Transcript Molecular Biology of the Cell

Polycomb
Complexes
&
Epigenetics
Adam Baim
MBB306
10/2/07
Chromatin Structure
Adapted from: Alberts, et al., Molecular Biology of the Cell, 4th Ed. (2002)
Polycomb (PcG) Complexes
• Chromatin-modifying transcriptional repressors
with key developmental roles:
– Embryonic development
– Adult stem cell renewal
– Deregulated in cancer
• PRC2
– subunits: EZH2, EED, SUZ12, RbAp48
– EZH2 is histone methyltransferase, acts upon H3K27
• PRC1
– subunits: BMI-1, CBX2,4,7,8, other proteins
– binds H3K27me3 and represses gene
Sparmann, et al (2006) Polycomb silencers control cell fate, development and cancer. Nature Reviews Cancer 6, 846-856
What’s known about Polycomb?
• Best known as epigenetic regulators of
homeotic (HOX) clusters (Ringrose, 2007)
• PRC1 has been shown to mediate
silencing by ubiquitylation of H2AK119; the
protein which reads this modification has
yet to be characterized (Schuettengruber,
et al., 2007)
• The H3K27me3 deposited by PRC2 might
directly repress tsc in absence of PRC1
(Schuettengruber, et al., 2007)
Trends in Polycomb Research
Number of Publications With "Polycomb" as Major Topic, Per Year
Data from ISI Web of Knowledge
250
Number of Publications
200
150
100
50
0
1970
1975
1980
1985
1990
Year
1995
2000
2005
2010
Overview of Paper
• In human embryonic fibroblasts:
– Examined changes in mRNA expression associated
with depletion of PRC1 and PRC2, to find potential
PcG target genes
– ChIP-on-chip analyses to confirm PcG targets:
• Isolated DNA sequences which bind to PRC1,2, and
H3K27me3 (ChIP)
• Hybridized labeled DNA to tiled array (allows for identification
of genes it contains which bind PRC1,2 and H3K27me3)
• In human neural progenitor cells:
– Evaluated PcG binding at genes involved in neural
differentiation
siRNA to Deplete Specific Proteins
• Create double-stranded
RNA copy of mRNA to be
blocked, transfect into
cells
• Internal machinery (e.g.,
RISC) uses this doublestranded RNA as template
for recognizing the gene’s
mRNA in vivo
• The mRNA is cleaved and
degraded, inhibiting
protein synthesis
Image: http://www.ohsu.edu/research/core/images/sirna_MWG-biotech.gif
Effects of PcG depletion on Expression
• Used siRNA to block
translation of EZH2, EED, and
SUZ12 (PRC2 subunits) and
BMI-1 (main PRC1 subunit)
• Confirmed inhibition (lack of
translated protein) via
Western blot (A)
• Extracted and labeled RNA
from PcG-inhibited cells and
control cells, hybridized
directly to Affymetrix gene
expression array
• Quantified changes in gene
expression (B,C)
– Selected 341 of the most
significantly changed genes as
potential PcG targets
Overview of Paper
• In human embryonic fibroblasts:
– Examined changes in mRNA expression associated
with depletion of PRC1 and PRC2, to find potential
PcG target genes
– ChIP-on-chip analyses to confirm PcG targets:
• Isolated DNA sequences which bind to PRC1,2, and
H3K27me3…“ChIP”
• Hybridized labeled DNA to tiled arrays (allows for identification
of genes which bind PRC1,2 and H3K27me3)…“chip”
• In human neural progenitor cells:
– Evaluated PcG binding at genes involved in neural
differentiation
Chromatin Immunoprecipitation (ChIP)
Protein
Authors used this technique to make
antibodies specific to SUZ12 (PRC2), CBX8
(PRC1), and H3K27me3
DNA that originally bound PRC1,2 and
H3K27me3 is now ISOLATED, but it needs to
be IDENTIFIED
http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mboc4/ch7f32.gif
http://imagecache2.allposters.com/images/ADVG/479.jpg
Immunoprecipitated DNA  Gene chip
• To validate specificity
of ChIP antibodies,
authors labeled this
DNA and hybridized it
to tiled gene chips
containing all genes
from the HOXA,
HOXB, HOXC, and
HOXD clusters
(known PcG binding
sites)
Adapted from: Alberts, et al., Molecular Biology of the Cell, 4th Ed. (2002)
HOXA Findings
• Heavy binding
of PRC1,2 and
H3K27me3
between
HOXA9 and
HOXA13 loci
• Proteins
“blanket” loci
rather than
binding
exclusively at
promoters
• αHA (-) control
Overview of Paper
• In human embryonic fibroblasts:
– Examined changes in mRNA expression associated
with depletion of PRC1 and PRC2, to find potential
PcG target genes
– ChIP-on-chip analyses to confirm PcG targets:
• Isolated DNA sequences which bind to PRC1,2, and
H3K27me3…“ChIP”
