Revisiting Polycomb Repression in Appendage Regeneration

项目摘要 成年斑马鱼能迅速再生被切断的鳍，包括复杂的骨骼，恢复到原来的大小， form.来源于损伤诱导的去分化成熟细胞的谱系限制性祖细胞增殖 重新分化以恢复失去的鳍组织因此，理解细胞状态转换的控制 在分化状态和祖细胞状态之间，保留细胞身份，是理解鲁棒性的核心。 附肢再生基因表达程序的深刻变化驱动去分化和再分化， 差异化状态转换。染色质景观被认为是稳定的祖细胞和 分化的状态程序和因此调节的染色质动力学可能是状态转换的基础。 此外，染色质机制被认为在表观遗传学上维持了指导细胞凋亡的网格状位置标识。 鳍大小恢复量的再生产物。多梳抑制复合物2（PRC 2）抑制 通过Ezh 1/Ezh 2催化的组蛋白H3（H3 K27 me）的赖氨酸-27甲基化的基因表达。PRC2/H3K27me 沉默分化细胞中的发育调控基因，稳定祖细胞状态程序， 保持细胞命运和位置的身份，包括Hox代码。我们之前将H3 K27 me的移除 上调组蛋白去甲基化酶对与起始鳍相关的广泛基因激活的标记 再生最近，我们针对ezh 1和ezh 2产生成年可行的PRC 2突变斑马鱼。引人注目的是， 尽管全球H3 K27 me 2/3水平大大降低，但仍正常发生多轮鳍再生 伴随着升高的活化相关的H3 K27乙酰化。因此，H3 K27 me的大部分不是 在鳍片再生过程中调节状态转换或保持单元身份所需的。这些 结果挑战了PRC 2/H3 K27 me 3在脊椎动物再生方面的教条。我们将奉行 探索性研究，以区分几种解释鳍再生如何进行的假设 没有这种主要的抑制性组蛋白修饰。对于目标#1，我们将分析全基因组蛋白 使用新的CUT&Tag技术在野生型和PRC 2突变体再生鳍中的修饰模式。我们将 使用RNA-Seq将PRC 2依赖性转录组与改变的染色质景观相关联。在目标2中， 我们将在实验中通过瞬时表达Ezh 2来绕过我们的ezh 1/ezh 2突变体的致死性， 使用mRNA注射和可诱导的转基因方法进行胚胎发育。我们将描述 再生缺陷，如果有的话，在衍生的无效PRC 2成人对定义关键H3 K27 me控制的调控 鳍再生网络。综合结果将为一个更大的项目研究提供中心前提 染色质动力学和器官再生的细胞转换。更广泛的影响包括指导使用 基于染色质的干扰剂以增强再生医学和作为儿科神经胶质瘤的治疗剂， 其他癌症和由破坏的PRC 2/H3 K27 me引起的先天性缺陷。

PROJECT SUMMARY Adult zebrafish rapidly regenerate amputated fins, including complex skeletons, back to their original size and form. Lineage-restricted progenitor cells derived from injury-induced, dedifferentiated mature cells proliferate and re-differentiate to restore lost fin tissue. Therefore, understanding the control of cell state transitions between differentiated and progenitor states, with retained cell identities, is central to understanding robust appendage regeneration. Profound changes in gene expression programs drive dedifferentiation and re- differentiation state transitions. Chromatin landscapes are assumed to stabilize both progenitor and differentiated state programs and therefore regulated chromatin dynamics likely underlie state transitions. Further, chromatin mechanisms are thought to epigenetically maintain grid-like positional identities that direct the fin size-restoring amount of regenerative outgrowth. Polycomb Repressive Complex 2 (PRC2) represses gene expression by Ezh1/Ezh2-catalyzed methylation of lysine-27 of histone H3 (H3K27me). PRC2/H3K27me silences developmental regulatory genes in differentiated cells, stabilizes progenitor state programs, and maintains cell fate & positional identities, including Hox codes. We earlier linked the removal of H3K27me marks by upregulated histone demethylases to widespread gene activation associated with initiating fin regeneration. Recently, we targeted ezh1 and ezh2 to generate adult viable PRC2 mutant zebrafish. Strikingly, multiple rounds of fin regeneration occur normally despite greatly reduced global H3K27me2/3 levels accompanied by elevated, activation-associated H3K27 acetylation. Therefore, the bulk of H3K27me is not required for the regulated state transitions or maintenance of cell identities during fin regeneration. These results challenge PRC2/H3K27me3 dogma in a compelling vertebrate regeneration context. We will pursue exploratory studies to distinguish between several hypotheses explaining how fin regeneration proceeds without most of this major repressive histone modification. For Aim #1, we will profile genome-wide histone modification patterns in wildtype and PRC2-mutant regenerating fins using new CUT&Tag technology. We will use RNA-Seq to correlate the PRC2-dependent transcriptome with altered chromatin landscapes. In Aim #2, we will experimentally bypass lethality of our ezh1/ezh2 mutants by transiently expressing Ezh2 during embryonic development using mRNA injections and inducible transgenic approaches. We will characterize regeneration defects, if any, in derived null PRC2 adults towards defining key H3K27me-controlled regulatory networks of fin regeneration. Combined outcomes will provide the central premise for a larger project studying chromatin dynamics and cell transitions of organ regeneration. Broader impacts include guidance on the use of chromatin-based perturbagens to enhance regenerative medicine and as therapeutics for pediatric gliomas, other cancers, and congenital defects caused by disrupted PRC2/H3K27me.