Transcript Dr. Huck

Stem cells and epigenetics
Huck-Hui Ng
Genome Institute of Singapore
17 July 2010
I. Stem cells
II. Epigenetics
Mouse pre-implantation development
1-cell
16-24 cell
morula
2-cell
Cavitating
morula
4-cell
E3.5
Blastocyst
8-cell
uncompacted
E4.5
Blastocyst
8-cell
compacted
TE
Ralston and Rossant, 2005; Chazaud et al., 2006; Ralston and Rossant, 2008
Mouse pre-implantation development
http://stemcells.nih.gov/
Embryonic stem cells
http://stemcells.nih.gov
Stem cells from blastocyst lineages
Trophoblast stem (TS)
cells are derived from
the trophectoderm
lineage
Embryonic stem (ES)
cells represent the
epiblast lineage
Ralston A, Rossant J. Genetic
regulation of stem cell origins in
the mouse embryo. Clin Genet
2005: 68: 106–112.
Extraembryonic
endoderm (XEN) cells
derive from the
primitive endoderm
lineage
The stem-cell hierarchy
Eckfeldt CE et al. Nat Rev Mol Cell Biol. (2005)
Waddington’s epigenetic landscape model
Embryonic stem cells
1) First isolated from mouse embryos in 1981.
Evans and Kaufman. Nature. (1981) 292:154-6.
Martin. Proc Natl Acad Sci U S A. (1981) 78:7634-8.
2) Gene targeting / Generation of transgenic mice
(Animal model for the study of gene functions in vivo)
3) Human ES cells
Thomson et al. Science. (1998) 282:1145-7
Reubinoff et al. Nat Biotechnol. (2000) 18:399-404.
How does the cell read genetic information?
http://images.crinet.com
Taatjes et al. (2004). Nat Rev Mol Cell Biol. 5(5):403-10
Sequence-specific transcription factor recruits multi-subunit
complexes that can modulate transcription and chromatin structure.
Oct4
 A POU transcription factor expressed by early embryo
cells and germ cells (Schöler et al, 1990. Nature).
 Required for the formation of pluripotent stem cells in
the mammalian embryo (Nichols et al, 1998. Cell).
 Required for the maintenance of pluripotency of ES
cells and controls lineage commitment (trophectoderm)
(Niwa et al, 2000. Nat Genet).
Chromatin Immunoprecipitation (ChIP)
HCHO crosslinking in living cells
Sonication
Immunoprecipitation
to enrich for binding sites
Formaldehyde:
 relatively non-specific high resolution crosslinker (2 Å)
 covalent crosslink is reversible (by heating in the presence
of Tris)
 fixation is extremely rapid
Cells are frozen in native state “snap shot”
The advantages of Chromatin IP
 allows one to probe the direct physical relationship
between DNA binding proteins and their DNA targets
 in vivo measurement of physical occupancy
(crosslinking in living cells)
 physiologically relevant targets (wild type context)
Mapping transcription factor binding sites
Aims:
1) How is the ES cell genome wired?
2) Are there cross-talks between the key signaling
pathways and the other transcription factors?
3) Can we infer the composition of multi-protein
complexes assembled on the chromatin?
Where do transcription factors bind in the genomic space?
* core factors
signaling
* effectors
*
*
*
*
*
*
*
*
self-renewal
regulator
High resolution localization of sites using ChIP-seq method
Oct4 binding
Oct4 gene (4.7 kb)
Binding profiles of 13
sequence specific
transcription factors
at Oct4 and Nanog loci
Colocalization of
transcription factor
binding sites at
Oct4 and Nanog
enhancers
Transcription factor relationship at
multiple transcription factor binding loci (MTL)
Oct4-centric
MTL
Myc-centric MTL
Oct4-centric MTL can enhance transcription
ES cell-specific enhanceosomes:
1) Regions densely bound by multiple transcription factors
(include Oct4, Sox2, Nanog, Smad1, STAT3 and others)
2) These sites are not commonly found at proximal promoter
regions (-500bp, +2,000bp)
3) Function as enhancers
4) Bound by co-activators (p300, CBP, NcoA3)
Clustering based on transcription factor binding sites
reveals five classes of genes
Nanog, Pou5f1, Sox2, Esrrb,
Klf4, c-Myc, n-Myc, Rif1, Sall4,
Tbx3, Tcf3, Tcfcp2l1, Zic3
Suz12 bound genes
Myc bound genes
ESC-specific
expression
constitutive
expression
Genes expressed in ES cells
Poorly expressed / silenced
Summary
Design principles of ES cell TF network
1) Regulatory loops for key transcription factors
Loh et al (2006). Nat Genet; Jiang et al (2008). Nat Cell Biol
2) Highly connected network
• Hotspots for transcription factor co-binding
- ES cell-specific enhanceosomes
• Nexus that integrate extracellular signaling and
intrinsic pathways
Chen et al (2008). Cell
3) Downstream targets of key TFs are important for ES cells
Rif1, Esrrb, Klf2, Klf4, Klf5
Loh et al (2006). Nat Genet; Jiang et al (2008). Nat Cell Biol
How can you de-differentiate a somatic cell?
