Fig. 4: The I1600M CHD6 mutation hinders co-factor recruitment at autophagy gene promoters. | Nature Communications

Fig. 4: The I1600M CHD6 mutation hinders co-factor recruitment at autophagy gene promoters.

From: Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

Fig. 4

a In silico rendering of the second putative SANT-SLIDE domain structure for wild-type and mutant CHD6 (based on the published Chd1 SANT-SLIDE structure7) and zoomed views around the I1600M mutation (bottom). b NanoDSF melting profiles (first derivative of the 350/330 nm ratio; n = 3 independent runs) of FLAG-tag purified wild-type (dark blue) or I1600M (light blue) full-length CHD6 along a temperature gradient. *: significantly different mean; P < 0.01, two-tailed unpaired Student’s t-test. Scattering profiles along the gradient reveal no difference in aggregation (inset). c Overexpression of wild-type or mutant CHD6 cloned into a piggybac vector (left) in iPSCs exemplified by Venus-GFP levels or anti-HA western blotting of CHD6 (right); β-tubulin provides a control. Bar, 25 μM. d Network representation of GO terms associated with proteins co-purifying with wild-type CHD6 in steady-state iPSCs. e Heatmaps showing enrichment or depletion of GO terms/pathways linked to CHD6-interacting proteins upon starvation, etoposide treatment or in steady-state wild-type and mutant iPSCs. f Western blot of SMARCC1 co-immunoprecipitating with overexpressed wild-type (wt) or mutant CHD6 (mut) in iPSCs; anti-HA and anti-β-tubulin blots provide a control. g Representative genome browser views for SMARCA4/-B1/-C1 ChIP-seq aligned to CHD6 ChIP-seq from wild-type (wt) and monoallelic mutant iPSCs (mut) around the DNAAF1 and LAMP1 promoters. h Line plots showing average SMARCA4/-B1/-C1 ChIP-seq signal profiles in the 4 kbp around CHD6 peaks; signal from mutCHD6 ChIP-seq provides a reference. i Venn diagram (left) showing overlap of genes bound by mutCHD6 and SMARCA4/-B1/-C1. Bar plots (right) showing significantly-enriched GO terms associated with the 243 shared genes. *: significantly more than expected by chance; P < 10−3, hypergeometric test. j Bar plots showing changes in SMARCC1 or ACTL6A ChIP-qPCR signal (fold enrichment over wt ±SD; n = 2 independent experiments) from heterozygous (white) or monoallelic mutant iPSCs (blue). k Heatmaps (left) showing changes in mRNA levels (log2) in response to iPSC starvation at genes co-bound by CHD6 and SMARCA4/-B1/-C1. Pearson’s correlation coefficients for each pair are shown. Bar plots (right) showing significantly-enriched GO terms associated with these genes.

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