Chromatin Structure & Gene activity part 2

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1 Chromatin Structure & Gene activity part 2

2 Figure Make sure that you understand it. It is good practice for identifying the purpose for various controls.

3 Chromatin remodeling Acetylation helps to make DNA more accessible but by itself it is not enough. Also need additional chromatin remodeling. What is it? Don t really know all of the types that fall under this heading. 1. movement of nucleosomes away 2. other ways to loosen their grip on the DNA. 3. changing the nucleosome so that other proteins can move it or loosen it. 4. changing the nucleosome so that other proteins will RECOGNIZE it. pg 410

4 Chromatin remodeling List of protein complex families SWI/SNF - SwitchSniff ISWI - limitation switch NuRD INO80 Produce nucleosome free regions around enhancers & promoters pg 410

5 human interferon-beta gene (IFN-ß) Dimitris Thanos human interferon-beta gene (IFN-ß) viral infection activates it. TF activators bind nucleosome free areas near promoter generating an enhanceosome.

6 Remodel IFN-ß overview Enhanceosome HATs N N N HATs acetylate N N N N ac ac ac ac N N N N represents enhanceosome Acetylated histones help attract CBP-RNAPII Holoenzyme. N CBP RNAPII Gg g TFIID N N N N ac ac ac ac pg 411 CBP has bromodomains and uses them to recognize acetylated histones.

7 Remodel IFN-ß overview SWI/SNF complex remodels nucleosome over the promoter, loosening it. CBP RNAPII Gg g TFIID ac ac g Gg NN N ac ac ac N ac ac ac TFIID binds to the TATA box and bends it, nucleosome moves 36 bp downstream. Transcript will now start. II AP RN IID TF CBP N N N ac ac ac ac ac N ac ac ac ac

8 Way too simple K8 acetylation of H4 recruits SWI/SNF complex K9 & K14 acetylation of H3 causes recruitment of TFIID

9 Histone code hypothesis/model Kinase binds enhanceosome & phosphoryaltes H3 S10. NOW GCN 5 can acetylate H3 K14. enhanceosome enhanceosome recruits GCN5 The GCN5 HAT acetylates H4 K8 & K3 K9 Histone code is complete & now proteins use it. Acetylated H4K8 is recognized by SWI/SNF. SWI/SNF remodels nucleosome. TFIID is attracted by acetylated H3 K9 and H3 K14. Nucleosome can now lets TFIID have access to the TATA box. which it binds. Then it bends the DNA and the nucleosome moves. Could figure this out by changing K s to A s and doing ChromIP.

10 How? The ChIP assay chromatin immunoprecipitation assay Allows one to determine which proteins and molecules are present on a transcriptional control region. Allows one to detect changes in abundance of proteins and molecules on a transcriptional control region.

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12 Agalioti et al 2002 Laboratory of Dimitris Thanos Fig th ed. Pattern is not random It has been postulated that phosphorylation of S10 is required for K14 acetylation. Time course supports this. TFIID binds after K14 is acetylated Time course of Sendai virus infection of HeLa cells. From the lab of Dimitris Thanos

13 Histone code hypothesis/model Kinase binds enhanceosome & phosphoryaltes H3 S10. NOW GCN 5 can acetylate H3 K14. enhanceosome enhanceosome recruits GCN5 The GCN5 HAT acetylates H4 K8 & K3 K9 Histone code is complete & now proteins use it. Acetylated H4K8 is recognized by SWI/SNF. SWI/SNF remodels nucleosome. TFIID is attracted by acetylated H3 K9 and H3 K14. Nucleosome can now lets TFIID have access to the TATA box. which it binds. Then it bends the DNA and the nucleosome moves. Could figure this out by changing K s to A s and doing ChromIP.

14 Histone code hypothesis/model Proteins associated with the enhancer modify the histones so that they are recognized by the General Transcription factor machinery.

15 Silencing Deacetylases can cause the condensation of chromatin. Condensation is the compression or packing that comes from many interactions between adjacent nucleosomes. Huge stretches are found in telomeres and centromeres. pg 416

16 c HAT M H3 M HDM M K4 Epigenesis M M Acetylation (activating) Deacetylation Deation Methylation (activating) M K9 A S10 P HDAC HMT K14 A K18 K23 A PK HDM A M K27 Phosphorylation (activating) S28 P M Deation Methylation (repressing) Dephosphorylation PP M K36 M HMT M K79 Histone tail basal transcript transcription; o heterochromat silenced (botto continuum of s (top right); repr Enrichment of and atio binding of tran or repressors (R transcriptional suggests that in subject to reac remains uncert modifications o ation an residues. H3 ph phosphorylatio is catalysed by reversed by his ation (w is catalysed by reversed by his phosphorylatio reversed by pro been identified residue. Panels Rev. Neurosci. R Tsankova, N, Renthal, W, Kumar, A, Nestler, EJ (2007) Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci, 8: MAY 2007 VOLUME 8

17 Histone ation Drosophila Repressed lys4 lys9 H3 lys20 H4 Repressors HP1 & polycomb can bind. Stimulated lys4 lys9 lys20 H3 H4 Now can be bound by the activator Brahma but not by the repressors HP1 and polycomb. Interpreted to mean that the proteins are interpreting a code. Methylated K9 has a different effect when it is with ated K4 and ated K20. pg 418.

18 Histone ation Drosophila pg 392 4th ed. Repressed lys4 lys9 H3 lys20 H4 Repressors HP1 & polycomb can bind. Stimulated lys4 lys9 lys20 H3 H4 Now can be bound by the activator Brahma but not by the repressors HP1 and polycomb. Interpreted to mean that the proteins are interpreting a code. Methylated K9 has a different effect when it is with ated K4 and ated K20. Interpreted to mean that the proteins are interpreting a code. Methylated K9 has a different effect when it is with ated K4 and ated K20. pg 418.

19 Modifications in one histone can affect (allow or deny) a modification in an another histone.

20 Histone ation Drosophila Repressed lys4 lys9 H3 lys20 H4 Repressors HP1 & polycomb can bind. Stimulated lys4 lys9 lys20 H3 H4 Now can be bound by the activator Brahma but not by the repressors HP1 and polycomb. Interpreted to mean that the proteins are interpreting a code. Methylated K9 has a different effect when it is with ated K4 and ated K20. Interpreted to mean that the proteins are interpreting a code. Methylated K9 has a different effect when it is with ated K4 and ated K20. pg 418.

21 How many characters are there in this code? More than 100 modifications have been detected by NMR. Many scientists find this to be disquieting because you can make a huge number of unique patterns (code words) with a 100 character alphabet.

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23 Figure Once transcription begins do all of the nucleosomes have to be bumped off?