Regulation of Gene Expression in Embryogenesis

Embryonic stem cells (ES cells) represent the earliest cell type of the mammalian embryo. ES cells can be maintained in culture and display the unique properties of self-renewal and pluripotency i.e., they have the ability to differentiate into many different cell types when exposed to specific signals. Understanding the molecular basis that gives rise to these properties has become an area of intense focus, particularly efforts to elucidate the transcriptional circuitry that defines the “stem cell state”. One factor that is essential for the viability of the early embryo is Fibroblast Growth Factor 4 (FGF4). Several years ago, we determined that FGF4 is expressed in ES cells and identified a specific enhancer that activates FGF4 gene transcription. Through detailed biochemical analyses, we showed that the FGF4 enhancer binds the ES cell-specific transcription factors Oct4 and Sox2 and directs the assembly of a ternary complex composed of these two factors on the enhancer DNA. In further studies, we also showed that active Sox2/Oct4 complex assembly results from protein-protein interactions between the DNA binding domains of each factor, but also requires a specific arrangement of the DNA binding sites as provided in the FGF4 enhancer DNA sequence.

Given that Sox2 and Oct4 are both required for enhancer activity, and that they are only co-expressed in ES cells and the early embryo (where FGF4 is also expressed), we had predicted that the Sox2/Oct4 complex represents a “combinatorial code” for the transcriptional activation of a subset of genes specifically in the early embryo. Accordingly, several laboratories have since demonstrated that transcription of the genes for the ES cell-specific factors UTF-1, Sox2, Oct4, and Nanog are all activated by the Oct4/Sox2 complex in ES cells and the early embryo. Nanog is of particular interest since it is itself a transcription factor and has been shown to be essential for maintaining the self-renewal properties of mouse ES cells. These and other studies have firmly established the Sox2/Oct4 complex as a “master regulator” defining the Embryonic Stem Cell state. However, genetic studies also indicate that Sox2 and Oct4 each must activate additional sets of genes independently of each other, presumably in conjunction with other protein partners. Furthermore, genes activated by Nanog are presently unknown, suggesting that there are additional important ES cell-specific regulatory complexes yet to be discovered.

Our current efforts are directed toward developing a novel, high-throughput method to functionally isolate enhancer elements that activate transcription specifically in ES cells. The large-scale identification and analysis of these enhancers will enable us to gain new insight into the DNA sequences and transcription factors directing gene expression in ES cells, and will provide a global view of their transcriptional circuitry. Through these studies we hope to reveal new Oct4 or Sox2 partnerships, Nanog target genes, or a role for novel transcription factor combinations in maintaining the stem cell state.