Esmaeili M et al. (2020), Chromatin accessibility and histone acetylation...

XB-ART-56750

Dev Biol 2020 Jun 01;4621:20-35. doi: 10.1016/j.ydbio.2020.02.013.

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Chromatin accessibility and histone acetylation in the regulation of competence in early development.

Esmaeili M , Blythe SA , Tobias JW , Zhang K , Yang J , Klein PS .

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As development proceeds, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner. While key inductive signaling pathways within competent cells are well-described at a molecular level, the mechanisms by which tissues lose responsiveness to inductive signals are not well understood. Localized activation of Wnt signaling before zygotic gene activation in Xenopus laevis leads to dorsal development, but competence to induce dorsal genes in response to Wnts is lost by the late blastula stage. We hypothesize that loss of competence is mediated by changes in histone modifications leading to a loss of chromatin accessibility at the promoters of Wnt target genes. We use ATAC-seq to evaluate genome-wide changes in chromatin accessibility across several developmental stages. Based on overlap with p300 binding, we identify thousands of putative cis-regulatory elements at the gastrula stage, including sites that lose accessibility by the end of gastrulation and are enriched for pluripotency factor binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence in the early gastrula while genes involved in mesoderm and neural crest development maintain accessibility at their promoters. Inhibition of histone deacetylases increases acetylation at the promoters of dorsal Wnt target genes and extends competence for dorsal gene induction by Wnt signaling. Histone deacetylase inhibition, however, is not sufficient to extend competence for mesoderm or neural crest induction. These data suggest that chromatin state regulates the loss of competence to inductive signals in a context-dependent manner.

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Species referenced: Xenopus laevis
Genes referenced: 1a11 axin2l barhl2 cdx2 chrd crebbp foxb2 foxh1.2 gs17 gsc hesx1 homer1 hoxa1 hoxd1 jarid2 lefty1 myod1 nodal3 odc1 pax3 pou3f2 pou5f3 pou5f3.2 pou5f3.3 sia1 sia2 snai1 snai2 tbxt tcf3 twist1 vegt wnt5b wnt8a
???displayArticle.antibodies??? H3f3a Ab24 H3f3a Ab26 H3f3a Ab39 IgG Ab1

