XB-IMG-176854
Xenbase Image ID: 176854
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Figure S5FHS SRCAP Defects Are in Part Due to Histone Variant H2A.Z.2 Dysfunction, Related to Figure 5.
A) Confocal microscopy of immunofluorescence staining in CNCCs with H2A.Z.1 or H2A.Z.2 endogenously V5- tagged; DAPI in blue, V5 (mouse antibody) in green. 10μm scale bar denoted.
(B) ChIP-seq traces for V5-tagged H2A.Z.1 (lines #1-#3 overlaid, shown in gray) and V5-tagged H2A.Z.2 (lines #1-#3 overlaid, shown in gray) at the SOX9 gene. Alignment to hg19, window at chr17: 69,940,012-70,184,231. H2A.Z.1 biased peaks shaded in purple, H2A.Z.2 biased peaks shaded in green, unbiased peaks shaded in gray.
(C) Identification of statistically significant H2A.Z.1 and H2A.Z.2 biased peaks. Plot of H2A.Z.1 ChIP enrichment versus H2A.Z.2 ChIP enrichment with identification of significant Z.1 sites (red), significant Z.2 sites (green), and not significant sites (grey). X-axis is mean counts, which are normalized reads for each site with an adjusted mean for a base factor.
(D) Association of H2A.Z.1 and H2A.Z.2 biased peaks with regulatory elements. Plot of H2A.Z.1 ChIP enrichment versus H2A.Z.2 ChIP enrichment with identification of promoters (red), enhancers (blue), and other sites (grey). X-axis is mean counts, which are normalized reads for each site with an adjusted mean for a base factor.
(E) All H2A.Z.1 and H2A.Z.2 biased peaks GREAT analysis - x-axis is distance to TSS in kb, y-axis is number of region-gene associations. TSS is indicated on graph (McLean et al., 2010).
(F) All H2A.Z.1 and H2A.Z.2 biased enhancers, as defined by chromatin modifications (Figure 4E) - GREAT analysis - x-axis is distance to TSS in kb, y-axis is number of region-gene associations. TSS is indicated on graph. Background set all identified enhancer regions from CNCCs (McLean et al., 2010).
(G) GO biological processes terms for H2A.Z.1 and H2A.Z.2 biased enhancers with description, binomial rank, p-value, and FDR (McLean et al., 2010). Processes relevant to FHS and neural crest development in red.
(H) GO phenotypes (human and mouse) terms for H2A.Z.1 and H2A.Z.2 biased enhancers with description, binomial rank, p-value, and FDR (McLean et al., 2010). Phenotypes relevant to FHS symptoms in red.
(I) CRISPR/Cas9 targeting strategy in hESCs for retargeting the V5-tagged H2A.Z.2 line with endogenous tagging and/or truncation of SRCAP gene using homologous recombination with ultramers to add FLAG-HA tag. SRCAP and H2A.Z.2 protein domains annotated as previously described.
(J) Validation of FLAG-HA tagged WT SRCAP and FHS mutant CNCCs by whole cell immunoprecipitation for FLAG with immunoblotting against HA tag. Immunoblot against input sample for V5 tag, endogenous SRCAP protein, and HSP90 as a loading control, with untargeted H9s as negative control.
(K) Association of statistically significant changes in H2A.Z.2 incorporation between WT and FHS MUT CNCCs with regulatory elements. Plot of H2A.Z.1 ChIP enrichment versus H2A.Z.2 ChIP enrichment with identification of TSSs/promoters (red) and other non-TSS sites including enhancers (blue). X-axis is mean counts, which are normalized reads for each site with an adjusted mean for a base factor.
Image published in: Greenberg RS et al. (2019) Copyright © 2019. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |