Examination of transcriptional networks reveals an important role for TCFAP2C, SMARCA4, and EOMES in trophoblast stem cell maintenance

Abstract

Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (FGF4, INHBA/NODAL/TGFB1) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells, we used chromatin immunoprecipitation with DNA microarray hybridization (ChIP-chip) analysis to investigate targets of the TFs-TCFAP2C, EOMES, ETS2, and GATA3-and a chromatin remodeling factor, SMARCA4. We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of TCFAP2C, SMARCA4, and EOMES at a significant number of genes, and transcriptional regulatory circuitry within the five factors. Moreover, RNAi depletion of Tcfap2c, Smarca4, and Eomes transcripts resulted in a loss of normal colony morphology and down-regulation of TS cell-specific genes, suggesting an important role for TCFAP2C, SMARCA4, and EOMES in TS cell self-renewal. Through genome-wide mapping and global expression analysis of five TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal.

Figure 1.

Analysis of TF binding in TS cells. (A) Distance of binding regions relative to the nearest transcriptional start site (TSS). (B) Number of target genes occupied by each TF or transcriptional regulator. (C) Enriched DNA binding motifs for TCFAP2C and GATA3 identified using CisFinder software (http://lgsun.grc.nia.nih.gov/CisFinder/). (D) TCFAP2C and GATA3 binding motifs are overrepresented in ChIP-enriched regions. Genomic sequences (10,000 bp) centered on ChIP-enriched TCFAP2C or GATA3 binding regions were extracted from the genome and evaluated for the number of TCFAP2C and GATA3 motifs. CisFinder software was used to generate position-frequency matrices (PFM) for TCFAP2C and GATA3 binding motifs. We assumed five false-positives per 10 kb of random sequence. (E) Gene Ontology (GO) functional annotation of bound genes was performed using ingenuity pathway analysis (IPA). GO terms for cellular location include extracellular space (EC), plasma membrane (PM), nucleus, unknown, and cytoplasm. (F) GO terms for biological function identified using IPA.

Figure 2.

Multiple transcription factor occupancy at target genes. (A) Number of target genes occupied by multiple factors. X-axis represents the number of factors per target gene, and the y-axis represents the number of genes occupied by multiple factors. (Black dots) Total targets occupied by at least N-factors. (B) Factor co-occupancy. Heat map colors reflect co-occurrence of factor pairs, where red indicates a higher and yellow indicates a lower number of genes. One major cluster includes factors TCFAP2C, SMARCA4, and EOMES. (C) Venn diagram showing the relationship between TCFAP2C, SMARCA4, and EOMES bound genes. (D) Max fold-change binding profiles for accumulated target genes of all factors were clustered to reveal binding patterns. Representative binding profiles of 3-TF and 4-TF target genes were centered on enrichment peaks [−500 bp, +500 bp] and clustered using Spotfire. (E) TF binding profiles were centered on enrichment peaks [−2 kb, +2 kb] and clustered using Spotfire. Average binding profiles of 5-TFs are also shown. (F) Promoter profiles of TCFAP2C (T), SMARCA4 (S), and EOMES (EO) common target genes were clustered. Note the enriched binding of TCFAP2C, SMARCA4, and EOMES relative to ETS2 (ET) and GATA3 (G) at target loci. Average binding profiles of TCFAP2C, SMARCA4, and EOMES at common target loci are also shown.

Figure 3.

Genomic view of TF binding in TS cells. TF binding site analysis at target gene loci. Representative profiles of TS cell–enriched genes occupied by four (A), three (B–F), two (G–J), and one (K) factor(s). MA enrichment values adjusted to log₂ and conservation are shown on the plot.

Figure 4.

Microarray expression analysis of undifferentiated and time-course differentiated TS cells. (A) Experimental design. TS cells were cultured in MEF-conditioned medium in the presence of FGF4. TS cell differentiation proceeded in the absence of FGF4 over 14 d. RNA was collected from TS cells at 0 h, 24 h, 48 h, and 72 h and on day 6, day 10, and day 14. (B) Hierarchical clustering analysis (HCA) of differentially expressed genes (greater than twofold). (C) HCA of k-means-clustered differentially expressed genes. (D) Patterns of gene expression identified using k-means clustering. (E) Landscape view of k-means-clustered genes. (F) PCA plot of the first two principal components describing most of the data variability. (G) PCA plot of differentially expressed genes clustered according to k-means. (H) Log₂ adjusted expression values of genes expressed in TS cells and differentiated cells. (I) Genes expressed in TS cells were down-regulated during differentiation. qRT-PCR expression analysis of genes enriched in undifferentiated TS cells or bound by at least one factor (Cdx2, Elf5, Eomes, Esrrb, Fgfr2, Mmp9, and Sox2). Data was normalized to Gapdh and then to the expression of TS cells cultured in the presence of FGF4 at 0 h. (J) Genes expressed in differentiated trophoblast and placental cells were up-regulated during TS cell differentiation. qRT-PCR expression analysis of trophoblast lineage-specific genes (Ascl2, Cd9, Cdkn1c, Ets2, Gata2, Gcm1, Hand1, Kitl, Prl3b1, Prl8a6, Rest, and Tpbpa), including genes also bound by at least one factor (Ets2, Gata2, Gcm1, Hand1, and Rest). (K) HCA of qRT-PCR gene expression data. Fold-change expression values were clustered with a green/black/red color scale. Expression values relative to Gapdh (Gene CT – Gapdh CT) were clustered with a red/orange/yellow color scale. (L) PCA of fold-change gene expression data obtained from qRT-PCR. Data plotted in the first two components reveal a stepwise progression of undifferentiated TS cells at 0 h to differentiated TS cells at day 14. (M) Confocal immunofluorescence analysis of TCFAP2C, SMARCA4, EOMES, and HAND1 expression in undifferentiated and differentiated TS cells. TCFAP2C and SMARCA4 expression is enriched in both undifferentiated and differentiated TS cells, EOMES expression is enriched in undifferentiated TS cells, and HAND1 expression is enriched in differentiated TS cells. TCFAP2C, SMARCA4, EOMES, and HAND1 are labeled in green, and Cytokeratin is labeled in red. Arrows indicate undifferentiated TS cells, and arrowheads indicate differentiated trophoblast cells.

