Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers

Abstract

Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.

Figure 1.

Differential H2AFZ and DNA methylation levels are linked to cell-type-specific FOXA1 recruitment to chromatin. Average FOXA1 (A), H3K4me2 (B), H2AFZ (C), and DNA methylation (D) enrichment levels at MCF7-specific, LNCaP-specific, or shared FOXA1-binding sites. Average signals were determined from tiling array data obtained in MCF7 and LNCaP cells as described in the Methods section except for H3K4me2 data in LNCaP cells, which were obtained from the ChIP-seq data of He et al. (2010). A random set of regions from chromosomes 8, 11, and 12 was also analyzed. (E) MeDIP-qPCR and H2AFZ ChIP-qPCR were performed in both MCF7 and LNCaP cells. For each analyzed FOXA1-binding site, the strongest enrichment was divided by the one obtained in the other cell type. A color code was used to show fold differences between the two cell types for both MeDIP and H2AFZ levels. Fold differences were inferred from data obtained in three independent experiments.

Figure 2.

Differential 5-methylcytosine levels within CpGs present at cell-type-specific FOXA1-binding sites. (A) Genomic DNA from MCF7 or LNCaP cells were subjected to HpaII digestion. The percentage of non-digested DNA was determined by qPCR using primers allowing for amplification of a DNA fragment encompassing a CCGG motif localized within cell-type-specific FOXA1-binding sites. Numbering of FOXA1-binding sites is consistent with Figure 1E. Results are means and standard deviations from three independent experiments. (B) The percentage of cytosine methylation for CpG dinucleotides found within 200 bp of the center of cell-type-specific FOXA1-binding sites was determined using bisulfite pyrosequencing. Two MCF7- and two LNCaP-specific sites were analyzed as described in the Methods section. Numbering of FOXA1-binding sites is consistent with Figure 1E. Results are means and standard deviations from a representative experiment performed in triplicates. (C) FOXA1 ChIP-qPCR experiments were performed in LNCaP and abl cells. Fold enrichments relative to a negative control region are indicated. Results are means and standards deviations from three independent experiments. (D) The percentage of cytosine methylation within CpG dinucleotides found near the center of UBE2C enhancers 1 and 2 was determined using bisulfite pyrosequencing in LNCaP and abl cells as indicated. Results are means and standard deviations from a representative experiment performed in triplicates.

Figure 3.

FOXA1 preferentially binds to DNA hypomethylated sites when ectopically expressed in MDA-MB231 cells. (A) Western blot assay showing FOXA1 expression in stably transfected MDA-MB231 cells (MDA-FOXA1). Note that only a faint non-specific band of lower molecular weight is observed in MDA-MB231 cells stably transfected with an empty vector (MDA-control) when the anti-FOXA1 antibody was used. (B) Peaks of FOXA1 enrichment were called using MAT at FDR 1% in MDA-FOXA1 cells and compared to those previously identified in MCF7 and LNCaP cells. Numbers of overlapping and non-overlapping binding sites are indicated on the Venn diagram. (C) ChIP-qPCR validation of eight sites identified by FOXA1 ChIP-chip in MDA-FOXA1. Fold enrichments relative to negative control regions are indicated. Results are from two independent experiments. (D) Average FOXA1-binding levels at MDA-MB231, MCF7-specific, or LNCaP-specific FOXA1 peaks were determined from ChIP-chip data obtained in MDA-FOXA1 cells. A random set of regions from chromosomes 8, 11, and 12 was also analyzed. (E) Average DNA methylation and H3K4me2 levels at FOXA1-binding sites from MDA-FOXA1 cells or at sites specific to MCF7 or LNCaP cells. MeDIP-chip or H3K4me2 ChIP-chip data were obtained in MDA-control or MDA-FOXA1 cells, as indicated. A random set of regions was used as control. (F) ChIP-qPCR experiments were performed in both MDA-control and MDA-FOXA1 cells to determine H3K4me1, 2, and 3 levels at FOXA1-binding sites validated in panel C. Fold enrichments relative to negative control regions are indicated. Results are from three independent experiments.

Figure 4.

