The maintenance of pluripotency and specification of cellular lineages during embryonic development are controlled by transcriptional regulatory networks, which coordinate specific sets of genes through both activation and repression. The transcriptional repressor RE1-silencing transcription factor (REST) plays important but distinct regulatory roles in embryonic (ESC) and neural (NSC) stem cells. We investigated how these distinct biological roles are effected at a genomic level. We present integrated, comparative genome- and transcriptome-wide analyses of transcriptional networks governed by REST in mouse ESC and NSC. The REST recruitment profile has dual components: a developmentally independent core that is common to ESC, NSC, and differentiated cells; and a large, ESC-specific set of target genes. In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog. We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype.

Oct4, Sox2, and Nanog are key components of a core transcriptional regulatory network that controls the ability of embryonic stem cells to differentiate into all cell types. Here we show that Zfp281, a zinc finger transcription factor, is a key component of the network and that it is required to maintain pluripotency. Zfp281 was shown to directly activate Nanog expression by binding to a site in the promoter in very close proximity to the Oct4 and Sox2 binding sites. We present data showing that Zfp281 physically interacts with Oct4, Sox2, and Nanog. Chromatin immunoprecipitation experiments identified 2,417 genes that are direct targets for regulation by Zfp281, including several transcription factors that are known regulators of pluripotency, such as Oct4, Sox2, and Nanog. Gene expression microarray analysis indicated that some Zfp281 target genes were activated, whereas others were repressed, upon knockdown of Zfp281. The identification of both activation and repression domains within Zfp281 suggests that this transcription factor plays bifunctional roles in regulating gene expression within the network.

Zfp206 (ZNF206 in human) encodes a zinc finger-and SCAN domain-containing protein that is highly expressed in pluripotent ESC. Upon differentiation of human and mouse ESC, Zfp206 expression is quickly repressed. Zfp206 was found to be expressed throughout embryogenesis but absent in adult tissues except testis. We have identified a role for Zfp206 in controlling ESC differentiation. ESC engineered to overexpress Zfp206 were found to be resistant to differentiation induced by retinoic acid. In addition, ESC with knocked-down expression of Zfp206 were more sensitive to differentiation by retinoic acid treatment. We found that Zfp206 was able to enhance expression from its own promoter and also activate transcription of the Oct4 and Nanog promoters. Our results show that Zfp206 is an embryonic transcription factor that plays a role in regulating pluripotency of embryonic stem cells.

It is well known that Oct4 and Sox2 play an important role in the maintenance of embryonic stem cell pluripotency. These transcription factors bind to regulatory regions within hundreds of target genes to control their expression. Zfp206 is a recently characterized transcription factor that has a role in maintaining stem cell pluripotency. We have demonstrated here that Zfp206 is a direct downstream target of Oct4 and Sox2. Two composite sox-oct binding sites have been identified within the first intron of Zfp206. We have demonstrated binding of Oct4 and Sox2 to this region. In addition, we have shown that Oct4 or Sox2 alone can activate transcription via one of these sox-oct elements, although the presence of both Oct4 and Sox2 gave rise to a synergistic effect. These studies extend our understanding of the transcriptional network that operates to regulate the differentiation potential of embryonic stem cells. Embryonic stem cells (ESCs)2 are derived from the inner cell mass of the blastocyst and exhibit both pluripotency and selfrenewing capabilities. For proper developmental outcome, ESCs must tightly regulate their differentiation status, and through continuing study, the molecular basis of that regulation process is beginning to emerge. Systematic, genome-wide interrogations have identified hundreds of genes, including several transcription factors, which have expression patterns tightly correlated with ES cell differentiation (1-6). Two key transcription factors, Oct4 and Sox2, have been identified that are crucial for maintenance of the pluripotent state of ESCs (7,8). ESCs lose the capacity to maintain pluripotency upon knockdown of expression of these transcription factors by RNA interference (9, 10). Gene knock-out studies confirm the importance of Oct4 and Sox2 for early embryonic development. It has been demonstrated by chromatin immunoprecipitation studies that Oct4 and Sox2 bind to a few thousand regulatory sites in the ES cell genome (11,12). It is likely that many of these target genes play a role in modulating ES cell differentiation. Indeed, the transcription factor Nanog, an established regulator of pluripotency, is transcriptionally regulated directly by Oct4 and Sox2 (13).Zfp206 is a transcription factor that is highly expressed in mouse and human ESCs and down-regulated upon differentiation (3, 14). Zfp206 contains a SCAN domain and 14 zinc-finger domains, which suggests that it may be a transcription factor that binds DNA directly. Zfp206 is expressed in the inner cell mass but not in trophectoderm, suggesting that it may play a role in establishing cell fate decisions regarding embryonic versus extraembryonic tissue (15). There is wide temporal and spatial distribution of RNA and protein in the early embryos, indicating that Zfp206 may regulate multiple cell fate decisions (14). Recent data have demonstrated that overexpression of Zfp206 promotes the formation of undifferentiated mouse ESC colonies in vitro (14). We have obtained similar results and further found that overexpression of Zf...