Fig. 4: Monoallelic CG-motif factor binding and CpG methylation after MSL2 loss.
a, The top 20 enriched transcription factors from enhancer and promoter motif analysis on the active allele of bi-to-mono genes in male CaBl and BlCa and female 9sCa Msl2-KO NPCs. The dot size shows the motif fold enrichment and the colour shows −log10[P]. Var, binding variant. b,c, ChIP–seq metagene plots of the indicated factors and histone acetylation at the TSS and the CpG methylation frequency at the TSS (left) of bi-to-mono genes and differentially methylated loci (DML, false-discovery rate (FDR) < 1 × 10−5) (right) in male CaBl (b) and BlCa (c) WT and Msl2-KO NPC clones. The log2[FC] values of ChIP–seq levels (IP/input) are displayed with the standard error (shadows). d, RT–qPCR analysis of Zfp422 mRNA levels in 6 h dBET-treated or DMSO-treated (mock) male CaBl and BlCa WT and Msl2-KO NPCs. mRNA levels relative to Rplp0 expression are shown. Significance was determined using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. Exact P values are as follows: CaBl: **P = 0.0067, *P = 0.0133; BlCa: ****P < 0.0001, ***P = 0.0003, *P = 0.0193. n = 3 independent experiments. Data are mean ± s.e.m. e, Anticorrelation between monoallelic CG-motif factor binding and CpG methylation in male CaBl Msl2-KO NPCs. The log2[FC] (allele 2/allele 1) in NRF1, SP1 and KANSL3 ChIP–seq signal (IP/input) (top) and the subtract (allele 2 − allele 1) of CpG methylation frequency (bottom). Allele-1-biased (magenta) and allele-2-biased genes (cyan) are shown. The diagrams were created using BioRender. f, The log2[FC] (KO/WT) in allelic NRF1 ChIP–seq signal (IP/input) (top) and CpG methylation (bottom) at genes with consistent bi-to-mono changes in male NPCs (Fig. 2a,d) separated into paternal (left; n = 16) or maternal (right; n = 30) change. Significance was determined using two-sided nonparametric Wilcoxon rank-sum tests; exact P values are indicated in the figure. Details on the box plots are provided in the Methods.
