Abstract
Embryonic stem (ES) cell pluripotency is regulated in part by transcription factor (TF) pathways that maintain self-renewal and inhibit differentiation. Stat3 and c-Myc TFs are essential for maintaining mouse ES cell self-renewal. c-Myc, together with Oct4, Sox2, and Klf4, is a reprogramming factor. While previous studies have investigated core transcriptional circuitry in ES cells, other TF pathways that promote ES cell pluripotency have yet to be investigated. Therefore, to further understand ES cell transcriptional networks, we used genome-wide chromatin immunoprecipitation and microarray analysis (ChIP-chip) to map Stat3 and c-Myc binding targets in ES cells. Our results show that Stat3 and c-Myc occupy a significant number of genes whose expression is highly enriched in ES cells. By comparing Stat3 and c-Myc target genes with gene expression data from undifferentiated ES cells and embryoid bodies (EBs), we found that Stat3 binds active and inactive genes in ES cells, while c-Myc binds predominantly active genes. Moreover, the transcriptional states of Stat3 and c-Myc targets are correlated with co-occupancy of pluripotency-related TFs, polycomb group proteins, and active and repressive histone modifications. We also provide evidence that Stat3 targets are differentially expressed in ES cells following removal of LIF, where culture of ES cells in the absence of LIF resulted in downregulation of Stat3 target genes enriched in ES cells, and upregulation of lineage specific Stat3 target genes. Altogether, we reveal transcriptional targets of two key pluripotency-related genes in ES cells – Stat3 and c-Myc, thus providing further insight into the ES cell transcriptional network.
Highlights
Pluripotent embryonic stem (ES) cells, derived from mammalian preimplantation embryos, can be cultured indefinitely in vitro and have the ability to differentiate into all somatic and germ cell lineages [1,2]
Stat3 is a downstream component of leukemia inhibitory factor (LIF) signaling, which is integral in maintaining ES cells in an undifferentiated state. c-Myc promotes ES cell self-renewal in the absence of LIF [11] and is a reprogramming factor [12,15]
By mapping genomic regions of Stat3 and c-Myc promoter binding we aim to evaluate the binding patterns of these transcription factor (TF) and how they are connected in the ES cell transcriptional network
Summary
Pluripotent embryonic stem (ES) cells, derived from mammalian preimplantation embryos, can be cultured indefinitely in vitro and have the ability to differentiate into all somatic and germ cell lineages [1,2]. LIF belongs to the interleukin-6 family of cytokines that signals through heterodimerization of receptors gp130 and LIF-R, resulting in activation of Jak, and phosphorylation of gp130 and LIF-R [3]. Subsequent Stat phosphorylation results in nuclear translocation and target gene transcriptional activation or repression [4]. Expression of Stat maintains self-renewal in the absence of LIF [7], while disruption of Stat results in ES cell differentiation [5]. While these studies demonstrate that Stat is essential for ES cell pluripotency, downstream targets of LIF/Jak/Stat signaling have not been fully identified
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