Identification of genes regulated by SOX17 during mammalian endoderm differentiation

Embryonic stem (ES) cells have the capacity to produce all cell types of the adult organism. A major goal of ES cell research is to differentiate them to specific cell types at high efficiency. Hepatocytes differentiated from ES cells could be used for cellular therapies or in vitro drug toxicity studies. Endoderm adjacent to the developing heart is induced to differentiate to hepatocytes by growth factors secreted from the cardiogenic mesoderm and the septum transversum mesenchyme. We cultured ES cells with these growth factors to direct them to a hepatocyte fate. After 18 days of differentiation, cultures expressed terminally differentiated hepatocyte markers. Using a dye that is selectively metabolized by hepatocytes, we observed that hepatocyte differentiation only occurs adjacent to cardiac contractile regions as occurs during normal development. We attempted to increase the efficiency of hepatocyte differentiation by increasing the amount of endoderm produced. However, specific markers of endoderm were lacking and we were limited to using Sox17 expression to assess endoderm formation in our cultures.;SOX17 is a transcription factor necessary for endoderm formation in multiple species. We used chromatin immunoprecipitation to generate markers of endoderm by identifying targets of SOX17 during endoderm differentiation. We identified novel regions regulated by SOX17 including a region in the first intron of zinc finger protein 202 (Zfp202), a region 3.5kb upstream of Tdgf1 (Cripto) and a region in the fourth intron of Wnt3. Further analysis of these regions demonstrated SOX17 binding to sites in these regions.;The region associated with Zfp202 activates transcription in heterologous promoter assays. This activation is dependent on a functional SOX17 site. Furthermore, Sox17 and Zfp202 co-expression in mouse embryos in the anterior foregut endoderm is consistent with SOX17 activation of Zfp202.;Two SOX17 sites within the region upstream of Cripto and one in the last intron of Wnt3 are involved in transcriptional repression. Sox17 expression did not overlap with Cripto or Wnt3 expression during normal development. This identifies a previously unknown role for SOX17 in transcriptional repression. This is the first unbiased identification of SOX17 binding regions which may further elucidate the molecular mechanisms of endoderm differentiation.