Chromatin and Transcription Chromatin Structure Chromatin Represses Transcription Nucleosome Positioning Histone Acetylation Chromatin Remodeling Histone Methylation CHIP Analysis Chromatin and Elongation Levels of Chromatin Structure 1
Histone and Nucleosomes Nucleosome Core Histone Fold 2
Tripartite Structure of Nucleosome Core Tripartite Structure and DNA 3
Nucleosome Structure Half-Nucleosomes 4
Tetranucleosome 5
30 nm Fiber Higher Order Structure Chromosome scaffold Scaffold Attachment Points Chromatin domains each loop 6
Nucleosomes and Transcription Lane 5: Nuclear ratio of histones to DNA Restriction enzyme demonstrate that remaining 25% transcription is due to population of DNA with naked promoter General Model Nucleosomes inhibit transcription If Histones get to genes first and organized chromatin forms, GTF cannot transcribe genes. If GTF get to genes first, latter addition of histones and chromatin formation doesn t interfer with transcription. 7
H1 Repression of Pol III Transcription Note: Two versions of 5S gene, somatic and oocyte specific. Naked versions of both genes are transcribed invitro Isolated genes from somatic cells only somatic gene transcribed invitro, oocyte gene not transcribed when bound to chromatin. Lane 1 Repressed Chromatin Lane 2: Accidentally derepressed Lane 1 Repressed Chromatin Lane 2 Salt derepressed Lane 3 Ion exchanged derepressed Lane 4 Pure H1 histone H1 and Activators Lanes 1 and 2 show activator enhances transcription 8 fold on naked DNA. (Mechanism?) Lanes 3 and 4 show a similar level of activation on chromatin made of core histones Lanes 5 and 6 show activator enhances transcription 200 fold stimulation on chromatin made of core histones and H1. Conclusion activators have a 25 fold stimulation due to anti repression (prevention of repression by H1 chromatin) 8
General DNase Sensitivity Globin gene is erythrocyte is more sensitive to DNase than same gene in MSB cell. Weintraub DNase Hypersensitivity 9
Hypersensitivity in Human Globin Genes Erythroleukemia Cells (Express globin genes) Human T Cells (globin genes not expressed) Nucleosome free zones on SV40 10
Activators and Chromatin Fry and Peterson, 2001 HAT Histone Acetyltransferase Part of Several CoActivators P300, P/CAF, TAF250 GCN5(SAGA Complex) Effect of Acetylation 1. Chromatin Structure 2. Target for Bromo-domain Proteins 11
Acetylation and Transcription Control Template Template 12 5S promoters with no terminator Tse et al 1998 Bromo Domain Many Transcription components contain bromodomains 1. TAF250 2. P300 3. SAGA 4. Others? Acetylated histones recruit transcription components. Acetylated Histones facilitate translocation of components 12
Acetylation/Bromodomains facilitates exchange of factors Repressors and histone deacetylases (HDAC) Effect of deacylation 13
Chromatin Remodeling Swi/Snf Complex Swi/Snf ATP ADP Loosens contact between DNA and Histone by incorporating extra DNA in Nucleosome/DNA intraction a DNA bulge. Effect of DNA Bulge caused by Swi/Snf Repositioning Events 14
Histone Methylation Histone Code ack-acetylated lysine, mer-methylated arginine, mek-methylated lysine, PS-phosphorylated serine, UK-ubiquinated lysine 15
CHIP (Chromatin Immunoprecipitation) Need an antibody specific to some modification or to some protein component Treat chromatin with formaldhyde to cross link and shear into small fragments Immunoprecipate all chromatin with structure recognized by antibody Use PCR to see if specific gene is present. Histone modifications at IFN-β 16
CHIP Analysis of PIC assembly Induced by GAL4 Assembly of PIC at IFN-β gene 17
Chromatin and Elongation FACT (Spt16+SSRP1) destabilize H2A/H2B dimer in advance of RNA polymerase Spt6 binds H3/H4 tetramer Nucleosome is reassembled after RNA polymerase has transcribed region. Other factors implicated 18