A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63

Abstract

Background: Open chromatin regions are correlated with active regulatory elements in development and are dysregulated in diseases. The BAF (SWI/SNF) complex is essential for development, and has been demonstrated to remodel reconstituted chromatin in vitro and to control the accessibility of a few individual regions in vivo. However, it remains unclear where and how BAF controls the open chromatin landscape to regulate developmental processes, such as human epidermal differentiation.

Results: Using a novel "on-plate" ATAC-sequencing approach for profiling open chromatin landscapes with a low number of adherent cells, we demonstrate that the BAF complex is essential for maintaining 11.6 % of open chromatin regions in epidermal differentiation. These BAF-dependent open chromatin regions are highly cell-type-specific and are strongly enriched for binding sites for p63, a master epidermal transcription factor. The DNA sequences of p63 binding sites intrinsically favor nucleosome formation and are inaccessible in other cell types without p63 to prevent ectopic activation. In epidermal cells, BAF and p63 mutually recruit each other to maintain 14,853 open chromatin regions. We further demonstrate that BAF and p63 cooperatively position nucleosomes away from p63 binding sites and recruit transcriptional machinery to control tissue differentiation.

Conclusions: BAF displays high specificity in controlling the open chromatin landscape during epidermal differentiation by cooperating with the master transcription factor p63 to maintain lineage-specific open chromatin regions.

Fig. 1

BAF controls the open chromatin landscape in epidermal differentiation. a Workflow of on-plate ATAC-seq analysis in control (CTRLi) and BRG1/BRM depletion (BAFi) conditions in primary human keratinocyte differentiation. b UCSC genome browser tracks showing replicates of ATAC-seq, generated from individual wells of differentiating human keratinocytes growing on a 96-well plate, in control (green tracks, n = 2) and BAF loss (red tracks, n = 2) conditions at the ZNF750 TF locus. Published DNase-seq in normal human epidermal keratinocyte (NHEK) cells from publically available ENCODE data (beige track) is also included as reference. An example of a decreased peak with BAF loss is shaded in blue, and an example of an increased peak is shaded in red. c Pie chart showing the distribution of the total 152,110 ATAC-seq peaks relative to gain and loss with BAFi. d–f Scatter plots showing the ATAC-Seq signal correlation between BAFi versus CTRLi, as well as between technical replicates. g Heatmap showing the cell-type specificity of both BAF-dependent and BAF-independent ATAC-seq peaks, as compared to DNase hypersensitive sites (DHS) open chromatin accessibility in 14 representative cell lines. h Boxplot showing the z-score distribution of BAF-dependent ATAC-seq peaks, as compared to all and gained ATAC-seq peaks. The lost ATAC-seq peaks with BAFi are highly enriched with keratinocyte-specific open chromatin regions (p < 10^-600, Kolmogorov–Smirnov test). TF transcription factor, ENCODE encyclopedia of DNA elements.

Fig. 2

BAF is enriched at and promotes open chromatin sites with p63 binding. a ATAC-seq summit-centered heatmap of ATAC-seq signal in control (CTRLi) and BAF-loss (BAFi) conditions, as well as BAF ChIP-seq signal in the same regions for all ATAC-seq peaks. The ATAC-seq peaks are sorted in BAF-independent, lost, and gained groups. b Average diagram of BAF ChIP-seq signal at BAF-independent, lost, and gained open chromatin regions. c Scatter plot demonstrating the relative enrichment of BAF binding at gained/lost ATAC-seq peaks. The p63-motif containing ATAC-seq peaks are highlighted in blue. d Bar graph showing the − Log10 p-value of TF motifs identified from a de novo Homer motif search, based on the ATAC-seq peaks gained or lost with BAFi. e Comparison of top motifs identified from the ATAC-seq analysis with a known p63 motif (weighted matrix). f Bar graph showing the percentage of ATAC-seq peaks with the p63 motif in both total ATAC-seq peaks as well as in BAFi peak loss. g Average diagram of genome-wide open chromatin accessibility at p63 and CTCF regions comparing CTRLi and BAFi conditions. h Venn diagram showing the overlap of gene sets controlled by p63 and BAF in keratinocyte differentiation. i Gene Ontology analysis of the 236 genes shared by BAF and p63. bp base pair, kb kilobase pair, TF transcription factor

Fig. 3

BAF regulates nucleosome positioning and recruits transcriptional machinery. a V-plot analysis of ATAC-seq fragments near p63 motif regions in both control (CTRLi) and BAF loss (BAFi) conditions. Schematic illustration of the V-plot analysis with phased nucleosome is included on the right. b Average diagram of nucleosome-free fragments (<100 bp) and mononucleosome fragment (180–247 bp) at p63 motif sites, demonstrating an average compaction of 40 bp at p63 sites with BAF loss. c UCSC genome browser tracks comparing ATAC-seq, H3K27Ac ChIP-seq, and Pol II ChIP-seq between CTRLi and BAFi, relative to p63 ChIP-seq at the HOPX TF locus. Decreased peaks with BAF loss are shaded with light blue. d–g Genome-wide average diagram of ChIP-seq (p63, H3K27Ac, Pol II, p300, and H3K27me3) comparing control to BAF loss. bp base pair, kb kilobase pair, TF transcription factor

Fig. 4

p63 is required to establish and to maintain open chromatin regions. a Heatmaps demonstrating chromatin accessibility at p63/CTCF motif sites in 15 different cell types in correlation with the p63/CTCF expression level. The accessibility to DNase I (ENCODE data) is demonstrated in red. RNA expression level (ENCODE data) of p63/CTCF in these cell types are include in parallel in blue. b Average diagram of total ATAC-seq signal at p63 sites comparing p63 loss (p63i) with control (CTRLi). c Average diagram of nucleosome-free fragments (<100 bp) at p63 motif regions, demonstrating p63 is required to maintain DNA accessibility at p63 motif regions. d Average diagram of mononucleosomal fragments (180–247 bp) comparing the nucleosome positioning at p63 sites between p63i and CTRLi conditions. Loss of p63 led to impaired nucleosome phasing and an average compaction of 25 bp at p63 motif sites. e Average diagram of predicted nucleosome binding probability based on DNA sequence composition in genomic regions centered by p63 motif, indicating that p63 motif sequences intrinsically favor nucleosome binding. bp base pair, ENCODE encyclopedia of DNA elements

Fig. 5

BAF and p63 cooperate in chromatin binding to reinforce open chromatin. a Venn diagram showing the overlap among ATAC-seq peaks, BAF ChIP-seq peaks, and p63 ChIP-seq peaks. b Proximity ligation analysis (PLA) in wild-type (CTRL, left), BAF-depleted (BAFi, middle), or p63-depleted (p63i, right) differentiating keratinocytes. Red represents PLA proximity signal for BAF and p63; blue is the Hoechst DNA stain. c Summit-centered heatmaps of 14,853 ATAC-seq peak regions co-bound with BAF and p63, showing ATAC-seq, p63 ChIP-seq, and BAF ChIP-seq signals in BAF or p63 loss and control conditions. d Average diagram of ATAC-seq, p63 ChIP-seq, and BAF ChIP-seq signals in BAF and p63 co-bound regions. bp base pair