Takahashi and Yamanaka, 2006. Cell
Yamanaka, 2007. Cell Stem Cell. 1(1):39-49
Transcription factors can specify
ES cell identity in non-stem cells
Oct4, Sox2,
Klf4, c-Myc
Takahashi K, Yamanaka S.
Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
Cell. 2006 Aug 25;126(4):663-76. Epub 2006 Aug 10.
Are different somatic cells amenable to
reprogramming?
Hochedlinger and Plath. Development 136, 509-523 (2009)
The path towards induced pluripotency
Hochedlinger and Plath. Development 136, 509-523 (2009)
II. Epigenetics
What is epigenetics?
References:
Li, E. (2002). Nature Reviews Genetics.
Chromatin modification and epigenetic reprogramming in
mammalian development.
Bird, A. (2002). Genes & Development.
DNA methylation patterns and epigenetic memory.
Bird, A. (2007). Nature.
Perceptions of epigenetics.
Epigenetics:
'outside conventional genetics'
The study of mitotically and / or meiotically heritable changes in gene
function that cannot be explained by changes in DNA sequence.
Russo, V.E.A., Martienssen, R.A., and Riggs, A.D. 1996. Epigenetic mechanisms
of gene regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Bird. 2002. DNA methylation patterns and epigenetic memory. Genes Dev. 16: 621.
There are two epigenetic systems that affect animal development and fulfill the
criterion of heritability: DNA methylation and the Polycomb-trithorax group (PcG/trx) protein complexes.
(Histone modification has some attributes of an epigenetic process, but the issue
of heritability has yet to be resolved.)
Yeast position effect variegation: sectored colonies
yeast chromosome
telomere
centromere
telomere
ADE2 (wildtype) gene at normal location on chromosome
wildtype white colony
telomere
centromere
telomere
ADE2 (wildtype) gene moved to location near telomere
red sectored colony
Yeast position effect variegation
Metastable states (on and off)
Single cell
Grown up colony
Epigenetic properties:
1) Mitotically heritable
2) Cannot be explained by changes in DNA sequence
Unifying definition of epigenetic
events:
The structural adaptation of chromosomal regions
so as to register, signal or perpetuate altered
activity states.
Bird (2007). Nature. 447, 396-398.
Expanded meaning of “epigenetics”
Nature 2003 Jan 23;421(6921):448-53
Controlling the double helix. Felsenfeld G, Groudine M.
Special features of nuclear architecture
in embryonic stem cells
Nuclear architecture in ES cells and
differentiating ES-derived cells
Meshorer E, Misteli T. Nat Rev Mol Cell Biol. 2006 Jul;7(7):540-6
Histone H3
Lys 4
methylation
(active mark)
Histone H3 Lys 9
methylation
(repressive mark)
Histone H3
acetylation
(active mark)
Histone H4
acetylation
(active mark)
Nature 2003 Jan 23;421(6921):448-53
Controlling the double helix.
Felsenfeld G, Groudine M.
Breathing Chromatin of ES cells
Open chromatin architecture permissive
for gene expression and pluripotency in ES cells ?
Undifferentiated
Differentiated
Meshorer E, Misteli T. Nat Rev Mol Cell Biol. 2006 Jul;7(7):540-6
Working model:
The crosstalk between the transcription factor network and
the epigenetic mechanism in the maintenance of pluripotency
Oct4
Genetic / biochemical
interactions with chromatin modifiers?
chromatin structure in ES cells ?
The roles of histone modifiers in ES cells
1. The roles of histone H3K9 demethylases
in ES cells
2. Oct4 and histone H3K9 methylase
Oct4’s target genes
Mapping of transcription factor binding sites in living cells
and study how the targets relate to ES cell properties
Oct4 ChIP-PET clusters mapped to Jmjd1a and Jmjd2c
Jmjd1a
Jmjd2c
Histone methylation is reversible
H3K36me2 demethylase
Different states of lysine methylation
histone H3 lysine methylases
Me1
Me2
Me3
histone H3 lysine demethylases
Zhang and Reinberg (2001) Genes Dev.
Histone H3 lys 9 demethylases
Jmjd1a
K9
Jmjd1a
K9
Jmjd2c
K9
K9
Demethylation of H3K9 me2
Jmjd2c
K9
K9
Demethylation of H3K9 me3
Cell. 2006 May 5;125(3):483-95.
