Figure 3. Identification of constitutively silenced Xp genes.
(A) Heatmap showing the 6 gene categories identified on the basis of their Xp silencing dynamics in both CM and MC crosses. Grey color, not detected. Sat1 and Pdzd11 in category 4 were removed from the downstream analyses due to strong inconsistency between CM and MC crosses.
(B) Boxplots showing the Xp silencing dynamics of 6 categories. N represents the number of genes in each category. The same N numbers are applied to all the boxplots below representing gene categories.
(C) Paternal expression fraction in each category at late 2C stage. P value was calculated by Mann-Whitney U test.
(D) Hierarchical clustering of 6 categories based on similarity in averaged silencing dynamics on Xp.
(E) Nascent RNA FISH of representative constitutive genes (white arrowheads) in late 2C embryos. Two alleles of early silenced gene Gnl3l (white arrows) are also labeled as an internal control. Scale bar: 5μm.
(F) Paternal fraction of reads from zygotic RNA transcripts in late 2C embryos by 4sU-seq. n=20 in category 4, n=16 in category 1 and n=9 in category 2+3. P values were calculated by Mann-Whitney U test.
(G) Linear distance of genes in each category to the Xist locus. *p=0.025, **p=0.001, ***p=0.010 and ****p<0.001 by Mann-Whitney U test.
(H) Interaction frequencies between Xist and genes of each category. P values calculated by Mann-Whitney U test.
(I) Relationship between gene categories and evolutionary age, as determined by location within evolutionary strata49. Left: Enrichment of each evolutionary stratum in indicated category. Right: Enrichment of each category in indicated evolutionary stratum. Significance of enrichment determined by fisher’s exact test.
(J) Xm hyperactivation parallel tracks Xp silencing. Maternal RPKM value for each gene is normalized to its male homolog at the same stage. P values were calculated by Wilcoxon Signed-Rank test. P > 0.05 between stages in Category 4 or 5.