine O C C C C HMT (Histone Methyl-Transferase) Histone demethylase C N C e-N-trimethyl-Lysine C C C N+ e e O C C C C

18 Histone methylation : Histone methyltransferases
Chromatin 9-98 Histone methylation : Histone methyltransferases Euchromatin methylation, PcG-dependent gene silencing Enhancer of Zeste Enhancer of Zeste H3 K27 Dm Trx, Ash1 Mammalian MLL1-3 Euchromatin methylation, gene activation trxG-dependent gene silencing

19 Histone demethylases 2 classes: LSD1 JmjC domain proteins Chromatin
9-98 2 classes: LSD1 JmjC domain proteins

20 Chromatin Histone demethylases 9-98 LSD1 demethylases target mono or dimethyl K4 and K9 of histone H3, depending on their interacting cofactor

21 Chromatin Histone demethylases 9-98 JmjC domain demethylases target many different lysines and are conserved

22 A reversible phenomenon with many dedicated components
Chromatin 9-98 Histone methylation A reversible phenomenon with many dedicated components Utx JMJD3

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24 Chromatin 9-98 Coffee break…

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