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...

Zfp206 (also named as Zscan10) belongs to the subfamily of C 2 H 2 zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 Å crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN–Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN–Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C 2 H 2 transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development.