The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation

Abstract

Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

Figure 1. Liver-specific enhancers retain MNase-accessible nucleosomes

(A) marker (M), MNase-untreated controls, Q (not warmed) and 0 (warmed), and low- or high-MNase digested DNAs on an agarose gel. (B) Signal tracks of MNase/ChIP/DNase-seq reads; two biological replicates for MNase-seq. Red boxes, sites of low MNase tag enrichment tested by qPCR in C; blue boxes, control sites. (C) Core histone ChIP-qPCR enrichment (means +/− SD of two biological replicates). (See also Figure S1)

Figure 2. Liver-specific enhancers retain MNase-accessible nucleosomes genome-wide

(A) Averaged profiles of low- and high-MNase-seq signal enrichments at, liver-specific enhancers ubiquitous enhancers, and active promoters. p-values by Wilcoxon rank-sum test at central 200 bp of enhancers and at upstream 200 bp from active TSS (pink box area). (B, E) Heatmaps of MNase-seqs and FoxA2 ChIP-exo at liver-specific enhancers, ubiquitous enhancers, and active promoters, rank ordered by low-MNase-seq read density. (C) Box and whisker plots show H3 and H2B ChIP-seq signal enrichment at central 200 bp of liver-specific and ubiquitous enhancers and at upstream 200 bp from active TSS. p-value by Wilcoxon rank sum test. (D) Gene expression levels of target genes of liver-specific and ubiquitous enhancers, based on predicted enhancer-promoter pairs (Shen et al., 2012). (See also Figure S2)

Figure 3. Pioneer factor FoxA2 and liver TFs are co-localized at accessible nucleosomes in liver-specific enhancers

(A) FoxA2 ChIP-exo tag 5’ end distribution centered by de novo FoxA motif midpoint and sorted by FoxA2 occupancy level. (B) Averaged profiles of low- and high-MNase-seq at TSS-distal FoxA2 binding sites, and (C) at CTCF binding sites overlapping with H3K27ac peaks. (D. E) Heatmaps of MNase-seqs, and FoxA2 ChIP-exo, C/EBβ and HNF4α ChIP-seq at MNase-accessible nucleosomes in liver-specific enhancers, rank ordered by low-MNase-seq read density. (F) Profiles of de novo motifs enrichment relative to midpoint of accessible nucleosome in enhancers. (G) Histogram of FoxA2 ChIP-exo binding locations relative to midpoint of accessible nucleosomes. The midpoint approximating the position of the respective nucleosome dyad axis. (See also Figure S3)

Figure 4. FoxA2 binds more with core histones than other liver TFs at enhancers

(A) Biological replicates of sonicated liver chromatin on agarose gel. (B) Examples of re-ChIP-qPCR target sites with MNase/ChIP-seq signals. (C) Box and whisker plots show re-ChIP-qPCR enrichment of core histone H2B and H3 over IgG at the promoter and enhancer target sites. Numbers in parentheses indicate number of sites tested. p-value by Wilcoxon rank sum test. (See also Figure S4)

Figure 5. FoxA2 binding is required to keep nucleosomes accessible in chromatin

Heatmap of (A) FoxA2 ChIP-exo in wild type, (B) low-MNase-seq, (C) high-MNase-seq, (D) low- over high-MNase-seq, and (E) H1 ChIP-seq in wild type (WT) and the FoxA1/A2 deletion mutant (Δ) at liver-specific enhancers, rank ordered by FoxA2 ChIP-exo read density. Box and whisker plots show at central 200 bp or 1000 bp of the top 25% FoxA2 occupied (FoxA2-strong) enhancers and at the bottom 25% FoxA2 occupied (FoxA2-weak) enhancers. p-values by Wilcoxon rank-sum test. (See also Figure S5–S7).

Figure 6. FoxA binding allows other liver-enriched TFs to bind

(A) FoxA2 ChIP-exo in wild type, and FoxA3, C/EBβ and HNF4α ChIP-seq signal tracks in wild type and the FoxA1/FoxA2 deletion mutant. (B–D) Heatmaps of FoxA2, FoxA3 and C/EBβ ChIP-seq in wild type (WT) and the FoxA1/A2 mutant (Δ) at FoxA2 binding sites overlapped with C/EBβ binding site in liver-specific enhancers, rank ordered by FoxA3 ChIP-seq signal in the FoxA1/A2 mutant. (E–G) Heatmaps of FoxA2, FoxA3 and HNF4α ChIP-seq in wild type and the FoxA1/A2 mutant at FoxA2 binding sites overlapped with HNF4α binding site in liver-specific enhancers, rank ordered by FoxA3 ChIP-seq read density in the FoxA1/A2 mutant. Box and whisker plots show at central 200 bp of the top 25% FoxA3 occupied (FoxA3-strong) enhancers and at the bottom 25% FoxA3 occupied (FoxA3-weak) enhancers. p-values by Wilcoxon rank-sum test. (See also Figure S6).

Figure 7. Maintenance of accessible nucleosomes by FoxA is required for liver-specific gene activation

(A, B) Tissue Expression analysis for > two-fold upregulated or downregulated genes in the FoxA1/A2 deletion mutant (Li et al., 2011) (See also Table 3). Number of enhancers that potentially link to the target genes (Shen et al., 2012) and number of FoxA2 ChIP-exo sites in the enhancers. (C, D) Box and whiskers plot show MNase accessibility (low/high MNase-seq) in wild type and the FoxA1/A2 mutant at central 200 bp of downregulated genes related FoxA2 sites and (D) at upregulated genes related FoxA2 sites (See also Figure S7). (E) Box and whiskers plot show FoxA3, C/EBβ and HNF4α ChIP-seq in wild type (WT) and the FoxA1/A2 mutant (Δ) at central 200 bp of downregulated genes related FoxA2 sites and (F) at upregulated genes related FoxA2 sites. p-values by Wilcoxon rank-sum test. (G) Models of nucleosome configuration at liver-specific enhancers. FoxA2 binding is required to displace linker histones, keep nucleosome accessible, and help recruitment of other liver enriched TFs, which stimulate liver gene transcription.