Transcript Twenty-five years of the nucleosome Kornberg and Lorch 1998, Cell

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 ATPdependent 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
Growth factor
Receptor
MAP kinase signal
transduction
pathway
Thus, Rsk-2
mutation
prevented H3
phosphorylation
Ras
Raf (MAPKKK)
MEK
(MAPKK)
ERK
RSK-2
H3
P
H3
( MAPK)
ExperimentInduce cells with growth factors,
Crosslink DNA+ proteins, then
immunoprecipitate with anti-Phospho-H3 Ab
Most genes H3
Crosslink,
nuclease
H3
c-fos
P H3
P
DNA
H3
Immunoprecipitate
with anti-Phospho-H3
Thus, known growthresponse genes are
bound by histones
with phosphorylated
H3
Agarose gel
Southern
Probe with labeled
c-fos DNA
C. Role in phosphorylation in cell division
1. Tetrahymena
# copies of
chromosomes
macronucleus
90
micronucleus
2
Mode of replication
in cell division
Pinching off
Normal mitosis
Affect of H3 mutation
at phosphorylation site
none
abnormal condensation
chromosome loss
Tetrahymena
(a protozoan)
Genetics, Russell, p6.
Tetrahymena- Histone H3 phosphorylation occurs
only in mitotic micronuclei
Macronuclei
Micronuclei
Mitotic (football shape)
2. Immunocytochemistry- observe phospho-H3 throughout
chromosomes during cell division
Thus, this must play a role is chromosome condensation during mitosis
3. Models1. 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 -
• activates transcription (coactivator)
• methylates proteins
• inactivation of methylation activity - lose
transcriptional activation
• methylates histone H3 in vitro
• what are CARM-1 targets??
coactivator
Steroid hormone receptor
CARM-1
p160
Me H3
+1
TATAA
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