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Twenty-five years of the nucleosome Kornberg and Lorch 1998, Cell 98: 285. HATs. HDACs. Important point-. P289- Although acetylation of histone tails may counteract condensation of nucleosomes in chromatin fibers, it is unlikely to disrupt the structure of the core particle for transcription
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Twenty-five years of the nucleosomeKornberg and Lorch 1998, Cell 98: 285.
HATs HDACs
Important point- • P289- Although acetylation of histone tails may counteract condensation of nucleosomes in chromatin fibers, it is unlikely to disrupt the structure of the core particle for transcription • Why? Tails are outside of the core, make little contribution to overall structure • Chromatin remodeling enzymes are likely responsible for nucleosome disruption
Chromatin remodeling • SWI/SNF proteins are chromatin remodelers • These disrupt nucleosome in an ATP-dependent fashion (ATPase activity) • Models- • displacement • octamer sliding
Histones- modifications and function Michael Hagmann 1999 Science 285:1203 • A. Phosphorylation of histones • two opposing functions reported • opening chromatin • condensing chromatin (cell division)
I. Phosphorylation • Immunocytochemistry with anti-phosphoH3 antibody • Phosphorylation of H3 observed during mitosis • Growth factor stimulation- observe ~100 speckels in cells, randomly distributed • correlates with # of genes that respond to growth factor stimuli. • Identify a 90 Kd protein Cellular and Molecular Genetics BLA510 Spring 2001 Gary A. Bulla, PhD
B. Coffin Lowry syndrome- mental retardation • and a defect in growth factor response • mutation identified in in Rsk-2 gene • Immunocytochemistry with anti-phosphoH3 Ab • -no speckles observed MAP kinase signal transduction pathway Thus, Rsk-2 mutation prevented H3 phosphorylation
c-fos Most genes H3 H3 H3 H3 DNA Crosslink, nuclease Immunoprecipitate with anti-Phospho-H3 Southern Agarose gel Probe with labeled c-fos DNA P P • Experiment- • Induce cells with growth factors, • Crosslink DNA+ proteins, then immunoprecipitate with anti-Phospho-H3 Ab Thus, known growth-response genes are bound by histones with phosphorylated H3
C. Role in phosphorylation in cell division 1.Tetrahymena
Tetrahymena (a protozoan) Genetics, Russell, p6.
Macronuclei Micronuclei Mitotic (football shape) Tetrahymena- Histone H3 phosphorylation occurs only in mitotic micronuclei
2. Immunocytochemistry- observe phospho-H3 throughout chromosomes during cell division Thus, this must play a role is chromosome condensation during mitosis 3. Models- 1. Phosphorylation + acetylation allows activation of gene expression, depending on context 2.Phospho-H3 loosens chromatin, enhancing transcription factor binding or mitotic factor binding
H3 Me II. Methylation CARM-1 - • activates transcription (coactivator) • methylates proteins • inactivation of methylation activity - lose transcriptional activation • methylates histone H3 in vitro • what are CARM-1 targets??
III. Acetylation- Bromodomain -100 AA found in ~30 chromatin associated proteins (inc. HATs) - may be binding motif for actetylated histones Acetylated lysine IV. Other modifications- ubiquitination, glycosylation
Methylation, phosphorylation and acetylation of histones OFF Histone H3 ON Bromodomain of a HAT Chromodomain of a chromatin remodeler Science 292:65, 2001
Is there a “Histone Code”? • Definition- “Covalent modifications of histones constitute an intricate pattern that creates a docking surface with which the modules of other proteins can interact” Shelley Berger, Wistar Institute Science 292:65, 2001