Detection of new genomic control elements is critical in understanding transcriptional regulatory networks in their entirety. We studied the genome-wide binding locations of three key regulatory proteins (POU5F1, also known as OCT4; NANOG; and CTCF) in human and mouse embryonic stem cells. In contrast to CTCF, we found that the binding profiles of OCT4 and NANOG are markedly different, with only approximately 5% of the regions being homologously occupied. We show that transposable elements contributed up to 25% of the bound sites in humans and mice and have wired new genes into the core regulatory network of embryonic stem cells. These data indicate that species-specific transposable elements have substantially altered the transcriptional circuitry of pluripotent stem cells.

Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. Yet, little is known about CTCF-associated higher order chromatin structures at a global scale. Here, we applied Chromatin Interaction Analysis by Paired-End-Tag sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, 1,480 cis and 336 trans interacting loci were identified with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive crosstalk between promoters and regulatory elements. This highly complex nuclear organization offers insights towards the unifying principles governing genome plasticity and function.

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis–antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis–antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis–antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis–antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis–antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis–antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.

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.

The transcription factor Zic3 is required for maintenance of ESC pluripotency. By genome-wide chromatin immunoprecipitation (ChIP-chip) in ESCs, we have identified 379 direct Zic3 targets, many of which are functionally associated with pluripotency, cell cycle, proliferation, oncogenesis, and early embryogenesis. Through a computational analysis of Zic3 target sequences, we have identified a novel Zic3 consensus binding motif (5 0 -CC C / T GCTGGG-3 0 ). ChIP results and in vitro DNA binding assays revealed that Zic3 binds with high affinity and specificity on the Nanog promoter. Here, we demonstrate that Zic3 functions as a transcriptional activator of the Nanog promoter in three ways:(a) Nanog transcript levels are sustained with Zic3 overexpression in differentiating ESCs, (b) Zic3 depletion in ESCs downregulates Nanog promoter activity, and (c) Zic3 overexpression leads to increased Nanog promoter activity. Furthermore, the activity of a mutant Nanog promoter with ablated Oct4/Sox2 binding is rescued by Zic3 overexpression to nearly wild-type levels. This indicates that Nanog is a positive transcriptional target of Zic3 in a mechanism that is independent of Oct4/Sox2 binding. Hence, we demonstrate an important pathway for regulation of Nanog expression in pluripotent ESCs through direct activation by Zic3. STEM CELLS

Zfp206(recently renamed Zscan10) encodes a zinc finger transcription factor specifically expressed in human and mouse embryonic stem cells (ESC). It has been shown that Zfp206 is required to maintain ESC in an undifferentiated, pluripotent state. Presented here are data showing that Zfp206 works together with two other transcription factors, Oct4 and Sox2, which are also essential regulators of ESC pluripotency. We show that Zfp206 binds to the Oct4 promoter and directly regulates Oct4 expression. Genome-wide mapping of Zfp206-binding sites in ESC identifies more than 3000 target genes, many of which encode transcription factors that are also targeted for regulation by Oct4 and Sox2. In addition, we show that Zfp206 physically interacts with both Oct4 and Sox2. These data demonstrate that Zfp206 is a key component of the core transcriptional regulatory network and together with Oct4 and Sox2 regulates differentiation of ESC.Pluripotency, the potential to give rise to all lineages of the developing embryo, is a unique and defining characteristic of mammalian embryonic stem cells (ESC).2 Pluripotent ESC, like the inner cell mass of the embryo from which they were derived, exist in a developmental state that is poised to respond to extracellular signals that specify unique patterns of cellular differentiation. ESC responding to extrinsic cues must undergo transitions from a self-renewing and pluripotent state to one of many alternative states of differentiation. Early genomics approaches have revealed transcriptional regulatory networks that are responsible for maintaining ESC pluripotency (1, 2). Two essential regulators of pluripotency are the transcription factors (TF) Oct4 and Sox2. Knockdown of these transcription factors results in loss of ESC pluripotency and induction of nonspecific differentiation (3). The importance of Oct4 and Sox2 in pluripotency is underscored by their ability to reprogram differentiated fibroblasts into induced pluripotent stemlike cells (4). More recently, thousands of direct target genes regulated by Oct4 and Sox2 have been identified through comprehensive, genome-wide chromatin immunoprecipitation studies (5, 6). Many of the Oct4/Sox2 targets are genes encoding other transcriptional regulators, including several that also have been found to play a role in regulating pluripotency, such as Nanog, Esrrb, Tcf3, Tcl1, Zfp281, Zic3, and Sall4 (3,7,[8][9][10][11][12].Zfp206 is another transcription factor that is specifically expressed in ESC (13,14) and is directly regulated by Oct4 and Sox2 (15). Zfp206 was implicated as a pluripotency factor because it was found highly expressed in undifferentiated ESC and the inner cell mass of the preimplantation embryo, but not in differentiated ESC or trophectoderm. Knockdown of Zfp206 expression induces ESC differentiation, whereas its sustained overexpression impedes retinoic acid induced differentiation of ESC, thus establishing that Zfp206 is a regulator of pluripotency (13). Zfp206 encodes a protein that contains 14 zinc fingers, although alte...

The differentiation of pluripotent embryonic stem cells is regulated by networks of activating and repressing transcription factors that orchestrate determinate patterns of gene expression. With the recent mapping of target sites for many transcription factors, it has been a conundrum that so few of the genes directly targeted by these factors are transcriptionally responsive to the binding of that factor. To address this, we generated genome-wide maps of the transcriptional repressor REST and five of its corepressors in mouse embryonic stem cells. Combining these binding-site maps with comprehensive gene-expression profiling, we show that REST is functionally heterogeneous. Approximately half of its binding sites apparently are nonfunctional, having weaker binding of REST and low recruitment of corepressors. In contrast, the other sites strongly recruit REST and corepressor complexes with varying numbers of components. Strikingly, the latter sites account for almost all observed gene regulation. These data support a model where productive gene repression by REST requires assembly of a multimeric ''repressosome'' complex, whereas weak recruitment of REST and its cofactors is insufficient to repress gene expression.