Abstract
The transcription factor FoxA2 is a master regulator of endoderm development and pancreatic beta cell gene expression. To elucidate the mechanisms underlying the activation of the FoxA2 gene during differentiation, we have compared the epigenetic status of undifferentiated human embryonic stem cells (hESCs), hESC-derived early endoderm stage cells (CXCR4+ cells), and pancreatic islet cells. Unexpectedly, a CpG island in the promoter region of the FoxA2 gene displayed paradoxically high levels of DNA methylation in expressing tissues (CXCR4+, islets) and low levels in nonexpressing tissues. This CpG island region was found to repress reporter gene expression and bind the Polycomb group protein SUZ12 and the DNA methyltransferase (DNMT)3b preferentially in undifferentiated hESCs as compared with CXCR4+ or islets cells. Consistent with this, activation of FoxA2 gene expression, but not CXCR4 or SOX17, was strongly inhibited by 5-aza-2'-deoxycytidine and by knockdown of DNMT3b. We hypothesize that in nonexpressing tissues, the lack of DNA methylation allows the binding of DNA methyltransferases and repressing proteins, such as Polycomb group proteins; upon differentiation, DNMT activation leads to CpG island methylation, causing loss of repressor protein binding. These results suggest a novel and unexpected role for DNA methylation in the activation of FoxA2 gene expression during differentiation.
Highlights
The transcription factor FoxA2 is a key mediator of endoderm development and pancreas gene expression
Analysis of CpG2a revealed a paradoxical pattern of methylation: low levels of methylation were observed in tissues that do not express FoxA2, and high levels were observed in FoxA2-expressing tissues (Fig. 1B)
We have identified a CpG island located in the promoter region of the FoxA2 gene that appears to directly regulate expression of the gene according to its methylation status
Summary
The transcription factor FoxA2 is a key mediator of endoderm development and pancreas gene expression. Development of multicellular organisms involves progression from pluripotency through multipotency, leading to terminal differentiation This process is mediated by transcription factors and epigenetic modifications that orchestrate the accompanying changes in gene expression; in turn this creates heritable cellular memories characteristic of both specific lineages and cell types (1, 2). We report that a CpG island in the promoter region of the FoxA2 gene paradoxically shows high levels of DNA methylation in endoderm lineage FoxA2-expressing tissues and low levels in nonexpressing tissues. Inhibition of methylation by 5-aza-2Ј-deoxycytidine (5-aza-dC) or directed DNMT3b knockdown led to decreased activation of FoxA2 during stem cell differentiation toward endoderm progenitors These results indicate that methylation of the CpG island plays a key role in FoxA2 gene activation in both early and late stages of FoxA2 expression
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