Microarray analysis of LIF/Stat3 transcriptional targets in embryonic stem cells.
Mouse embryonic stem (ES) cells can be propagated in vitro while retaining their properties of pluripotency and self-renewal under the continuous presence of leukemia inhibitor factor (LIF). An 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. 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 LIF deprivation or, more specifically, 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 colony formation as well as germ layer marker 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, upregulation of growth factors such as the transforming growth factor-beta relative Lefty1 or transcriptional regulators such as Id1 and 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.
Sekkaï
Dalila
D
INSERM U362, Institut Gustave-Roussy, 94805 Villejuif Cedex, France. dsekkai@igr.fr
Gruel
Gaëtan
G
Herry
Magali
M
Moucadel
Virginie
V
Constantinescu
Stefan N
SN
Albagli
Olivier
O
Tronik-Le Roux
Diana
D
Vainchenker
William
W
Bennaceur-Griscelli
Annelise
A
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D056970:Co-Repressor Proteins; D015536:Down-Regulation; D033041:Embryo Research; D005786:Gene Expression Regulation; D015850:Interleukin-6 / Q000235:genetics* / Q000378:metabolism*; D051663:Kv1.2 Potassium Channel; D053605:Leukemia Inhibitory Factor; D051379:Mice; D046228:Microarray Analysis; D009154:Mutation; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000235:genetics* / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D015854:Up-Regulation
D056970:Co-Repressor Proteins; D015850:Interleukin-6; D051663:Kv1.2 Potassium Channel; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D050796:STAT3 Transcription Factor; C089462:Tle5 protein, mouse; D014157:Transcription Factors