The BAF chromatin complex component SMARCC1 does not mediate GLI transcriptional repression of Hedgehog target genes in limb buds

Abstract

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes.

Keywords: BAF; GLI3; Hedgehog; Limb bud; SWI/SNF; Transcriptional repression.

Figure 1.. SMARCC1 binds to active limb enhancers and GLI binding regions.

A. SMARCC1 binding regions in anterior and posterior halves of E11.5 wild-type forelimbs identified by CUT&RUN (n=3). B. SMARCC1 and previously identified H3K27ac binding in E11.5 forelimbs are co-enriched. C. Most VISTA limb enhancers are bound by SMARCC1. D. Intersection of all anterior SMARCC1-binding regions with previously identified E10.5 GLI3 binding regions. E. GLI3 anterior SMARCC1 binding at select GLI target genes.

Figure 2.. SMARCC1 has GLI-independent roles in regulating chromatin accessibility.

A, B. Differential ATAC-seq on E11.5 control (PrxCre⁻;Smarcc1^c/c; n=2) and PrxCre⁺;Smarcc1^c/c (n=3) anterior forelimbs. Normalized enrichment plot indicates ATAC enrichment for all regions (grey; n=100,188) and all GLI enhancers (magenta; n=309). C. ATAC fold enrichment in control vs. PrxCre⁺;Smarcc1^c/c samples across all ATAC accessible regions (grey) and GLI enhancers (magenta) showing significantly decreased accessibility in mutants (Wilcoxon signed rank test; ***p<2.2e-16). D. Approximately half of GLI enhancers have altered ATAC accessibility in PrxCre⁺;Smarcc1^c/c forelimbs. E. Tracks showing GLI3 binding (green), and ATAC accessibility in control and mutant samples.

Figure 3.. GLI3 does not recruit or maintain SMARCC1 at enhancers.

A. SMARCC1 binds to a minority of H3K27ac-enriched (H3K27ac⁺), promoter-proximal GLI-bound enhancers (GLI-enh.) at E9.25 (21–24s). B,C. Representative tracks showing GLI3 and H3K27ac enrichment at previously defined nascent GLI enhancers with (B) and without (C) SMARCC1 binding (magenta) at E9.25. D. SMARCC1 binding determined by CUT&RUN on control (Gli3^+/+; n=3) and Gli3^−/−(n=4) anterior forelimbs at E11.5 (40–43s). E. Intersection of significant (FDR<0.05) SMARCC1 enrichment at GLI enhancers in control and Gli3^−/− forelimbs. F. Fold enrichment of SMARCC1 at GLI enhancers in control and Gli3^−/− forelimbs (Wilcoxon signed rank test). G,H. Representative tracks showing SMARCC1 binding at GLI enhancers.

Figure 4.. Smarcc1 is not required for GLI-repression.

A-H. Sox9 expression in control (n=3; A,E), PrxCre⁺;Smarcc1^c/c (n=4; B,F), Shh^−/− (n=4; C,G), and PrxCre⁺;Smarcc1^c/c; Shh^−/− (‘DKO’) (n=3; D,H) fore- (A-D) and hindlimbs (E-H) at E12.5. Scale bars denote 10mm. I-K. Proposed model showing BAF-independent GLI transcriptional repression.