Abstract
Nanog, Sox2, and Oct4 are transcription factors all essential to maintaining the pluripotent embryonic stem cell phenotype. Through a cooperative interaction, Sox2 and Oct4 have previously been described to drive pluripotent-specific expression of a number of genes. We now extend the list of Sox2-Oct4 target genes to include Nanog. Within the Nanog proximal promoter, we identify a composite sox-oct cis-regulatory element essential for Nanog pluripotent transcription. This element is conserved over 250 million years of cumulative evolution within the eutherian mammals. A Nanog proximal promoter-EGFP (enhanced green fluorescent protein) reporter transgene recapitulates endogenous Nanog mRNA expression in embryonic stem cells and their differentiated derivatives. Sox2 and Oct4 interaction with the Nanog promoter was confirmed through mutagenesis and in vitro binding assays. Electrophoretic mobility shift assays indicate that the Sox2-Oct4 heterodimer forms more efficiently on the composite element within Nanog than the similar element within Fgf4. Using chromatin immunoprecipitation, we show that Oct4 and Sox2 bind to the Nanog promoter in living mouse and human embryonic stem cells. Furthermore, by specific knockdown of Oct4 and Sox2 mRNA by RNA interference in embryonic stem cells, we provide genetic evidence for a link between Oct4, Sox2, and the Nanog promoter. These studies extend the understanding of the pluripotent genetic regulatory network within which the Sox2-Oct4 complex are at the top of the regulatory hierarchy.
Highlights
Nanog is a homeobox-containing transcription factor with an essential function in maintaining the pluripotent cells of the inner cell mass and in the derivation of embryonic stem cells (ESCs)1 from these [1]
Sequence Comparisons Identify Conserved Non-coding Sequences—As Nanog is expressed in both mouse and human pluripotent cells, we reasoned the pluripotent transcription of this gene is maintained through the functional conservation of cis-regulatory elements, and concomitantly, the location and sequence of these elements would be conserved through purifying selection
We have characterized the proximal promoter of Nanog and established that the region from Ϫ289 to ϩ117 relative to the transcription start site contains sufficient cis-regulatory information to recapitulate endogenous Nanog expression, at least as seen in undifferentiated ESCs and their differentiated derivatives
Summary
Nanog is a homeobox-containing transcription factor with an essential function in maintaining the pluripotent cells of the inner cell mass and in the derivation of embryonic stem cells (ESCs) from these [1]. Overexpression of Nanog is capable of maintaining the pluripotency and self-renewing characteristics of ESCs under what normally would be differentiation-inducing culture conditions [2] Concomitant with this essential function in pluripotent cell maintenance is its restricted expression pattern. The POU domain-containing Oct and the HMG domaincontaining Sox are two other transcription factors known to be essential for normal pluripotent cell development and maintenance [5, 6] Both have independent roles in determining other cell types [6, 7], at least part of their function in pluripotent cells is via a synergistic interaction between the two to drive transcription of target genes. We describe a cis-regulatory module conserved throughout the eutherian mammals that is capable of recapitulating endogenous Nanog expression; at the core of this module is a composite oct-sox element necessary for pluripotent expression
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