Repression of Nanog Gene Transcription by Tcf3 Limits Embryonic Stem Cell Self-Renewal

Abstract

The dual function of stem cells requires them not only to form new stem cells through self-renewal but also to form lineage-committed cells through differentiation. Embryonic stem cells (ESC), which are derived from the blastocyst inner cell mass, retain properties of self-renewal and the potential for lineage commitment. To balance self-renewal and differentiation, ESC must carefully control the levels of several transcription factors, including Nanog, Sox2, and Oct4. While molecular mechanisms promoting transcription of these genes have been described, mechanisms preventing excessive levels in self-renewing ESC remain unknown. By examining the function of the TCF family of transcription factors in ESC, we have found that Tcf3 is necessary to limit the steady-state levels of Nanog mRNA, protein, and promoter activity in self-renewing ESC. Chromatin immunoprecipitation and promoter reporter assays showed that Tcf3 bound to a promoter regulatory region of the Nanog gene and repressed its transcriptional activity in ESC through a Groucho interaction domaindependent process. The absence of Tcf3 caused delayed differentiation of ESC in vitro as elevated Nanog levels persisted through 5 days of embryoid body formation. These new data support a model wherein Tcf3-mediated control of Nanog levels allows stem cells to balance the creation of lineage-committed and undifferentiated cells.

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

“…Transcriptional profiling of Tcf3 2/2 ESCs showed that Tcf3 ablation caused strongly opposite effects on gene expression compared with Oct4 knockdown and Nanog knockdown (Loh et al 2006;Yi et al 2008). It is notable that the effect on Oct4-regulated genes was measured in Tcf3 2/2 cells, which did not display elevated Oct4 protein levels (Pereira et al 2006;Yi et al 2008), and that knockdown of Nanog in Tcf3 2/2 cells did not reduce the levels of stem cell genes examined (Yi et al 2011). Thus, the effects of Tcf3 ablation went beyond just Nanog and Oct4 stimulation and appeared to directly affect the genes co-occupied by Tcf3/Oct4/Sox2/Nanog.…”

“…All four Tcf/Lef proteins are expressed in mESCs (Pereira et al 2006;Kelly et al 2011); comparing Tcf/Lef mRNA levels with quantitative RT-PCR assays revealed that Tcf3 was by far the most abundant (Pereira et al 2006). Awealth of data shows that Tcf3 plays an important role in regulating the self-renewal and differentiation of mESCs; however, some data reveal b-catenin activities that are independent of a Tcf3 -b-catenin transactivator complex.…”

“…Perhaps the best illustration of co-occupancy came from ChIP-seq experiments, which showed that of the 1369 known genes identified as Tcf3-bound, 1173 were also bound by Oct4, and 942 were bound by each, Tcf3, Oct4, Sox2, and Nanog (Marson et al 2008). Other studies showed Oct4 and Nanog mRNA and protein levels were increased when Tcf3 was knocked down or ablated (Pereira et al 2006;Cole et al 2008;Tam et al 2008;Salomonis et al 2010;Wagner et al 2010). Transcriptional profiling of Tcf3 2/2 ESCs showed that Tcf3 ablation caused strongly opposite effects on gene expression compared with Oct4 knockdown and Nanog knockdown (Loh et al 2006;Yi et al 2008).…”

“…Loss-of-function experiments showed that Tcf3 mediates important effects on the self-renewal of mESCs. Initial experiments showed that genetic ablation of Tcf3 allowed Tcf3 2/2 mESCs to self-renew in serum-containing media without LIF/Jak/Stat3 signaling (Pereira et al 2006;Yi et al 2008;Yi and Merrill 2010;Wray et al 2011). Tcf3 2/2 mESCs also displayed delayed differentiation in EB assays and colony-based differentiation assays (Pereira et al 2006;Yi et al 2008;Wray et al 2011).…”

Defining the nature of human pluripotent stem cell progeny

“…Of the 53 genes that were higher in PSC derivatives (in red), 22 were also strongly expressed in undifferentiated PSCs relative to somatic cells (indicated with asterisk). This list included LIN28B, DPPA4, and TCF7L1 (TCF3), all of which are known to play a role in ESCs and in very early mammalian development [28,[30][31][32][33]. Furthermore, 35 genes were downregulated in PSC derivatives compared to tissue-derived cells (in green), perhaps reflecting a state of incomplete specification, regardless of the cell type generated.…”