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ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage.

ABSTRACT: Pluripotency, the capacity for indefinite self-renewal and differentiation into diverse cell types is a unique state exhibited by embryonic stem (ES) cells. Transcriptional regulators, such as Oct4, are critical for pluripotency, but the role of epigenetic modifiers remains to be fully elucidated.Here, we show that ERG-associated protein with SET domain (ESET), a histone methyltransferase enzyme, maintains pluripotency through repression of Cdx2, a key trophectoderm determinant, by histone H3 lysine 9 trimethylation (H3K9me3) of the promoter region. Notably, this repression is mediated through the synergistic function of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4. ESET localises to the promyelocytic leukaemia (PML) nuclear bodies and is SUMOylated in ES cells. Interaction of ESET with Oct4 depends on a SUMO-interacting motif (SIM) in Oct4, which is critical for the repression of Cdx2.Loss of ESET or Oct4 results in strikingly similar phenotypes both in ES cells with their differentiation into trophectoderm cells, and in early embryos where there is a failure of development of the pluripotent inner cell mass (ICM) of blastocysts. We propose that SUMOylated ESET-Oct4 complex is critical for both the initiation and maintenance of pluripotency through repression of differentiation, particularly of the trophectoderm lineage by epigenetic silencing of Cdx2.

SUBMITTER: Yeap LS

PROVIDER: S-EPMC2763847 | biostudies-other | 2009 Oct

REPOSITORIES: biostudies-other

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ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage.

Yeap Leng-Siew LS Hayashi Katsuhiko K Surani M Azim MA

Epigenetics & chromatin 20091007 1

<h4>Background</h4>Pluripotency, the capacity for indefinite self-renewal and differentiation into diverse cell types is a unique state exhibited by embryonic stem (ES) cells. Transcriptional regulators, such as Oct4, are critical for pluripotency, but the role of epigenetic modifiers remains to be fully elucidated.<h4>Results</h4>Here, we show that ERG-associated protein with SET domain (ESET), a histone methyltransferase enzyme, maintains pluripotency through repression of Cdx2, a key trophect ...[more]

PMID: 19811652

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Project description:ObjectivesInsulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development.MethodsTo explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs.ResultsInterestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ?33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Ppar?, Fabp4, C/ebp?, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development.ConclusionWe report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.

Project description:Hepatoma-derived growth factor (HDGF) is a nuclear protein with both mitogenic and angiogenic activity that is highly expressed in the developing heart and vasculature. To date, the mechanism underlying the function of HDGF is unknown. Oligonucleotide microarray analysis was used to gain insights into HDGF function. Adenoviral expression of HDGF significantly (> or =2-fold) downregulated a large group (66) of genes, and increased expression of a relatively small number of genes (9). Two groups of target genes that are involved in cardiovascular development and transcriptional regulation, including the skeletal/cardiac muscle specific SET and MYND domain containing 1 (SMYD1) gene, were validated by real time PCR. This suggested that HDGF could function as a transcriptional repressor. In a one-hybrid system, GBD-HDGF significantly repressed reporter gene activity in a dose-dependent manner. This demonstrated that HDGF has transcriptional repressive activity. Moreover, in G-7 myoblast cells, over-expression of a GFP-HDGF fusion specifically downregulated SMYD1 mRNA expression and the activity of the human SMYD1 promoter. HDGF repressed SMYD1 gene transcription through interaction with a transcriptional corepressor C-terminal binding protein (CtBP). Over-expression of CtBP potentiated the trans-repressive activity of HDGF; on the other hand, knocking down CtBP attenuated the trans-repressive effect of HDGF. HDGF binds CtBP through a non-canonical binding motif (PKDLF) within the PWWP domain, as mutation of DL to AS abolished HDGF and CtBP interaction, and diminished the trans-repressive effect of HDGF without affecting DNA binding. Finally, fluorescent microscopy showed that HDGF induced the nuclear accumulation of CtBP, suggesting that HDGF forms a transcriptional complex with CtBP. Taken together, our data demonstrate that HDGF functions as a transcriptional repressor of the SMYD1 gene through interaction with the transcriptional corepressor CtBP. Because of moderate conservation of the CtBP binding motif in HDGF family members, trans-repressive activity mediated by CtBP may be a common function among HDGF proteins.

Dataset Information

ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage.

Publications

ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage.

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