Chromatin Accessibility Analysis Reveals Distinct Functions for HDAC and EZH2 Activities in Early Regeneration

Xenopus tropicalis tadpoles have the capability to scarlessly regenerate appendages including the limb and tail. Following injury, complex genetic processes must be activated and inactivated with high spatial and temporal resolution to result in a properly patterned appendage. Functional studies have established that histone modifying enzymes that act to close chromatin are required for regeneration, but the genomic regions sensitive to these activities are not established. Here we show that early inhibition of HDAC or Ezh2 activity results in incomplete regeneration. To identify the impact that each of these perturbations has on chromatin accessibility, we applied an assay for transposase accessible chromatin (ATAC-seq) to HDAC or Ezh2 inhibited regenerating tadpoles. We find that neither perturbation results in a global increase in chromatin accessibility, but that both inhibitors have targeted effects on chromatin accessibility and gene expression. Upon HDAC inhibition, promoter regions and regulatory regions neighboring genes associated with neuronal regeneration are precociously accessible, whereas following EZH2 inhibition we find gene bodies and regulatory regions associated with regulation of the immune response and apoptosis are preferentially accessible. Together this suggests distinct roles for EZH2 activity and HDAC activity in appendage regeneration. ATAC-seq timecourse analysis of chromatin dynamics of HDAC inhibited or EZH2 inhibited regenerated tadpole tails and their vehicle controls. Samples in duplicate.

热带爪蟾（Xenopus tropicalis）的蝌蚪具备对肢体与尾部等附肢进行无瘢痕再生的能力。机体遭受损伤后，需以极高的时空分辨率精准激活并关闭复杂的遗传程序，方可形成结构与模式均正常的附肢。已有功能研究证实，介导染色质闭合的组蛋白修饰酶对再生过程不可或缺，但这类酶所作用的基因组敏感区域尚未明确。本研究发现，早期抑制组蛋白去乙酰化酶（HDAC）或Ezh2活性会导致再生过程不完全。为明确这两种扰动对染色质可及性的影响，我们对经HDAC或Ezh2抑制剂处理的再生蝌蚪开展了转座酶可及性染色质测序（ATAC-seq）分析。结果显示，两种抑制剂均未引发染色质可及性的全局升高，但二者均对染色质可及性与基因表达具有靶向调控作用。在HDAC抑制条件下，与神经元再生相关基因邻近的启动子区域及调控区域会提前获得可及性；而在EZH2抑制后，与免疫应答调控及细胞凋亡相关的基因本体区域及调控区域则呈现出优先可及性。综上，本研究结果表明EZH2与HDAC活性在附肢再生过程中发挥着截然不同的生物学功能。本数据集包含HDAC抑制、EZH2抑制的再生蝌蚪尾部及其溶剂对照组的染色质动态变化的ATAC-seq时序分析数据，所有样本均设置双份生物学重复。