Epigenetic program involved in the establishment of FOXA1-dependent enhancers during cellular differentiation. (A) Western blot assay showing induction of FOXA1 expression after stimulation of P19 cells with retinoic acid (RA) for 48 h. (B) FOXA1 ChIP-qPCR experiments were performed in P19 cells stimulated (+) or not (−) with RA for 48 h or 120 h. Fold enrichments relative to a negative control region are indicated. Results are from three independent experiments. (C) H3K4me1, 2, and 3 ChIP-qPCR experiments performed and analyzed as in B. (D) The percentage of cytosine methylation for CpG dinucleotides found within 200 bp of the center of FOXA1-binding sites was determined using bisulfite pyrosequencing. Shown is the average methylation levels of all CpGs analyzed for a given binding site. (E) RT-qPCR experiments performed in P19 cells stimulated (+) or not (−) with RA for 48 h or 120 h. Expression of analyzed genes was normalized using Rplp0 and is shown as fold induction relative to expression in undifferentiated P19 cells, which was set to 1. Results are from two independent experiments performed in duplicates. Note that, as expected, expression of the pluripotency transcription factor Nanog was strongly reduced upon induction of P19 cell differentiation with RA (data not shown). (ND) Not detectable.

Figure 5.

Kinetics of FOXA1 recruitment and epigenetic switch at enhancers during the course of P19 cell neural differentiation. (A) FOXA1 and H3K4me2 ChIP-qPCR as well as bisulfite-pyrosequencing experiments were performed in unstimulated or RA-treated P19 cells and analyzed as in Figure 4. For DNA methylation data, the percentage of cytosine methylation for CpG dinucleotides found within 200 bp of the center of FOXA1-binding sites is indicated. Note that significant DNA demethylation (p < 0.05) was observed for all analyzed CpGs except CpG 2 of site 3. On the other hand, no significant decreases in cytosine methylation levels were obtained at control CpGs localized within the Vtn promoter (inset). These CpGs had been previously identified as methylated in both undifferentiated and RA-stimulated P19 cells (A Sérandour and G Salbert, unpubl.). (B) Bisulfite-pyrosequencing was used to monitor the methylation status of CpGs found in site 1 within DNA issued from FOXA1 ChIP. Input DNA was used as a reference in these experiments. P19 cells were harvested and processed for ChIP assays after 6, 12, 18, 24, 48, or 120 h of RA treatment, as indicated. Results are means and standard deviations from two independent experiments performed in duplicates.

Figure 6.

FOXA1-binding sites are DNA methylation- and cell-context-sensitive enhancers. (A) Reporter assays were performed using in vitro methylated or unmethylated enhancers cloned within a CpG-free luciferase reporter vector and transfected in MCF7 or LNCaP cells. “Empty” refers to the control reporter construct lacking an enhancer. Numbering of FOXA1-binding sites is consistent with Figure 1E. Results are means and standard deviations from a representative experiment performed in triplicates. (B) Reporter assays were performed using unmethylated reporter constructs that were transfected in MCF7 or LNCaP cells. Results are means and standard deviations from a representative experiment performed in triplicates. (C) Reporter assays were performed using unmethylated reporter constructs that were transfected in HEK293 cells. Results are means and standard deviations from a representative experiment performed in triplicates. (D) Reporter assays were performed using unmethylated reporter constructs that were transfected in MDA-control or MDA-FOXA1 cells, as indicated. The day before transfection, 2.5 μg/mL tetracycline was added to the cells. Results are means and standard deviations from a representative experiment performed in triplicates. For all experiments, luciferase activities are expressed relative to that obtained for the control reporter plasmid lacking an enhancer, which was set to 1.

Figure 7.

Model depicting the proposed dynamic TF binding and epigenetic hierarchy that governs establishment of FOXA1-dependent enhancers. FOXA1 collaborates with additional pioneer factors (e.g., GATA family factors) to trigger transcriptional competency of specific enhancers. We propose that this involves the initiation of an epigenetic switch consisting of DNA demethylation and induction of H3K4 methylation. These epigenetic changes allow for subsequent recruitment of transcriptional regulatory effectors, among which are nuclear receptors (NR) that directly regulate gene expression in response to environmental cues. See the Discussion section for further details.