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	Fig. 1. Characterization of Chromatin Accessibility at the onset of Gastrulation A. Distribution of ATAC-seq peaks at the gastrula stage. ATAC-seq was performed on ectodermal explants from early gastrula (stage 10) embryos (3 biological replicates). Sequences were aligned to the xenLae2 genome and peaks were called with MACS2 and annotated with HOMER. Peaks that had 0 reads in 1 or more of the replicates (1.3% of total peaks called) were excluded from this analysis. B. Number of accessible regions in each chromosome. Homeologous chromosomes (â€œLâ€ â€‹= â€‹long, â€œSâ€ â€‹= â€‹short chromosome) are represented in the same color. An additional 6805 peaks mapped to scaffolds that have not been assigned to a chromosome. C. Correlation between promoter accessibility and gene expression in stage 10 ectodermal explants. Log2 of mRNA expression detected by microarray (Livigni et al., 2013) was plotted vs. log2 of normalized mean ATAC reads for peaks within 1 â€‹kb of the TSS. The data were divided into 4 quadrants based on the median value for each axis so that an equal number of data points was used to analyze each quadrant. D. Genes from each quadrant in panel D were subjected to functional annotation using DAVID, which showed limited enrichment for terms with FDR <0.01, except for the group with higher ATAC reads and lower expression levels (lower right quadrant in D). The -Log10 of the FDR is plotted on the horizontal axis.
	Fig. 2. Dorsal genes are inaccessible at the onset of Gastrulation A. Embryos were cultured in 0.1xMMR and Wnt signaling was activated by exposing embryos to a 10â€“12 â€‹min pulse of 0.3M LiCl at the indicated stages. Activation of Wnt signaling in cleavage stage embryos leads to radial dorsalization (center). Activation of Wnt signaling at the late blastula stage leads to posteriorization (right). Controls are untreated stage 33 tadpoles. For this and following experiments, â‰¥ 80% of embryos treated with LiCl at the cleavage stage showed dorsalization at tadpole stages, with complete lack of trunk structures, no obvious somites, expanded and frequently circumferential eyes, and frequently expanded cement gland (DAI â€‹â‰¥ â€‹8 (Kao and Elinson, 1988; Karimi et al., 2018)). Similarly, â‰¥80% of embryos exposed to LiCl at the late blastula stage demonstrated anterior truncation/posteriorization, with reduced or absent forebrain and small or absent cement gland, frequently with small or absent eyes and microcephaly. B. Wnt signaling was activated by exposure to LiCl at cleavage stage (stage 6) or the blastula stage (stage 9) and expression of dorsal Wnt target genes sia1 and nodal3.1 and non-dorsal Wnt target genes (hoxa1, hoxd1, and cdx2) was measured by RT-qPCR in whole embryos at the midgastrula stage (stage 10.5). Gene expression in all samples was normalized to ODC and then expression in embryos exposed to LiCl at stage 6 (blue bars) and stage 9 (orange bars) was normalized to untreated (grey) and are presented as mean values for fold change in expression for 5 biological replicates except for nodal3.1 which had 3 replicates. Error bars represent standard error of the mean (SEM). C. Representative sequencing tracks for the dorsal Wnt target genes sia1 and nodal3.1, the organizer marker Gsc, and non-dorsal Wnt target genes hoxa1, hoxd1, and cdx2. Y-axis represents normalized reads scaled by 1,000,000/total alignments. Scale bar represents 1 â€‹kb.
	Fig. 3. Repressive histone modifications H3K9me3 and H3K27me3 are not associated with loss of competence A. Model for chromatin accessibility mediating loss of competence. Left: Open chromatin state predicted to be associated with competence. â€œAcâ€ represents histone acetylation, typically associated with active or accessible chromatin. Right: Chromatin inaccessibility predicted to be associated with loss of competence. â€œMeâ€ represents histone methylation at sites that are typically associated with repressive states or closed chromatin, such as H3K9me3 or H3K27me. B. ChIP-qPCR for H3K27me3 (orange) at Wnt target genes sia1 and nodal3.1 in dorsal versus ventral halves of gastrula stage (stage 10) embryos. IgG controls in grey. Embryos were cultured in 0.1xMMR until gastrula stage, fixed, dissected into dorsal and ventral halves using the dorsal blastopore lip as a marker, and subjected to ChIP. C. ChIP-qPCR for H3K27me3 in whole embryos at the sia1, nodal3.1, and gsc promoters was also performed at early (st 10) and late (st 12.5) gastrula stages. âˆ—p â€‹< â€‹0.05 for comparison of H3K27me3 to IgG background control (one-tailed t-test); â€œn.s.â€ indicates not significant. D. ChIP-qPCR for H3K9me3 (green) at promoters for sia1 and nodal3.1, as well the control genes 1a11 and xretrop(L), in dorsal and ventral halves of gastrula stage embryos. IgG controls in grey. Embryos cultured, dissected, and collected as in B. Data in B - D represent means of 3 biological replicates and error bars show SEM.
	Fig. 4. Histone deacetylase activity suppresses competence for dorsal induction A. Embryos were cultured with and without 100 â€‹nM Trichostatin A (TSA) beginning at the 64-cell stage and collected at the onset of gastrulation. ChIP-qPCR was performed for H3K9ac (blue) at the sia1 and nodal3.1 promoters. IgG controls are in grey. B. Embryos were treated with or without 100 â€‹nM TSA beginning at the 32â€“64 â€‹cell stage and exposed to LiCl in the late blastula stage as in Fig. 2. Ectodermal explants were dissected at the onset of gastrulation and harvested immediately to measure expression of sia1 and nodal3.1 as in Fig. 2. As sia1 and nodal3.1 were frequently undetectable in explants from control embryos, it was not always possible to calculate a fold-increase relative to control. The histogram shows the mean of two replicates for which sia1 was detectable in controls. Regression analysis found a significant increase in both sia1 and nodal3.1 expression in explants treated at stage 9 with TSA and lithium compared to lithium alone (for sia1, mean Î”Î”Ct â€‹= â€‹2.09 with p â€‹< â€‹0.0001; for nodal3.1, mean Î”Î”Ct â€‹= â€‹2.65, p â€‹< â€‹0.0001) or TSA alone (for sia1, mean Î”Î”Ct â€‹= â€‹1.67 with p â€‹= â€‹0.0004; for nodal3.1, mean Î”Î”Ct â€‹= â€‹1.65 with p â€‹= â€‹0.008); there was no significant interaction between lithium and TSA. C. Embryos were exposed to LiCl and TSA as in panel B and harvested at the onset of gastrulation to measure whole embryo expression of sia1 and nodal3.1. D. Embryos were exposed to LiCl and TSA as above and ventral marginal zones were dissected at the onset of gastrulation and harvested immediately to measure expression of sia1 and nodal3.1.
