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Twenty-five years of the nucleosome Kornberg and Lorch 1998, Cell 98: 285.

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 nucleosome Kornberg and Lorch 1998, Cell 98: 285.

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  1. Twenty-five years of the nucleosomeKornberg and Lorch 1998, Cell 98: 285.

  2. HATs HDACs

  3. 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

  4. Chromatin remodeling • SWI/SNF proteins are chromatin remodelers • These disrupt nucleosome in an ATP-dependent fashion (ATPase activity) • Models- • displacement • octamer sliding

  5. Histones- modifications and function Michael Hagmann 1999 Science 285:1203 • A. Phosphorylation of histones • two opposing functions reported • opening chromatin • condensing chromatin (cell division)

  6. 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

  7. 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

  8. 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

  9. C. Role in phosphorylation in cell division 1.Tetrahymena

  10. Tetrahymena (a protozoan) Genetics, Russell, p6.

  11. Macronuclei Micronuclei Mitotic (football shape) Tetrahymena- Histone H3 phosphorylation occurs only in mitotic micronuclei

  12. 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

  13. 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??

  14. 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

  15. Methylation, phosphorylation and acetylation of histones OFF Histone H3 ON Bromodomain of a HAT Chromodomain of a chromatin remodeler Science 292:65, 2001

  16. 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

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