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

3. HATs HDACs

4. 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

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

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

7. I. Phosphorylation Cellular and Molecular Genetics BLA510 Spring 2001 Gary A. Bulla, PhD 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

8. 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

9. Experiment- Induce cells with growth factors, Crosslink DNA+ proteins, then immunoprecipitate with anti-Phospho-H3 Ab Immunoprecipitate with anti-Phospho-H3 DNA Crosslink, nuclease H3 P P Most genes c-fos Southern Agarose gel Probe with labeled c-fos DNA Thus, known growth- response genes are bound by histones with phosphorylated H3

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

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

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

13. 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

14. 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?? Me H3

15. III. Acetylation- Bromodomain -100 AA found in ~30 chromatin associated proteins (inc. HATs) - may be binding motif for actetylated histones IV. Other modifications- ubiquitination, glycosylation Acetylated lysine

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

17. 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” Science 292:65, 2001 Shelley Berger, Wistar Institute