	Fig. 5. Chromatin Accessibility and histone deacetylation do not correlate with loss of competence at later stages of development A. Representative sequencing tracks for the mesoderm genes tbxt, wnt8a, and myod1 at stage 10. The ATAC-seq data are normalized to total reads. Y-axis represents the number of normalized reads with the scale chosen for optimal visualization of peaks for each gene. Scale bar represents 1 â€‹kb in all panels. B. Diagram of explant assays for mesoderm and neural crest induction. On the left is a blastula embryo showing where ectodermal explants are dissected (dashed lines). In the center are explants that can be subjected to different treatments (FGF or Wnts) to induce mesoderm or neural crest, respectively. On the right are the stages at which the ectodermal explant loses competence for a given inductive signal. C. For mesoderm induction, explants were dissected at the late blastula stage and cultured in FGF or control buffer and harvested at stage 13 for analysis of tbxt expression by RT-qPCR. A separate group of embryos was treated with or without 100 â€‹nM TSA beginning at the late blastula stage (stage 9) until ectodermal explants were dissected at the gastrula stage. Explants were similarly cultured with or without FGF at early gastrula (stage 10.5) and harvested at stage 13 to measure induction of tbxt. All groups are normalized to untreated controls. D. Representative sequencing tracks for neural crest genes (snai1, snai2, twist) during competence (stage 10) and at loss of competence (stage 12). The ATAC-seq data are normalized to total reads. Y-axis represents the number of normalized reads with the scale chosen for optimal visualization of peaks for each gene. Scale bar represents 1 â€‹kb. E. RT-qPCR measuring gene expression of neural crest genes (snai1, snai2, twist) normalized to housekeeping gene ODC. Each blastomere of 2-cell embryos was injected in the animal pole with mRNAs encoding Chordin (0.5ng/blastomere) and THVGR (10pg/blastomere). Ectodermal explants were dissected at the late blastula stage (stage 9) and cultured until early gastrula (stage 10). A subset of explants were cultured in control buffer and then treated with dexamethasone at early gastrula stage (stage 10) to induce THVGR and activate Wnt signaling. The rest of the ectodermal explants were cultured in 100 â€‹nm TSA. Of the TSA treated explants, a subset was treated with dexamethasone at early gastrula (stage 10) or at late gastrula (stage 12.5). Explants were cultured until stage 22 when they were collected and expression for snai1, snai2, twist was measured by RT-qPCR. Data in panels C and E represent 3 biological replicates and error bars are SEM.
	Fig. 6. Differential Chromatin Accessibility at early versus late gastrulation A. Correlation of ATAC peaks within 1 â€‹kb of TSS at stage 10 vs stage 12 (FDR â€‹< â€‹0.05). B. Volcano plot of Log2 fold change in ATAC promoter peaks at stage 12/stage 10 versus -Log10 of false discovery rate (FDR). Dotted lines indicate the threshold for genes selected for functional annotation. C. Genes associated with ATAC peaks within 1 â€‹kb of the TSS that decrease by â€‹â‰¥ â€‹2 fold at stage 12 were submitted for functional annotation through DAVID, which revealed marked enrichment of DNA-templated transcription factors. Uniprot keywords are ranked according to false discovery rate with a cutoff FDR <0.01. The horizontal axis shows -Log10 of the FDR. A similar analysis of promoter peaks that increased â‰¥2 fold did not identify any enriched categories. D. Representative tracks for chordin (chrd), foxb2, and the temporal marker gs17 showing dynamic chromatin accessibility at the respective promoters.
	Fig. 7. Putative Cis-regulatory Modules in gastrula stage embryos A. Venn diagram of accessible regions at stage 10 (blue circle) versus p300 ChIP-seq peaks (yellow circle); overlap represents putative cis-regulatory elements. p300 peaks were determined by re-mapping ChIP-Seq data from (Session et al., 2016) to the xenlae2 genome. B. Accessible peaks within intergenic and intronic regions that were also bound by p300 (pCRMs) at stage 10 and which showed reduced accessibility at stage 12 were identified; genes with TSSs within 100 â€‹kb of these peaks (2074) were subjected to functional gene annotation using DAVID. Uniprot keywords are ranked according to false discovery rate with a cutoff FDR <0.01. C. Representative tracks for foxh1.2, hesx1, vegt, and lefty comparing p300-bound sites (top) to ATAC-seq peaks at stage 10 (middle) and stage 12 (lower tracks). Scale bar â€‹= â€‹1 â€‹kb. D. Transcription factor motifs associated with stage 10 pCRMs identified by HOMER (Heinz et al., 2010). Sites are ranked by -log10[p-value] (compared to genomic background); those with p â€‹â‰¤ â€‹10âˆ’170 are shown. E. Frequency of transcription factor motifs in pCRMs (blue) that showed reduced accessibility at stage 12 (Î”pCRMs) compared to genomic background (orange) and to all pCRMs (grey). p-values for the respective comparisons are shown to the right.
	Supplementary Figure 1: Chromatin accessibility by ATAC-Seq (X. laevis gastrula) vs Dnase-Seq (X. tropicalis, MBT) ATAC-Seq tracks from stage 10 (early gastrula) Xenopus laevis ectodermal explants (GSE GSE76059) were compared to Dnase-Seq tracks from stage 8.5 (MBT) Xenopus tropicalis embryos from (Gentsch et al 2019; GSE113186). Tracks were viewed in IGV. Scale bars indicate 1kb. Y-axes were adjusted for each gene to allow side-by-side comparisons and do not reflect relative intensity of signals between different genes, species, stage, or method of analysis. TSS: transcription start site. “Xl”: Xenopus laevis; “Xt: Xenopus topicalis.

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