Microarray analysis of LIF/Stat3 transcriptional targets in ES cells

¹ INSERM U362, Institut Gustave Roussy, Villejuif, France
² CEA LGRH, Evry, France
³ Ludwig Institute for Cancer Research and Christian de Duve Institute of Cellular Pathology, Brussels, Belgium

^* To whom correspondence should be addressed. E-mail: dsekkai{at}igr.fr.

	Abstract

Mouse ES cells can be propagated in vitro while retaining their properties of pluripotency and self-renewal under the continuous presence of LIF. Essential role has been attributed to subsequent activation of the Stat3 transcription factor in mediating LIF self-renewal response. To date however, downstream target genes of Stat3 in ES cells are still unknown. In order to isolate these genes, we performed a microarray-based kinetic comparison of LIF stimulated (undifferentiated) ES cells versus ES cells induced to differentiate by shutting down Stat3 activity through either I) LIF deprivation or more specifically, ii) expression of a Stat3 dominant negative mutant. In each case, we chose the earliest time at which ES cells lose their self renewal properties as illustrated by a decrease in the number of embryoid bodies and blast cell colonies formation as well as germ layer markers expression. Comparison of the two independent approaches revealed similarly regulated genes that are likely to be involved in the Stat3 effects on ES cell self-renewal. For instance, up regulation of growth factors such as the TGF- relative Lefty1 or transcriptional regulators such as Id1, Id2 and down regulation of the groucho-like protein Aes1 (grg5) were found. Promoter analysis of the aes1 gene revealed three functional Stat3 consensus sites as shown by luciferase assays. Furthermore, chromatin immunoprecipitation experiment demonstrated that Stat3 is recruited to the promoter of aes1 in ES cells. These data demonstrated that the aes1 gene is a direct transcriptional target of Stat3 in ES cells.

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