Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells

Abstract

Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation.

Figure 1.

Tcf3, Oct4, and Nanog co-occupy the genome in mouse ES cells. (A) Tcf3 binds to known target genes. Examples of previously known Tcf3-bound regions are displayed as unprocessed ChIP-enrichment ratios for all probes within the chromosomal region indicated below the plot. The gene is depicted below the plot, and the TSS and direction are denoted by an arrow. (B) Tcf3, Oct4, and Nanog display nearly identical binding profiles. Analysis of ChIP–chip data from genes bound by Tcf3, Oct4, or Nanog reveals that the three factors bind to similar genomic regions at all promoters. Regions from −8 kb to +2 kb around each TSS were divided into bins of 250 bp. The raw enrichment ratio for the probe closest to the center of the bin was used. If there was no probe within 250 bp of the bin center then no value was assigned. For genes with multiple promoters, each promoter was used for analysis. The analysis was performed on 3764 genes, which represents 4086 promoters. Promoters are organized according to the distance between the maximum Tcf3-binding ratio and the TSS. (C) Tcf3, Oct4, and Nanog bind in close proximity at target genes. Plots display unprocessed ChIP-enrichment ratios for all probes within the chromosomal region indicated below the plot. The gene is depicted below the plot, and the TSS and direction are denoted by an arrow.

Figure 2.

Tcf3, Oct4, and Nanog bind the promoters of Tcf3, Oct4, Sox2, and Nanog. Plots display unprocessed ChIP-enrichment ratios for all probes within the chromosomal region indicated below the plot. The gene is depicted below the plot, and the TSS and direction are denoted by an arrow.

Figure 3.

Tcf3 is an integral component of the core regulatory circuitry of ES cells. (A) Tcf3 forms an interconnected autoregulatory loop with Oct4, Sox2, and Nanog. Proteins are represented by ovals and genes are indicated by rectangles. (B) Model showing a key portion of the regulatory circuitry of mES cells where Oct4, Sox2, Nanog, and Tcf3 occupy both active and silent genes. The evidence that Oct4, Nanog, and Tcf3 occupy these genes is described here; Sox2 occupancy is inferred from previous studies in human ES cells (Boyer et al. 2005). Evidence that the transcriptionally silent genes are occupied by Polycomb Repressive Complexes is from Boyer et al. (2006), and unpublished data and that these genes have stalled RNA polymerases is from Guenther et al. (2007) and Stock et al. (2007). Proteins are represented by ovals and genes are indicated by rectangles.

Figure 4.

Knockdown of Tcf3 and activation of the Wnt pathway in mES cells reveal a role for Tcf3 in repression of target genes and a role in regulating pluripotency. (A) Tcf3 knockdown results in up-regulation of target genes. The effect of Tcf3 knockdown on gene expression was measured by hybridization of labeled RNA prepared from Tcf3 knockdown cells against RNA prepared from cells infected with nonsilencing control lentivirus at 48 h post-infection. A heat map of biological replicate data sets of expression changes was generated where genes are ordered according to average expression change. Tcf3 target genes have a higher average expression change than the average for all genes upon knockdown of Tcf3. (B) Wnt CM results in up-regulation of Tcf3 target genes. The effect of Wnt activation on gene expression was measured by hybridization of labeled RNA prepared from mES cells grown in Wnt CM against RNA prepared from cells grown in mock CM. A heat map of biological replicate data sets of expression change upon addition of Wnt CM where genes are ordered according to average expression change of replicates. Tcf3 target genes have a higher average expression change than the average for all genes upon addition of Wnt CM. (C) Tcf3 knockdown results in increased expression of Oct4, Sox2, and Nanog. Real-time PCR demonstrates that Oct4, Sox, and Nanog have increased expression upon knockdown of Tcf3. Values are normalized to Gapdh transcript levels, and fold change is relative to cells transfected with a nonsilencing hairpin. (D) Tcf3 knockdown results in increased staining for Oct4 and Nanog. Immunofluorecence was performed on mES cells grown one passage off of feeders that were either infected with Tcf3 knockdown lentivirus or infected with nonsilencing control lentivirus. Cells were fixed with 4% paraformaldehyde 96 h post-infection. Cells were stained with Oct4, Nanog, and DAPI. Images for Oct4 and Nanog staining were taken at 40× magnification and an exposure time of 300 msec. Tcf3 KD 1 and KD 2 represent different knockdown hairpin constructs. Tcf3 KD 2 is the virus also used in A, C, E, and F. (E) Tcf3 knockdown results in a significant increase of Oct4 staining. Quantification of Oct4 staining was performed in cells infected with Tcf3 or Gfp knockdown virus. (F) Tcf3 knockdown cells proliferate over more passages in the absence of LIF. Relative cell numbers of ES cells transfected with Tcf3 or control virus through multiple passages off of feeders in the presence or absence of LIF. Identical cell numbers were initially plated, and cells were split 1:12 every 2–3 d. Cells were counted at each passage and values for cells grown in the absence of LIF were normalized to cells grown in the presence of LIF.

Figure 5.

Model depicting the influence of Wnt pathway components on pluripotency and differentiation in ES cells. ES cells are poised between the pluripotent state and any of a number of differentiated states. Oct4, Sox2, and Nanog act to promote the pluripotent state (depicted by an arrow). Tcf3 can exist in an activating complex with β-catenin or a repressive complex with Groucho (Reya and Clevers 2005). Under standard growth conditions, the Wnt pathway is only active at low levels (Supplemental Fig. S4; Dravid et al. 2005; Yamaguchi et al. 2005; Lindsley et al. 2006; Ogawa et al. 2006; Anton et al. 2007; Takao et al. 2007). Therefore, Tcf3 is mainly in a repressive complex promoting differentiation (depicted by a thick arrow), although some Tcf3 associates with β-catenin to activate target genes and promote pluripotency (depicted by a thin arrow). In Tcf3 knockdown cells, there is no influence from Tcf3 on cell state. Thus, the balance is tipped toward maintaining pluripotency. Upon Wnt stimulation, the balance again tips toward maintaining pluripotency as more Tcf3 associates with β-catenin in an activating complex (depicted by a thick arrow). This model is not meant to imply that Wnt or Tcf3 are themselves pluripotency factors, but rather that they can influence cell state in the presence of other pluripotency factors, such as Oct4, Sox2, and Nanog.