Fig. 6: MommeD43 results in depleted H3K27me3 on the inactive X chromosome.

a ChIP-seq track of H3K27me3 in Smchd1^{MommeD43-GFP/MommeD43-GFP} vs Smchd1^GFP/GFP primary female NSCs at the Hoxb cluster, n = 3 biological replicates per genotype. The track shows no differences in H3K27me3 localization (ChIP-seq is not a directly quantitative technique, and so the difference in peak height between the two tracks might not be of biological significance). b Scatter plot of log-transformed normalized H3K27me3 ChIP-seq counts in Smchd1^{MommeD43-GFP/MommeD43-GFP} vs Smchd1^GFP/GFP NSCs around (±2.5 kb, then merged if within 1 kb) peaks determined from both datasets. The Pearson coefficient indicates very high positive correlation. c MA plot showing normalized CTCF ChIP-seq values over peaks. On the x axis, Smchd1^{MommeD43-GFP/MommeD43-GFP} - Smchd1^GFP/GFP log2 fold change, and on the y axis the average between them. Highlighted are peaks showing statistically significant differential binding, in cyan for Smchd1^{MommeD43-GFP/MommeD43-GFP}, and in magenta those found in Smchd1^del/del, both compared to Smchd1^GFP/GFP (FDR 0.1), n = 3 biological replicates per genotype. d Representative images of three independent immunofluorescence staining experiments with anti-H3K27me3 (green), anti-SMCHD1 (orange), and DAPI (DNA stain, cyan) in primary female Smchd1^+/+ (top) and Smchd1^{MommeD43/MommeD43} (bottom) NSCs. The arrow points to the inactive X chromosome which is characterized by very high levels of both H3K27me3 and SMCHD1. e–g Scoring (mean ± SD) of the volume and total levels of H3K27me3 and Smchd1 of the inactive X chromosome in female Smchd1^fl/fl, Smchd1^del/del, Smchd1^+/+, and Smchd1^{MommeD43/MommeD43} (ENU mutant and CRISPR mutant) primary female NSCs. Levels of H3K27me3 and Smchd1 are defined as the total intensity of fluorescence inside the volume of the inactive X, normalized by the total nuclear volume of each scored cell, then by the mean of all datapoints in its respective control. The inactive X chromosome is defined as the high H3K27me3 region in a semi-automated approach using Imaris to define a closed surface of highest green fluorescence in each nucleus. MommeD43 abbreviated to MD43. The percentage displayed is the ratio between each Smchd1 variant and its respective control. N provided for each sample in the figure. Unpaired t tests two-tailed p values displayed for each comparison.