Figure 5.

GSEA analysis of TF occupied genes and histone H3 acetylation (AcH3) in TS cells. GSEA analysis of TF target genes in undifferentiated and differentiated TS cells (A); Cdx2- (B), Eomes- (C), and Gata3-overexpressing (D) ES cells; and TS cells and ES cells (E). The majority of target genes are expressed higher in undifferentiated compared with differentiated TS cells, and Cdx2-, Eomes-, and Gata3-overexpressing ES cells compared with control ES cells, and TS cells compared with ES cells. The x-axis represents enrichment of gene expression in TS cells at 0 h through 14 d of differentiation (A); Cdx2- (B), Eomes- (C), or Gata3-overexpressing (D) ES cells compared with control ES cells; or TS cells compared with ES cells (E). Target genes are represented as a black and white heat map below the plot. (F–J) GSEA analysis of multiple versus single factor-occupied genes in TS cells. Expression of genes that are common targets of three and two factors is more enriched in TS cells (F); Cdx2- (G), Eomes- (H), and Gata3-overexpressing (I) ES cells; and TS cells compared with genes occupied by a single factor (J). (K–O) Distribution of enrichment scores of multiple factor-occupied genes in TS cells (K); Cdx2- (L), Eomes- (M), and Gata3-overexpressing (N) ES cells; and TS cells (O). (P) GSEA of AcH3 associated genes in undifferentiated and differentiated TS cells. Expression of AcH3 associated genes is enriched in undifferentiated TS cells. The x-axis represents enrichment of gene expression in TS cells at 0 h through 14 d of differentiation. Inset shows the distribution of enrichment scores of AcH3 associated genes in TS cells. (Q) Association between AcH3 marks and binding of TFs. (R) Profiles of AcH3 marks were centered on enrichment peaks [−2 kb, +2 kb] and clustered. (S) Heat map of TS cell transcriptome data sorted by the level of gene expression in TS cells at 0-h genomic profiles of AcH3 marks at genes expressed at low (T) and high (U) levels in undifferentiated versus differentiated TS cells. MA enrichment values adjusted to log₂ and conservation are shown on the plot.

Figure 6.

TCFAP2C, SMARCA4, and EOMES are required for TS cell self-renewal. (A) Tcfap2c, Smarca4, and Eomes siRNAs used to deplete Tcfap2c, Smarca4, and Eomes mRNA in TS cells. Control siRNA, Tcfap2c siRNA, Smarca4 siRNA, Eomes siRNA, Tcfap2c/Smarca4/Eomes siRNA, Tcfap2c/Smarca4 siRNA, Tcfap2c/Eomes siRNA, or Smarca4/Eomes siRNA were nucleofected into TS cells every 2 d for 6 d. (B) Normal colony morphology was lost in TS cells nucleofected with Tcfap2c siRNA, Smarca4 siRNA, Eomes siRNA, Tcfap2c/Smarca4/Eomes siRNA, Tcfap2c/Smarca4 siRNA, Tcfap2c/Eomes siRNA, or Smarca4/Eomes siRNA compared with TS cells nucleofected with control siRNA. (C) Immunofluorescence analysis of TS cells conucleofected with Tcfap2c/Smarca4/Eomes siRNA. TCFAP2C, SMARCA4, and EOMES expression was down-regulated in TS cells nucleofected with Tcfap2c/Smarca4/Eomes siRNA compared with TS cells nucleofected with control siRNA. Cytoplasm was stained with an antibody specific to Cytokeratin (Cytok) and nuclei were stained with Hoechst 33258. Scale bars, 100 μM. (D) qRT-PCR expression analysis of Tcfap2c, Smarca4, and Eomes siRNA TS cells with control TS cells. Expression of TS cell enriched genes were down-regulated following nucleofection of Tcfap2c, Smarca4, and Eomes siRNA. (E) Heat map summary of qRT-PCR results.

Figure 7.

Transcriptional regulation of target genes in TS cells. (A) Model for integration of TCFAP2C, SMARCA4, and EOMES with core transcriptional machinery in TS cells. Colored circles represent the investigated factors, and arrows represent direction of transcriptional regulation. (B) Extended transcriptional network in TS cells integrating the investigated five factors with downstream target genes. Open circles represent target genes. (C) Transcriptional regulation within the five factors, including autoregulation of TCFAP2C and EOMES. (D) Summary of genes coexpressed or uniquely expressed in TS cells, ES cells, and their differentiated progeny.