• Hybridized labeled DNA to tiled arrays (allows for identification
of genes which bind PRC1,2 and H3K27me3)…“chip”
• In human neural progenitor cells:
– Evaluated PcG binding at genes involved in neural
differentiation
Identification of genes from ChIP DNA
• Represented the 341 putative PcG target gene
loci found in the mRNA expression assay on
tiled gene chips as:
|------------15kb------------GENE------5kb----|
• Hybridized these chips with the same ChIP DNA
used in the HOX experiment (i.e., the DNA that
binds PRC1,2, and H3K27me3
• Hybridization suggested that PRC1,2 and
H3K27me3 bound within the gene loci of 43
individual genes
“Bell enrichments”
binding is greatest at
particular points in the
gene locus
Depleted
Subunit
“Blanket enrichments”
binding occurs
throughout the gene
Changes in expression for the 43 genes
suggested as PcG/H3K27me3 targets by
the ChIP on chip experiment
Antibody binding for specific genes
“Genome-wide identification of human promoters bound by PcG”
• Experiment repeated on chips containing probes
for 24,275 promoters, each consisting of:
|-------------1300bp----------TSS-----200bp---|
=“Transcription Start Site”
• PcGs bound promoters for 70% of the genes
identified in the previous tiled array
• Of the promoters found to bind PcGs, there was
a significant overlap between those bound by
PRC1, PRC2, and those which were H3K27me3
– The authors believe this is “…consistent with the fact
that PRC1 is dependent on PRC2-mediated K27 trimethylation for its ability to bind chromatin”
Suggests there is significant overlap
in regions where SUZ12 (PRC2),
CBX8 (PRC1), and H3K27me3 are
bound
Overview of Paper
• In human embryonic fibroblasts:
– Examined changes in mRNA expression
associated with depletion of PRC1 and PRC2
– ChIP-on-chip analyses:
• Isolated DNA sequences which bind to PRC1,2, and
H3K27me3…“ChIP”
• Hybridized labeled DNA to tiled arrays (allows for
identification of genes which biund PRC1,2 and
H3K27me3)…“chip”
• In human neural progenitor cells:
– Evaluated PcG binding at genes involved in
neural differentiation
Many of the PcG target genes suggested
in the genome-wide experiment regulate
neuronal differentiation
PcGs in Neural Differentiation
• In human embryonic neural
progenitor cells, authors
selected four genes with
known roles in neuronal
differentiation
– Experiments showed
progressive decrease in PcG
binding and H3K27me3 during
differentiation (induced by
retinoic acid)
– Differentiation requires
displacement of PcGs which
repress differentiation-guiding
genes
– PcGs replaced when
differentiation is finished (e.g.,
HOX genes)
An Unusual Result
• Authors then examined a set genes which are highly
expressed except during differentiation, when they are
strongly silenced
– ChIP experiments showed presence of PcGs and H3K27me3 at
promoters of these genes in the undifferentiated state, when genes
are being expressed
– PcGs and H3K27me3 are only slightly increased during
differentiation, when the genes are silenced
Looking again at HOXA
• Authors found binding of EZH2 (PRC2) and CBX8 (PRC1)
with H3K27me3 throughout the HOXA locus, both BEFORE
and AFTER differentiation
Conjecture
• The authors postulate that
PcG target genes are “preprogrammed to be repressed
upon appropriate cell fate
signals,” explaining
transcription in spite of
PRC1,2 binding and
H3K27me3
• Support: PcG and trithorax
proteins found at polycomb
binding sites and HOX
promoters before expression
levels are established by
segmentation factors
Key Findings
• Only a small number of PcG target genes found in the
global-analysis were affected by PcG depletions
– Hypothesis: Majority of ~1000 known PcG target genes are
silenced in embryonic fibroblasts, perhaps due to EZH2’s ability
to methylate H1K26
– Alternative Hypothesis: fibroblasts lack transcriptional activators
necessary for expression of tissue-specific genes
• ChIP-on-chip revealed 43 novel PcG target genes with
“important regulatory roles in mesenchymal
differentiation and development.”
– Neuronal, bone, and sex differentiation; muscle development,
hematopoiesis
• PcG binding to target genes “does not strictly correlate
with transcriptional silencing.”
End.
All figures from Bracken, et al.,
2006 except where stated
otherwise