Cell. 2006 May 5;125(3):467-81.
Nature. 2006 Jul 20;442(7100):312-6.
Differentiation of ES cells leads to
reduction of Jmjd1a and increase in H3K9Me2
 Jmjd1a
 β-tubulin
 H3K9Me2
 H3
Western
Differentiation of ES cells leads to
reduction of Jmjd2c and increase in H3K9Me3
 Jmjd2c
 β-tubulin
 H3K9Me3
 H3
Western
Jmjd1a and Jmjd2c are induced in reprogrammed fibroblasts
Takahashi K, Yamanaka S. Cell. 2006 Aug 25;126(4):663-76
Oct4 directly regulates Jmjd1a and Jmjd2c
A
B
Oct4 binds to intronic sequences of Jmjd1a and Jmjd2c
Depletion of Jmjd1a induces H3K9Me2 but not H3K9Me3
Western
Jmjd1a
K9
K9
Depletion of Jmjd2c induces H3K9Me3 but not H3K9Me2
Western
Jmjd2c
K9
K9
Depletion of Jmjd1a and Jmjd2c induces ES cell differentiation
Changes in expression of ES cell and differentiation markers
upon Jmjd1a or Jmjd2c depletion
Actions of JmjC histone demethylases and their roles in ES cells
histone
modifiers
Global effects
Large scale regulation
e.g. heterochromatization
Localized effects
Regulation of specific promoters
e.g. targeted recruitment
TF
coating
transient
interactions
gene
Tcl1 is regulated by Jmjd1a
Tcl1:
 T-cell lymphoma breakpoint 1
 enhances Akt kinase activity and
induces its nuclear translocation
 a self-renewal regulator in ES cells
(Ivanova (2006). Nature; Matoba (2006). PLoS ONE)
Jmjd1a regulates the expression and H3K9Me2 of Tcl1
A
B
C
D
Oct4 binding at Tcl1 promoter is dependent on Jmjd1a
Tcl1
promoter
Tcl1
Oct4
Jmjd2c regulates the expression and H3K9Me3 of Nanog
Jmjd2c binds to Nanog promoter
A
B
C
Increased H3K9Me3 at Nanog promoter leads
to increased binding of co-repressors (HP1 and KAP1)
A
B
C
Interface between genetic and epigenetic regulation
Oct4
Jmjd1a
Jmjd2c
Tcl1
Tcl1
Nanog
Nanog
Maintenance of ES cells
guardian of
self-renewal genes
Model: role of histone demethylases
in maintaining ES cells
Open chromatin
Low H3K9 methylation
 Jmjd1a
 Jmjd2c
 Jmjd1a
 Jmjd2c
Oct4
Sox2
Nanog
Self-renewal
Condensed chromatin
High H3K9 methylation
Summary
1. Oct4 directly regulates Jmjd1a and Jmjd2c, which are
upregulated in ES cells.
2. Depletion of Jmjd1a and Jmjd2c leads to global increase
in H3K9Me2 and H3K9Me3, respectively.
3. Depletion of Jmjd1a leads to ES cell differentiation.
4. Novel regulatory pathways used by Oct4 to maintain the
expression of its downstream targets (Tcl1 and Nanog).
Histone H3 lys 9 methylase
Jmjd2c
K9
Jmjd2c
K9
Eset
K9
Eset
Methylation of H3K9 me3
Mol Cell. 2003 Aug;12(2):475-87.
Knockout of Eset
Mol Cell Biol. 2004 Mar;24(6):2478-86.
Depletion of Eset induces differentiation
Tcfap2a, Tcfap2c and Cdx2 are repressed by Eset
H3K9me3
H3K9me2
Eset occupancy
Eset depleted cells express Cdx2 and Cdh3
Eset interacts with Oct4 and
recruitment of Eset is dependent on Oct4
Model for regulation of cell fate by Oct4 through Eset
Summary
1. Eset interacts with Oct4.
2. Depletion of Eset leads to ES cell differentiation.
3. Eset regulates H3K9Me2 and H3K9Me3 of Tcfap2a and
Tcfap2c.
4. Oct4 may control pluripotency through Eset
(selective recruitment of Eset by Oct4 to lineage specific
genes).
Overview
1. Oct4 directly controls the expression of histone
H3 lysine 9 demethylases (Jmjd1a and Jmjd2c).
2. Oct4 recruits Eset (histone H3 lysine 9 methylase)
to repress trophectoderm lineage and genes.
Oct4
Jmjd1a Jmjd2c
Suppress H3K9
methylations
and promote
expression of
self-renewal genes
Eset
Mediates H3K9
methylations
and repress
trophectoderm
lineage
Cellular potency in development
and reprogramming
Hemberger et al (2009). Nat. Rev. MCB