Heterozygous Mutations in SMARCA2 Reprogram the Enhancer Landscape by Global Retargeting of SMARCA4

Abstract

Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.

Keywords: AP-1; SMARCA2; SWI/SNF complex; chromatin remodeling; neural differentiation; neural progenitor cells.

Figure 1.. SMARCA2 mutations impair neural progenitor differentiation.

A. Schematic diagram of SMARCA2 ATPase domain with conservation score. B. Conservation analysis comparing NCBRS SNVs with normal SNVs. Error bars = mean ± SD, n=28 (NCBRS), 24 (Normal) C. Structure of yeast SNF2 domain and DNA with NCBRS mutations labeled in red. D. Light microscopy of SMARCA2^+/+, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. E. Cell confluency of WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. **: p<0.01, * p<0.05 Error bars = mean ± SD, n=4. F. Cell cycle analysis of WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs as measured by propidium iodide staining. Error Bar = mean ± SD, n=3. G and H. Immunofluorescence of WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs stained with PAX6 and NESTIN (left), or SOX2 and Ki67 (right), along with DAPI. Scale bar=50μm. I. Quantitation of the percent of positively stained cells in G and H, Error Bar = mean ± SD, n=3. J. Immunofluorescence of WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ neurons stained with TUJ1, MAP2, and DAPI. Scale bar=50μm. K. Quantitation of the number of positively stained cells per field in J, Error Bar = mean ± SD, n=3. See also Figure S1.

Figure 2.. SMARCA2 mutant NPCs have altered gene expression.

A. Scatterplot of average log2 Fragments Per Kilobase of transcripts per Million mapped reads (FPKM) mRNA expression level against log2 fold change (FC) in expression of SMARCA2^K755R/+/WT (top) or SMARCA2^R1159Q/+/WT NPCs (bottom). B. Hierarchical clustering of log2 FPKM of differentially expressed genes (DEGs) in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. C. Overlap of upregulated and downregulated DEGs in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. D. Significance of gene ontology (GO) analysis of genes that are downregulated (top) or upregulated (bottom) in both SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. E. FPKM of SWI/SNF subunits in WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. Error bars = mean ± SD, n=2. F. Immunoblotting of protein level of SWI/SNF subunits in WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. G. Quantitation of western blots as represented in F, Error bars = mean ± SD, sample size see method. H. SMARCA2 immunoprecipitation in WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs and immunoblotting for the indicated SWI/SNF subunits. See also Figure S2.

Figure 3.. Changes in chromatin accessibility in SMARCA2 mutant NPCs are correlated with the binding of SMARCA4.

A. Scatterplot of average Log2 ATAC-seq tag density of WT against SMARCA2^K755R/+ (top), WT against SMARCA2^R1159Q/+ (middle) and SMARCA2^K755R/+ against SMARCA2^R1159Q/+ (bottom). B. Heat map of log2 ATAC tag density at differentially accessible sites in WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. C. Scatterplot of average Log2 ChIP-seq tag density of WT against SMARCA2^K755R/+ (top), WT against SMARCA2^R1159Q/+ (middle) and SMARCA2^K755R/+ against SMARCA2^R1159Q/+ (bottom). D. Heat map of SMARCA2 and SMARCA4 ChIP-seq tags at sites with increased or decreased accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs, clustered as in B. E and F. Histogram of Normalized ChIP-seq signal for SMARCA2 or SMARCA4 at sites that decreased (E, n=15,791) or increased (F, n=19,203) accessibility in SMARCA2^K755R/+ or SMARCA2^R1159Q/+ NPCs. G. Scatterplot of log2 fold change (FC) of SMARCA2 against SMARCA4 ChIP density in SMARCA2^K755R/+ (Left) or SMARCA2^R1159Q/+ (Right) versus WT. H. Pie chart of composition of sites with decreased (down) or increased (up) accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs compared to WT NPCs. See also Figure S2 and S3.

Figure 4.. SOX3-dependent neural enhancers are lost in SMARCA2 mutant NPCs.

