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

• 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

Cellular and Molecular Genetics BLA510 Spring 2001Gary 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

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

Growth factor

Receptor Ras

Raf (MAPKKK)

MEK (MAPKK)

ERK ( MAPK)

RSK-2H3 H3

P

MAP kinase signal transduction

pathway

Thus, Rsk-2 mutation prevented H3 phosphorylation

Experiment-Induce cells with growth factors,Crosslink DNA+ proteins, then immunoprecipitate with anti-Phospho-H3 Ab

Immunoprecipitatewith anti-Phospho-H3

DNA

Crosslink, nuclease

H3 P H3

P H3H3

Most genes c-fos

SouthernAgarose gel Probe with labeled c-fos DNA

Thus, known growth-response genes are bound by histones with phosphorylated H3

C. Role in phosphorylation in cell division

1. Tetrahymena

# copies of chromosomes

Mode of replication in cell division

macronucleus

micronucleus

90

2

Pinching off none

Normal mitosis abnormal condensation chromosome loss

Affect of H3 mutation at phosphorylation site

Genetics, Russell, p6.

Tetrahymena(a protozoan)

Macronuclei

Tetrahymena- Histone H3 phosphorylation occurs only in mitotic micronuclei

Micronuclei Mitotic (football shape)

2. Immunocytochemistry- observe phospho-H3 throughout chromosomes during cell divisionThus, 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

II. Methylation

CARM-1 -

p160CARM-1

TATAA

Steroid hormone receptor+1

coactivator

• activates transcription (coactivator)

• methylates proteins

• inactivation of methylation activity - lose transcriptional activation

• methylates histone H3 in vitro

• what are CARM-1 targets??

Me H3

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

OFF

ON

Histone H3

Bromodomain of a HAT

Chromodomain of a chromatin remodeler

Methylation, phosphorylation and acetylation of histones

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”

Science 292:65, 2001

Shelley Berger, Wistar Institute