Progressive methylation of <i>POU5F1</i> regulatory regions during blastocyst development

Abstract

The <i>POU5F1</i> gene encodes one of the 'core' transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5' regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere with <i>POU5F1</i> expression and function in <i>in vitro</i>-derived cell lines. Also, its permanent inactivation in differentiated cells depends on <i>de novo</i> methylation of its promoter. However, little is known about the epigenetic regulation of <i>POU5F1</i> expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of the <i>POU5F1</i> 5' upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5' regulatory region and the first exon accompanying differentiation and the gradual repression of <i>POU5F1</i> Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.