A. Enrichment of sites that increased or decreased accessibility in both SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs for histone modifications and enhancer groups in WT NPCs. B. Histogram of normalized ChIP-seq tags for H3K4me and H3K27ac at the NPC active enhancers (n=4,977) that lose accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. C. Significance of de novo motif enrichment on sites that lose accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. D. The SOX3 and RFX1 motifs identified in C. E. Proportion of decreased accessible sites in SMARCA2^K755R/+ or SMARCA2^R1159Q/+ NPCs containing indicated motif. F. Overlap of SOX2, SOX3, and SMARCA4 ChIP-seq binding sites. G. Proportion of NPC super enhancers (SEs) or Brain (cingulate gyrus) super enhancers that are bound by SMARCA4, or SMARCA4, SOX2, and SOX3. H. Histogram of normalized ChIP-seq tags for SMARCA4 in WT NPCs transfected with siRNAs against control or SOX2 and SOX3 at SOX3 binding sites that lose chromatin accessibility (n=3,190). I. Immunoblot of SOX2/3 in WT, SMARCA2^K755R/+, SMARCA2^R1159Q/+ NPCs. J-N. Histogram of normalized ChIP-seq tags for SOX2 (J), SOX3 (K), SMARCA4 (L), SMARCA2 (M) or ATAC-seq tags (N) in WT, SMARCA2^K755R/+ or SMARCA2^R1159Q/+ NPCs at SOX3 binding sites that lose accessibility (n=3,190). O. Fraction of all Differentially Expressed Genes (DEGs) from SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs bound by SOX2, SOX3, or both. P. Fraction all DEGs from SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs down- or up-regulated in SOX2/SOX3 knockdown NPCs. See also Figure S4.

Figure 5.. SMARCA2 mutations lead to de novo activation of astrocyte-specific enhancers.

A. Heat map of log2 ATAC tag density or ChIP-seq tags at increased accessible sites in WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs defined as having no accessibility (De novo) or a peak of accessibility (Poised) in NPCs. B. Rank of percent overlap of sites that increased or decreased accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs with super enhancers from 59 tissues in the dbSUPER database. C. Histogram of normalized ChIP-seq tags for H3K4me (top) and H3K27ac (bottom) at the DE sites that gain accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs (n=9,441). D. Proportion of astrocyte Super Enhancer (SE)-associated genes with increased accessibility and gene expression in SMARCA2 mutant NPCs. E. Proportion of SMARCA2 mutant upregulated genes that are selectively expressed in astrocytes and have increased accessibility in SMARCA2 mutant NPCs.

Figure 6.. Pioneering activity of AP-1 retargets SWI/SNF complexes to de novo enhancers.

A. Enrichment of de novo motifs on sites that gain accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. B. De novo FRA1 (bZIP) motif identified in A. C. (Bottom) Enrichment (-log p value) of motifs +/− 50bp of AP-1 sites with increased accessibility in SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. (Top) Percent of AP-1 sites with increased accessibility that have the indicated motif. D. FPKM of RNA-seq of AP-1 family members in WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. Error bars = mean ±SD,n=2. *FDR < 0.05. E. Immunoblot and quantitation of FRA2 in WT, SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs. F. Heat map of FRA2 ChIP-seq tags at differentially accessible sites clustered as in Figure 3B. G. FRA2 co-immunoprecipitation of SMARCA4, AM-SMARCA2, and AM-SMARCA2 R1159Q in WT NPCs. H-J. Histogram of normalized ChIP-seq tags for SMARCA4 (H), SMARCA2 (I), and ATAC-seq tags (J) at DE sites that gain FRA2 binding in both SMARCA2^K755R/+ and SMARCA2^R1159Q/+ NPCs (n=7,312). K-M. Histogram of normalized ATAC-seq tags (K), or ChIP-seq tags for SMARCA4 (L) and SMARCA2 (M) in SMARCA2^K755R/+ NPCs transfected with siRNAs against control or FRA1/FRA2/JUNB at DE sites that gain FRA2 binding in SMARCA2^K755R/+ NPCs (n=10,900). N. Proportion of genes with increased expression, accessibility, and FRA2 binding in SMARCA2 mutant NPCs. See also Figure S5.