Tissue-specific SMARCA4 binding at active and repressed regulatory elements during embryogenesis

Abstract

The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development.

Figure 1.

Overview of Smarca4^FLAG mouse generation and SMARCA4 binding site mapping in vivo. A knock-in mouse strain carrying a FLAG epitope at the carboxy terminus of SMARCA4 was engineered and used to perform ChIP-seq from a panel of embryonic tissues. For each ChIP-seq data set, the total number of identified enriched SMARCA4 regions is indicated near the corresponding coverage map (schematics).

Figure 2.

ChIP-seq identifies SMARCA4-enriched regions genome-wide. (A) Genomic distribution of SMARCA4-enriched regions. (B) Tissue-specific SMARCA4 enrichment in TSS versus distal regions in each data set. The bar graph illustrates the lower percentage of tissue-specific SMARCA4-bound regions among the proximal sites (TSS) versus distal sites by indicating the number of tissues in which a specific SMARCA4 region is significantly enriched (red, present in one tissue; dark green, present in seven tissues). (C,D) Heatmaps displaying SMARCA4 coverage across three tissues (forebrain, limb, and heart). Each row of heatmap represents one enhancer, with coverage plotted across the 10 kb surrounding enrichment peak. (C) SMARCA4 enrichment proximal to TSSs. (D) SMARCA4 enrichment at distal regions. (E) Selection of six reproducible SMARCA4-bound enhancers at E11.5, illustrating SMARCA4 activity across different tissues. Reproducible predicted patterns are indicated by red arrows while unpredicted reproducible tissue-specific activity is indicated by yellow arrows. (Fb) Forebrain; (Mb) midbrain; (Hb) hindbrain; (Nt) neural-tube; (Lb) limbs; (Ht) heart. The reproducibility of the indicated patterns is indicated for each embryo (number of observed/total number of embryos).

Figure 3.

Proximal SMARCA4 enrichment is associated with active and bivalent promoters. (A,B) Heatmaps displaying coverage for SMARCA4 and informative histone marks (H3K4me3 and H3K27me3) for proximal regions. Each row of heatmap represents one enhancer, with coverage plotted across the 10 kb surrounding enrichment peak. SMARCA4 enrichment at proximal regions for forebrain (n = 10,737) (A) and limb (n = 10,193) (B). (C,D) Gene expression profile associated with SMARCA4 proximal regions classified as active or bivalent. RNA-seq data were generated for E11.5 forebrain and limb and the distribution of expression levels of the nearest gene was compared for SMARCA4-enriched regions in forebrain (C) and limb (D).

Figure 4.

Histone marks associated with activation and repression co-localize with SMARCA4. (A,B) Heatmaps displaying coverage for SMARCA4 and informative histone marks for proximal (H3K4me3 and H3K27me3) and distal (H3K4me1, H3K27ac, H3K27me3) regions. Each row of heatmap represents one enhancer, with coverage plotted across the 10 kb surrounding enrichment peak. SMARCA4 enrichment at distal regions for forebrain (n = 12,269) (A) and limb (n = 6918) (B). (C) Classified regulatory groups based on histone signatures. (*) Bivalent SMARCA4 elements show a mix of enrichment patterns. (D) Differential enrichment for functional annotation terms associated with distal SMARCA4 regions categorized by histone signature. Shown are the top five enriched “Biological process terms” specific to distal forebrain (blue) and limbs (green) differentially marked SMARCA4 regions. (E,F) Transcription associated with SMARCA4 regions that can be classified as active or repressed based on co-occurring histone marks. RNA-seq data were generated for E11.5 forebrain and limb and the distribution of expression levels of the nearest gene was compared for SMARCA4-enriched regions in forebrain (E) and limb (F) classified by histone signature.

Figure 5.

SMARCA4 marks developmental enhancers in an active or repressed state. (A) Intersection of forebrain SMARCA4-enriched regions with 1747 published mouse sequences tested in the transgenic assay shows that SMARCA4 marks enhancers and the histone signature at SMARCA4-enriched loci predicts tissue-specific activity. (Positive) Reproducible enhancer activity in forebrain; (other) reproducible enhancer activity in a tissue other than forebrain; (negative) no detectable enhancer activity in vivo. (B) Representative examples of regions with characteristic forebrain and limb chromatin state showing predicted tissue specificity in transgenic assays. One representative embryo is displayed for each region and the reproducibility of the observed pattern is indicated (number of observed/total number of embryos) for each. The predicted activity in forebrain (fb) is shown both in the side views (red arrows) and top views (the angle from which the image was captured is indicated on the side views by a black arrow).