Fig. 7: Genome-wide investigation of H4S47 O-GlcNAcylation function at origins.
From: H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins
a–c, Detection of activated origins by SNS-seq. HEK293T cells were prepared as in Figure 1a. Heat maps (a) and box plots (b) show the signals of SNS among the centers of all peaks (n = 48,835) in H4WT and H4WT cells treated with PUG. P values were calculated by unpaired, two-tailed t-tests using R. For box plots, the bottom, middle line, and top of the box indicate the 25th, 50th, and 75th percentiles, respectively, and whiskers indicate minimum and maximum values (b). Genome tracks show the signals of SNS. PCCA, ZNF644, GAU1, and GRM5-AS1 are RefSeq gene names (c). d, Analysis of H4 O-GlcNAcylation at replication origins by Re-ChIP. Sequential ChIP analyses using specific antibodies that recognize O-GlcNAc modification (RL2 antibody) and H4 were conducted in HEK293T cells. IgG was used as a negative control. Published replication origins, as well as their flanking regions (indicated by the distance from the origin; kb)15, were measured by quantitative PCR. The y axis is the ratio of DNA in immunoprecipitate to that in input. OR, origin. TOP1: n = 3, MCM4: n = 3, ChrX (ENm006): n = 4 biologically independent experiments. e, Impact of H4S47 O-GlcNAcylation on MCM2 pS53 at replication origins. HEK293T cells, prepared as in Figure 1a, were analyzed by Re-ChIP, in which antibodies against Flag that recognize Flag-H4 and MCM2 pS53 were added sequentially. The y axis is the ratio of Flag-H4 and MCM2 pS53 Re-ChIP signals to the corresponding Flag-H4 ChIP signals. TOP1: n = 4, MCM4: n = 5, ChrX (ENm006): n = 6 biologically independent experiments. Two-way ANOVA with Tukey’s test, means ± s.d., n.s., non-significant (d,e). f, Venn diagram showing SNS-seq peaks identified in HEK293T cells that were impaired with mutated H4S47 or were unimpaired. g, Analysis of the distribution of SNS peaks for replication timing. Histogram shows the distribution of replication timing values for H4S47A impaired peaks (n = 17,037) and unimpaired peaks (n = 20,553) identified by SNS-seq in HEK293T cells. RT, replication timing.
