Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells.

The histone H3 Lys 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using chromatin immmunoprecipitation (ChIP) and sequencing (ChIP-seq) analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single-cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and, subsequently, placental tissues. Coimmunoprecipitation and ChIP assays further demonstrate that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our results suggest that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor.

[1]  R. Beddington,et al.  An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. , 1989, Development.

[2]  H. Schöler,et al.  Oct‐4: Gatekeeper in the Beginnings of Mammalian Development , 2001, Stem cells.

[3]  J. Tung,et al.  Dazl Functions in Maintenance of Pluripotency and Genetic and Epigenetic Programs of Differentiation in Mouse Primordial Germ Cells In Vivo and In Vitro , 2009, PloS one.

[4]  Janet Rossant,et al.  Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst , 2005, Development.

[5]  Janet Rossant,et al.  Interaction between Oct3/4 and Cdx2 Determines Trophectoderm Differentiation , 2005, Cell.

[6]  Jian Huang,et al.  Analysis of Transcription Factor AP-2 Expression and Function During Mouse Preimplantation Development1 , 2006, Biology of reproduction.

[7]  Clifford A. Meyer,et al.  Model-based Analysis of ChIP-Seq (MACS) , 2008, Genome Biology.

[8]  Guoji Guo,et al.  Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo. , 2008, Genes & development.

[9]  X. Chen,et al.  The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells , 2006, Nature Genetics.

[10]  S. Orkin,et al.  An Extended Transcriptional Network for Pluripotency of Embryonic Stem Cells , 2008, Cell.

[11]  Xi Chen,et al.  Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. , 2007, Genes & development.

[12]  M. Surani,et al.  Genetic and Epigenetic Regulators of Pluripotency , 2007, Cell.

[13]  B. Panning,et al.  The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation , 2006, Nature Cell Biology.

[14]  G. Fuhrmann,et al.  Tex19, a Mammalian‐Specific Protein with a Restricted Expression in Pluripotent Stem Cells and Germ Line , 2008, Stem cells.

[15]  J. Rossant,et al.  Genetic regulation of stem cell origins in the mouse embryo , 2005, Clinical genetics.

[16]  Hiroaki Kitano,et al.  The PANTHER database of protein families, subfamilies, functions and pathways , 2004, Nucleic Acids Res..

[17]  H. Cedar,et al.  G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis , 2006, Nature Cell Biology.

[18]  Karl Mechtler,et al.  Loss of the Suv39h Histone Methyltransferases Impairs Mammalian Heterochromatin and Genome Stability , 2001, Cell.

[19]  N. D. Clarke,et al.  Integration of External Signaling Pathways with the Core Transcriptional Network in Embryonic Stem Cells , 2008, Cell.

[20]  H. Schöler,et al.  Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4 , 1998, Cell.

[21]  A. Smith,et al.  Embryo-derived stem cells: of mice and men. , 2001, Annual review of cell and developmental biology.

[22]  Megan F. Cole,et al.  Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells , 2006, Cell.

[23]  En Li,et al.  Suv 39 h-Mediated Histone H 3 Lysine 9 Methylation Directs DNA Methylation to Major Satellite Repeats at Pericentric Heterochromatin , 2003 .

[24]  Dustin E. Schones,et al.  High-Resolution Profiling of Histone Methylations in the Human Genome , 2007, Cell.

[25]  T. Kouzarides.  Histone methylation in transcriptional control. , 2002, Current opinion in genetics & development.

[26]  W. Reik,et al.  Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5 , 2008, Nature Cell Biology.

[27]  J. Zeitlinger,et al.  Polycomb complexes repress developmental regulators in murine embryonic stem cells , 2006, Nature.

[28]  O. Ohara,et al.  Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity , 2008, Development.

[29]  J. Miyazaki,et al.  Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells , 2000, Nature Genetics.

[30]  J. Rossant.  Stem Cells and Early Lineage Development , 2008, Cell.

[31]  S. Elledge,et al.  CDYL bridges REST and histone methyltransferases for gene repression and suppression of cellular transformation. , 2008, Molecular cell.

[32]  T. Jenuwein,et al.  The many faces of histone lysine methylation. , 2002, Current opinion in cell biology.

[33]  T. Nottoli,et al.  Transcription factor AP-2gamma is essential in the extra-embryonic lineages for early postimplantation development. , 2002, Development.

[34]  D. Loebel,et al.  Lineage choice and differentiation in mouse embryos and embryonic stem cells. , 2003, Developmental biology.

[35]  G. Daley,et al.  Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos , 2008, Nature Genetics.

[36]  H. Kato,et al.  G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. , 2002, Genes & development.

[37]  M. Boiani,et al.  Production of cloned mice by somatic cellnuclear transfer , 2006, Nature Protocols.

[38]  J. Rossant,et al.  Promotion of trophoblast stem cell proliferation by FGF4. , 1998, Science.

[39]  H. Schöler,et al.  Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. , 1994, Developmental biology.

[40]  H. Schöler,et al.  The Caudal‐Related Protein Cdx2 Promotes Trophoblast Differentiation of Mouse Embryonic Stem Cells , 2006, Stem cells.

[41]  Sheng Zhong,et al.  A core Klf circuitry regulates self-renewal of embryonic stem cells , 2008, Nature Cell Biology.

[42]  Hong Lei,et al.  Histone H3-K9 Methyltransferase ESET Is Essential for Early Development , 2004, Molecular and Cellular Biology.

[43]  J. W. Pendleton,et al.  Homeobox genes in mouse development. , 1991, Critical reviews in eukaryotic gene expression.

[44]  Soumen Paul,et al.  GATA3 Is Selectively Expressed in the Trophectoderm of Peri-implantation Embryo and Directly Regulates Cdx2 Gene Expression* , 2009, The Journal of Biological Chemistry.

[45]  Yi Zhang,et al.  mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression. , 2003, Molecular cell.

[46]  Tsutomu Ohta,et al.  Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. , 2005, Genes & development.

[47]  Allen D. Delaney,et al.  Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing , 2007, Nature Methods.

[48]  Hans R. Schöler,et al.  New type of POU domain in germ line-specific protein Oct-4 , 1990, Nature.

[49]  T. Mikkelsen,et al.  Genome-wide maps of chromatin state in pluripotent and lineage-committed cells , 2007, Nature.

[50]  Megan F. Cole,et al.  Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells , 2005, Cell.

[51]  Yi Zhang,et al.  Molecular cloning of ESET, a novel histone H3-specific methyltransferase that interacts with ERG transcription factor , 2002, Oncogene.

[52]  G. Maul,et al.  Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation. , 2003, Genes & development.

[53]  A. Mortazavi,et al.  Genome-Wide Mapping of in Vivo Protein-DNA Interactions , 2007, Science.

[54]  Irving L. Weissman,et al.  Association of reactive oxygen species levels and radioresistance in cancer stem cells , 2009, Nature.

[55]  N. Gray,et al.  Deletion of the Pluripotency-Associated Tex19.1 Gene Causes Activation of Endogenous Retroviruses and Defective Spermatogenesis in Mice , 2008, PLoS genetics.

[56]  J. Rossant.  Lineage development and polar asymmetries in the peri-implantation mouse blastocyst. , 2004, Seminars in cell & developmental biology.