OCT3/4 Regulates Transcription of Histone Deacetylase 4 (Hdac4) in Mouse Embryonic Stem Cells Russell C. Addis, 1 Megana K. Prasad, 2 Robert L. Yochem, 3 Xiangcan Zhan, 3 Timothy P. Sheets, 3 Joyce Axelman, 3 Ethan S. Patterson, 4 and Michael J. Shamblott 3 * 1 Department of Cell and Developmental Biology, and Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 2 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 3 Department of Neurology, Johns Hopkins University School of Medicine and Hugo W. Moser Research Institute at the Kennedy Krieger Institute, Baltimore, Maryland 4 Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri ABSTRACT OCT3/4 is a POU domain transcription factor that is critical for maintenance of pluripotency and self-renewal by embryonic stem (ES) cells and cells of the early mammalian embryo. It has been demonstrated to bind and regulate a number of genes, often in conjunction with the transcription factors SOX2 and NANOG. In an effort to further understand this regulatory network, chromatin immunoprecipitation was used to prepare a library of DNA segments specifically bound by OCT3/4 in undifferentiated mouse ES (mES) cell chromatin. One segment corresponds to a region within the first intron of the gene encoding histone deacetylase 4 (Hdac4), a Class II histone deacetylase. This region acts as a transcriptional repressor and contains at least two functional sites that are specifically bound by OCT3/4. HDAC4 is not expressed in the nuclei of OCT3/4þ mES cells and is upregulated upon differentiation. These findings demonstrate the participation of OCT3/4 in the repression of Hdac4 in ES cells. J. Cell. Biochem. 111: 391–401, 2010. ß 2010 Wiley-Liss, Inc. KEY WORDS: EMBRYONIC STEM CELLS; OCT3/4; HDAC4; PLURIPOTENCY; TRANSCRIPTIONAL REGULATION E mbryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. ES cells and cells with ES-like properties have been derived from a number of mammalian species including mouse [Evans and Kaufman, 1981; Martin, 1981] and human [Thomson et al., 1998]. ES cells are capable of indefinite self-renewal in vitro and can differentiate into a wide variety of cell and tissue types, making them important tools for the study of development, cell differentiation, and gene function. Traditionally, mouse ES cells (mES) have been classified as pluripotent due to their ability, in chimeric animals, to form all tissues of the adult but failure to produce extraembryonic cell types of the trophectoderm and primitive endoderm lineages [Beddington and Robertson, 1989; Rossant, 2007]. This broad capacity to differentiate is also demonstrated by formation of embryoid bodies (EBs) in vitro and teratomas following transplantation into syngeneic or immunocompromised mice. The capacity of ES cells to participate so broadly in the differentiation of almost all of the specialized cell types of the body and retain indefinite capacity for self-renewal is both remarkable and rare. The list of cell types that share most of these properties includes embryonal carcinoma cells [Andrews et al., 1982; Andrews, 1984], embryonic germ cells [Resnick et al., 1992; Labosky et al., 1994; Shamblott et al., 1998] and, most recently, induced pluripotent stem (iPS) cells [Takahashi and Yamanaka, 2006; Okita et al., 2007; Yu et al., 2007]. Gene knockout studies have pointed to three factors, OCT3/4 [Nichols et al., 1998], SOX2 [Avilion et al., 2003], and NANOG [Chambers et al., 2003; Mitsui et al., 2003], that play essential roles in maintaining pluripotency in mouse embryos and ES cells. These factors have been shown to co-occupy promoter regions of hundreds of genes and to collectively maintain and regulate ES cell pluripotency [Boyer et al., 2005; Loh et al., 2006]. Identification of genes bound by OCT3/4 is a highly specific method to further understand how pluripotency is established and maintained in ES cells. OCT3/4 binding sites are often located in close proximity to SOX2 binding sites, but this is not always the case [Pesce and Scholer, 2001] and SOX2 is expressed in other tissues, frequently associated with stem or progenitor populations [Zappone et al., 2000; Muta et al., 2002; Ferri et al., 2004]. NANOG is specifically Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 111:391–401 (2010) 391 *Correspondence to: Michael J. Shamblott, 707 North Broadway, Suite 518, Baltimore, MD 21205. E-mail: shamblott@kennedykrieger.org Received 28 January 2010; Accepted 12 May 2010  DOI 10.1002/jcb.22707  ß 2010 Wiley-Liss, Inc. Published online 19 May 2010 in Wiley Online Library (wileyonlinelibrary.com).