ATAC-seq identifies accessible chromatin domains in gastrula stage Xenopus laevis

In the embryo, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner, and the mechanisms for the loss of responsiveness are not well understood. To test the hypothesis that loss of competence is associated with changes in chromatin accessibility, we adapted the assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) for use in Xenopus laevis to evaluate genome-wide changes in chromatin accessibility in early and late gastrula stages. ATAC-seq was performed on presumptive ectoderm (animal caps) explanted at the blastula stage and cultured until siblings reached early (stage 10) or late (stage 12) gastrula stage. Approximately 70,000 accessible regions were identified at intergenic regions, introns, promoters, and transcription termination sites at both stages. Accessibility decreased from stage 10 to stage 12 at 279 promoters, including developmental regulators such as Foxh1, chordin, and VegT. Accessible chromatin domains overlap extensively with previously reported p300 binding sites, consistent with putative cis-regulatory modules (pCRMs), and these pCRMs are enriched for binding motifs for pluripotency factors, including Sox, Oct/Pou, and KLF binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence, but inhibition of histone deacetylases increases acetylation at these promoters and extends competence for dorsal gene induction by Wnt signaling. In contrast, the promoters for genes involved in mesoderm and neural crest development remain open through gastrulation, and histone deacetylase inhibition does not 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. ATAC-seq performed on ectodermal explants from stage 10 (early gastrula) and stage 12 (late gastrula).

在胚胎发育进程中，具备感应能力的组织会以时空受限的方式解读诱导信号，而组织丧失响应能力的具体机制目前尚未完全阐明。为验证“感应能力丧失与染色质可及性改变相关”这一科学假说，我们将转座酶可及性染色质高通量测序（ATAC-seq）适配至非洲爪蟾（Xenopus laevis）实验体系，以检测早期与晚期原肠胚阶段的全基因组染色质可及性变化。本实验以囊胚阶段分离的预定外胚层（动物帽，animal caps）组织为实验材料，将其体外培养至同期胚胎分别达到早期（10期）或晚期（12期）原肠胚阶段后，对其开展ATAC-seq检测。在两个发育阶段，研究人员均在基因间区、内含子、启动子及转录终止位点处鉴定得到约7万个染色质可及区域。在279个启动子区域中，染色质可及性从10期至12期出现下降，其中包含Foxh1、chordin、VegT等发育调控因子的启动子。上述染色质可及区域与此前报道的p300结合位点存在广泛重叠，这与推定顺式调控模块（pCRMs）的特征高度吻合；且这些pCRMs显著富集多能性因子的结合基序，包括Sox、Oct/Pou及KLF结合基序。背侧Wnt靶基因的启动子在组织丧失感应能力后不再具备可及性，而抑制组蛋白去乙酰化酶可提升这些启动子区域的乙酰化水平，并延长Wnt信号介导的背侧基因诱导所需的感应能力。与之形成对比的是，参与中胚层及神经嵴发育的基因启动子在整个原肠胚阶段始终保持可及状态，且抑制组蛋白去乙酰化酶无法延长中胚层或神经嵴诱导所需的感应能力。上述实验数据表明，染色质状态以环境依赖的方式调控组织对诱导信号响应能力的丧失过程。本研究的ATAC-seq实验样本取自10期（早期原肠胚）及12期（晚期原肠胚）的外胚层分离组织。