Stem cells and epigenetics Huck-Hui Ng Genome Institute of Singapore 17 July 2010
I. Stem cells II. Epigenetics
1-cell 2-cell 4-cell 8-cell uncompacted 8-cell compacted Mouse pre-implantation development 16-24 cell morula Cavitating morula E3.5 Blastocyst E4.5 Blastocyst 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 Extraembryonic endoderm (XEN) cells derive from the primitive endoderm lineage Ralston A, Rossant J. Genetic regulation of stem cell origins in the mouse embryo. Clin Genet 2005: 68: 106–112.
The stem-cell hierarchy Eckfeldt CE et al. Nat Rev Mol Cell Biol. (2005)
Embryonic stem cells • 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) • 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? Taatjes et al. (2004). Nat Rev Mol Cell Biol. 5(5):403-10 http://images.crinet.com 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 vivomeasurement of physical occupancy (crosslinking in living cells) physiologically relevant targets (wild type context)
Mapping transcription factor binding sites • Aims: • 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?
core factors signaling effectors self-renewal regulator * * * * * * * * * * Where do transcription factors bind in the genomic space?
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
ES cell-specific enhanceosomes: • Regions densely bound by multiple transcription factors • (include Oct4, Sox2, Nanog, Smad1, STAT3 and others) • These sites are not commonly found at proximal promoter • regions (-500bp, +2,000bp) • Function as enhancers • 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 Poorly expressed / silenced Genes expressed in ES cells
Summary • Design principles of ES cell TF network • 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)
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: 6-21. There are two epigenetic systems that affect animal development and fulfill the criterion of heritability: DNA methylation and the Polycomb-trithorax group (Pc-G/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 centromere telomere telomere ADE2 (wildtype) gene at normal location on chromosome wildtype white colony centromere telomere 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: • Mitotically heritable • 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 9 methylation (repressive mark) Histone H3 acetylation (active mark) Histone H4 acetylation (active mark) Histone H3 Lys 4 methylation (active mark) Nature 2003 Jan 23;421(6921):448-53 Controlling the double helix. Felsenfeld G, Groudine M.
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.
K9 Jmjd1a Jmjd1a K9 K9 Histone H3 lys 9 demethylases Demethylation of H3K9 me2 K9 K9 Jmjd2c Jmjd2c Demethylation of H3K9 me3 K9 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 Control -LIF +RA Jmjd1a β-tubulin H3K9Me2 H3 Western
Differentiation of ES cells leads to reduction of Jmjd2c and increase in H3K9Me3 Control -LIF +RA Jmjd2c β-tubulin H3K9Me3 H3 Western