The goal of this study is to broaden our understanding of a gene regulatory network that plays key roles during embryogenesis. Recent studies argue strongly that the transcription factors Sox2 and Oct-3/4 each exhibit important hallmarks of master regulators that orchestrate mammalian embryogenesis and the self-renewal of embryonic stem (ES) cells. Sox2 and Oct-3/4 have been shown to work together cooperatively to coordinate the transcription of at least 6 genes (FGF-4, UTF1, Fbx15, Nanog, Sox2 and Oct-3/4) expressed during embryogenesis and by ES cells. These genes are referred to as Sox2:Oct-3/4 target genes. The work proposed here is based on 3 novel findings and hypotheses. First, we recently identified 19 additional genes that are likely to be part of the Sox2:Oct-3/4 gene regulatory network. Given that 4, and possibly 5, of the 6 known Sox2:Oct-3/4 target genes are essential for embryogenesis it is our central hypothesis that the majority of Sox2:Oct-3/4 target genes play critical roles during embryogenesis, and that many are required for the self-renewal of ES cells. Second, the expression of the 6 known Sox2:Oct-3/4 target genes was shown to be very heavily dependent on closely spaced DNA binding sites for Sox2 and Oct-3/4 in the enhancer of each gene. These and other studies led us to hypothesize that the Sox2:Oct-3/4 gene regulatory network is controlled coordinately by a shared set of co-activators, at least some of which are themselves regulated during differentiation and, thus, are key players in cell fate determination. Third, although Sox2, in combination with Oct-3/4, is known to stimulate the expression of Sox2:Oct-3/4 target genes in stem cells, we determined that at least 5 of the 6 Sox2:Oct-3/4 target genes, including the Sox2 gene itself, are inhibited when Sox2 is overexpressed. These findings led to our hypothesis that Sox2:Oct-3/4 target genes are regulated carefully by a negative, as well as a positive, feedback loop to ensure that these genes are expressed at levels required for embryogenesis.
Three Specific Aims are proposed to test these hypotheses. 1) Examine a newly identified set of putative Sox2:Oct-3/4 target genes for their effects on the self-renewal of ES cells and mammalian development. 2) Examine the roles of specific co-activators in the expression of Sox2:Oct-3/4 target genes. 3) Determine the molecular mechanisms by which Sox2 overexpression inhibits the expression of Sox2:Oct-3/4 target genes. Together, these studies will provide a mechanistic understanding of a critical gene regulatory network that orchestrates embryogenesis and controls the self-renewal of ES cells. As such, these studies will provide novel insights and have significant impact on developmental biology, regenerative medicine and cancer biology.

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National Institute of General Medical Sciences (NIGMS)
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Development - 2 Study Section (DEV2)
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Haynes, Susan R
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University of Nebraska Medical Center
Internal Medicine/Medicine
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Rizzino, Angie (2013) Concise review: The Sox2-Oct4 connection: critical players in a much larger interdependent network integrated at multiple levels. Stem Cells 31:1033-9
Ormsbee Golden, Briana D; Wuebben, Erin L; Rizzino, Angie (2013) Sox2 expression is regulated by a negative feedback loop in embryonic stem cells that involves AKT signaling and FoxO1. PLoS One 8:e76345
Wuebben, Erin L; Mallanna, Sunil K; Cox, Jesse L et al. (2012) Musashi2 is required for the self-renewal and pluripotency of embryonic stem cells. PLoS One 7:e34827
Mallanna, Sunil K; Rizzino, Angie (2012) Systems biology provides new insights into the molecular mechanisms that control the fate of embryonic stem cells. J Cell Physiol 227:27-34
Cox, Jesse L; Mallanna, Sunil K; Ormsbee, Briana D et al. (2011) Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells. J Cell Sci 124:2654-65
Chakravarthy, Harini; Ormsbee, Briana D; Mallanna, Sunil K et al. (2011) Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery. FASEB J 25:206-18
Ji, Ming; Rao, Enyu; Ramachandrareddy, Himabindu et al. (2011) The miR-17-92 microRNA cluster is regulated by multiple mechanisms in B-cell malignancies. Am J Pathol 179:1645-56
Mallanna, Sunil K; Rizzino, Angie (2010) Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells. Dev Biol 344:16-25
Mallanna, Sunil K; Ormsbee, Briana D; Iacovino, Michelina et al. (2010) Proteomic analysis of Sox2-associated proteins during early stages of mouse embryonic stem cell differentiation identifies Sox21 as a novel regulator of stem cell fate. Stem Cells 28:1715-27
Cox, Jesse L; Rizzino, Angie (2010) Induced pluripotent stem cells: what lies beyond the paradigm shift. Exp Biol Med (Maywood) 235:148-58

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