Figure 4. Hdac1 maintains differential H3 acetylomes between germ layers.

(A) Embryos treated with 100 nM Trichostatin A (TSA) displaying gastrulation defects 24 hr post-fertilization. Asterisk denotes the dorsal side containing the early blastopore lip. (B) Western blot analyses showing various histone acetylation modifications affected by HDAC inhibition. Anti-H3 is used as a loading control. (C) Clustered heatmap depicting signals of pan-H3Kac at st10.5 in whole embryos (WE), animal cap (AC), and vegetal mass (VG) cells. The signals are shown in a window of 5 kb centered on the summits of combined AC and VG peaks presented in descending order within each cluster. (D) Stacked bar graph representing proportions of localized (LC) versus non-localized (NL) pan-H3Kac signals found at Hdac1 cis-regulatory module (CRM) clusters in Figure 3C. A Hdac1 peak is considered to exhibit localized pan-H3Kac if it overlaps with either AC- or VG-specific pan-H3Kac peaks (Cluster B or C in C); a Hdac1 peak is considered to exhibit non-localized pan-H3Kac if it overlaps with pan-H3Kac peaks shared between AC and VG (Cluster A in C). NP: not overlap with any pan-H3Kac peak. (E) Spike-in normalized pan-H3Kac signals across Hdac1 CRM clusters (clusters in Figure 3C) in Dimethy Sulfoxide (DMSO)- or TSA-treated AC explants. ${\displaystyle \overline{\mathrm{\Delta }}}$ represents the log2 scaled average differences of spike-in normalized pan-H3Kac signals between DMSO- and TSA-treated AC explants. Randomized genomic regions (n = 23,442) are used as the negative control. (F) Fold changes (FC) of pan-H3Kac signals at Hdac1 CRM clusters (clusters in Figure 3C) in DMSO- or TSA-treated AC explants. Red dotted line denotes the level of zero. *** denotes p < 0.001 (Student’s t-test). (G) Fold changes (FC) of pan-H3Kac signals in Cluster I of Hdac1 CRM clusters (clusters in Figure 3C) for each spatial CRM category. d denotes effect size calculated by Cohen’s d.

Figure 4—figure supplement 1. Trichostatin A (TSA)-mediated HDAC inhibition does not alter Hdac1 genomic occupancy.

(A) Embryos treated with 10 mM valproic acid (VPA) display gastrulation defects at 24 hr post-fertilization. (B) Western blot analyses showing protein levels of Hdac1 and Hdac2 upon TSA treatment in st9 and st10.5 embryos. b-Tubulin is used as a loading control. (C, D) Pairwise Pearson correlation analyses comparing Hdac1 ChIP-seq signals of (C) st9 and (D) st10.5 DMSO- and TSA-treated embryos. (E, F) Pairwise Pearson correlation analyses comparing Hdac1 ChIP-seq signals of (E) st9 and (F) st10.5 DMSO- and TSA-treated embryos among different sample batches. WT denotes two biological replicate samples from time-course experiments. (G, H) Differential ChIP-seq analysis of (G) st9 and (H) st10.5 Hdac1 ChIP-seq between DMSO- and TSA-treated embryos. Red dots denote significant and gray dots denote nonsignificant differential irreproducibility discovery rate (IDR) peaks.

Figure 4—figure supplement 2. Differential H3 acetylomes are established in different germ layers.

(A) A schematic diagram of dissection experiments to separate animal cap tissues (AC) and vegetal mass tissues (VG) for ChIP-seq or RNA-seq. Late blastula stage explants were cultured ~1–1.5 hr to the early gastrula stage before being harvested for analyses. (B) Venn diagram comparing pan-H3Kac peaks in AC and VG. (C) Transcripts per million (TPM) expression of genes associated with distinct spatial pan-H3Kac clusters in Figure 4C. (D, E) Transcripts per million (TPM) expression of genes associated with (D) AC-specific pan-H3Kac and (E) VG-specific pan-H3Kac peaks across three germ layers. p-Values are calculated by Student’s t-test. (F) Genome browser visualization of pan-H3Kac signals at known ectodermal (foxi1, kctd15, lhx5, sox2), mesodermal (foxd3, myf5, myf6, tbxt), endodermal (foxa4, mixer, mix1, sox17a, sox17b.1, sox17b.2, vegt), and ‘housekeeping’ (h3.3, rbbp7, tbp) genes in whole embryos, AC, and VG explants. Y-axis values represent scaled track height. Black bars denote an interval of 2 kb.

Figure 4—figure supplement 3. Germ-layer-specific H3 acetylomes require HDAC activity.

(A) Spike-in normalized pan-H3Kac signals across Hdac1 cis-regulatory module (CRM) Clusters (clusters in Figure 3C) in DMSO- or Trichostatin A (TSA)-treated vegetal mass (VG) explants. ${\displaystyle \overline{\mathrm{\Delta }}}$ represents the log2 scaled average differences of spike-in normalized pan-H3Kac signals between DMSO- and TSA-treated VG explants. Randomized genomic regions (n = 23,442) are used as the negative control. (B, C) Clustered heatmaps depicting signals of pan-H3Kac and Hdac1 at st10.5 (B) animal cap (AC) and (C) VG explants treated with DMSO and TSA. The signals are scaled using spike-in normalization factors and shown in a window of 5 kb centered on the summits of pan-H3Kac peaks in descending order within each cluster. (D) Fold changes (FC) of pan-H3Kac signals at Hdac1 CRM Clusters (clusters in Figure 3C) in DMSO- or TSA-treated VG explants. Red dotted line denotes the level of zero. *** denotes p < 0.001 (Student’s t-test). (E, F) Pan-H3Kac signals at Hdac1 CRM Clusters (clusters in Figure 3C) in DMSO- or TSA-treated (E) AC and (F) VG explants. Values for Y-axis are log2 transformed. Red dotted line denotes the level of zero. Student’s t-test is used to obtain p-values. (G, H) Scatter plots representing the anti-correlation between fold changes of pan-H3Kac signals and ranked endogenous pan-H3Kac signals in Hdac1-bound CRM Cluster I and Cluster III of AC or VG explants shown in Figure 3C. Gray lines depict linear regression curves generated from a linear model. Both X and Y axes’ values are log2 transformed. (I) Fold changes (FC) of pan-H3Kac signals representing each spatial CRM category in Cluster III (clusters Figure 3C). d denotes effect size calculated by Cohen’s d.