The underlying epigenetic mechanisms that govern cell-fate determination and pluripotency during early embryonic development are poorly understood. The long-term goal is to better understand the chromatin-based mechanisms that regulate gene expression changes between days 2.5 (8-cell) to 6.5 (pre-gastrulation) of mouse embryonic development. These stages of development in a mouse model correspond to the stages of development in humans when a large percentage of embryos are lost to either failed implantation or early miscarriage. The overall objective of this application is to investigate the specific role of the Brg1 chromatin-remodeling protein in trophectoderm development and pluripotency. Brg1 is a critical chromatin-remodeling enzyme that is implicated in human cancer and mammalian development. Brg1-deficient embryos arrest around the blastocyst stage, undergo perturbations in gene expression, and exhibit defects in the ICM and trophectoderm. Moreover, disruption of Brg1 function in embryonic stem (ES) cells results in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes, and upregulation of differentiation genes.
The specific aims of this application are to test the hypotheses that: 1) Brg1 is required for epigenetic silencing of Oct4 and Nanog expression in the trophectoderm. 2) Brg1 and Nanog promote embryonic pluripotency through epigenetic regulation of Oct4 and Klf5. To address these questions a battery of molecular, cellular, and biochemical assays will be performed in vitro and in vivo. These will include microinjection of siRNAs and mRNAs, immunocytochemistry, chromatin immunoprecipitation (ChIP) assays, chromatin- remodeling assays, and real-time qPCR analysis. The proposed studies will enhance our basic knowledge of early mammalian development. Moreover, these studies are relevant to clinical infertility, human oncology, and stem cell differentiation for cell replacement therapies.
In the long-term the proposed studies will lead to better methodologies for selecting high quality embryos and/or protocols for augmenting embryo viability (eg, optimization of embryo culture protocols). Enhanced embryo viability in assisted eproduction technologies (ART) will lead to reduced numbers of embryos transferred per cycle and correspondingly reduced multiple pregnancy rates;and furthermore, proposed studies on the role of Brg1 in early embryos and ES cells have direct implications in human oncology and ES cell differentiation for cell replacement therapies. Collectively, these studies are highly relevant to the mission of the NICHD.
|Sengupta, Satyaki; Lingnurkar, Raj; Carey, Timothy S et al. (2015) The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency. J Biol Chem 290:14462-75|
|Carey, Timothy S; Cao, Zubing; Choi, Inchul et al. (2015) BRG1 Governs Nanog Transcription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation. Mol Cell Biol 35:4158-69|
|Cao, Zubing; Carey, Timothy S; Ganguly, Avishek et al. (2015) Transcription factor AP-2? induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage. Development 142:1606-15|
|Paul, Soumen; Knott, Jason G (2014) Epigenetic control of cell fate in mouse blastocysts: the role of covalent histone modifications and chromatin remodeling. Mol Reprod Dev 81:171-82|
|Carey, Timothy S; Choi, Inchul; Wilson, Catherine A et al. (2014) Transcriptional reprogramming and chromatin remodeling accompanies Oct4 and Nanog silencing in mouse trophoblast lineage. Stem Cells Dev 23:219-29|
|Knott, Jason G; Paul, Soumen (2014) Transcriptional regulators of the trophoblast lineage in mammals with hemochorial placentation. Reproduction 148:R121-36|
|Choi, Inchul; Carey, Timothy S; Wilson, Catherine A et al. (2013) Evidence that transcription factor AP-2? is not required for Oct4 repression in mouse blastocysts. PLoS One 8:e65771|
|Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V R et al. (2013) Trophoblast lineage cells derived from human induced pluripotent stem cells. Biochem Biophys Res Commun 436:677-84|
|Choi, Inchul; Carey, Timothy S; Wilson, Catherine A et al. (2012) Transcription factor AP-2? is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis. Development 139:4623-32|
|Luo, Jiesi; Suhr, Steven T; Chang, Eun Ah et al. (2011) Generation of leukemia inhibitory factor and basic fibroblast growth factor-dependent induced pluripotent stem cells from canine adult somatic cells. Stem Cells Dev 20:1669-78|
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