Revisiting Polycomb Repression in Appendage Regeneration

重新审视附肢再生中的多梳抑制

• 批准号: 10742697
• 负责人: KRYN STANKUNAS
• 金额: $ 40.56万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10742697
• 关键词: Acetylation; Adult; Amputation; Animals; Applications Grants; Back; Biological Assay; Bone Injury; Bypass; Cell Differentiation process; Cell Maintenance; Cell Proliferation; Cells; Childhood Glioma; Chromatin; Code; Compensation; Complex; Congenital Abnormality; Craniofacial Abnormalities; Defect; Deposition; Development; Embryonic Development; Epigenetic Process; Excision; Fertilization; Gene Activation; Gene Expression; Histone H2A; Histone H3; Histones; Human; Injections; Injury; Knowledge; Link; Lysine; Malignant Childhood Neoplasm; Malignant Neoplasms; Messenger RNA; Methylation; Modification; Natural regeneration; Organ; Organogenesis; Outcome; PRC1 Protein; Pattern; Polycomb; Regenerative Medicine; Regenerative research; Regulator Genes; Repression; Research; Residual state; Resources; Role; Site; Skeleton; Technology; Testing; Therapeutic; Tissues; Transgenes; Transgenic Organisms; United States National Institutes of Health; Vertebrates; Visit; Zebrafish; appendage; design; epigenome; epigenomics; experimental study; gene regulatory network; gene repression; genome-wide; histone demethylase; histone modification; in vivo; insight; limb injury; limb regeneration; mutant; novel; novel strategies; organ regeneration; progenitor; programs; regeneration model; regenerative; repaired; restraint; self-renewal; skeletal; spine bone structure; stem cells; transcriptome; transcriptome sequencing; virtual

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.

项目概要 成年斑马鱼能够迅速再生被截断的鳍，包括复杂的骨骼，恢复到原来的大小 形式。来自损伤诱导的去分化成熟细胞的谱系限制祖细胞增殖 并重新分化以恢复丢失的鳍组织。因此，了解细胞状态转换的控制 分化状态和祖细胞状态之间的关系，以及保留的细胞身份，对于理解鲁棒性至关重要 附肢再生。基因表达程序的深刻变化驱动去分化和重新分化 微分状态转换。染色质景观被认为可以稳定祖细胞和 不同的状态程序以及因此调节的染色质动力学可能是状态转变的基础。 此外，染色质机制被认为可以在表观遗传上维持网格状的位置特征，从而指导 再生生长的鳍尺寸恢复量。 Polycomb 抑制复合物 2 (PRC2) 抑制 通过 Ezh1/Ezh2 催化组蛋白 H3 (H3K27me) 赖氨酸 27 甲基化来表达基因。 PRC2/H3K27me 沉默分化细胞中的发育调节基因，稳定祖细胞状态程序，以及 维持细胞命运和位置身份，包括 Hox 代码。我们之前链接了 H3K27me 的删除 通过上调组蛋白去甲基化酶来标记与起始鳍相关的广泛基因激活 再生。最近，我们以 ezh1 和 ezh2 为目标，产生了成年可存活的 PRC2 突变斑马鱼。引人注目的是， 尽管整体 H3K27me2/3 水平大大降低，但多轮鳍再生正常发生 伴随着与激活相关的 H3K27 乙酰化升高。因此，H3K27me的大部分不是 鳍再生过程中调节状态转换或维持细胞身份所需。这些 结果在令人信服的脊椎动物再生背景下挑战了 PRC2/H3K27me3 教条。我们将追求 探索性研究区分几种解释鳍再生如何进行的假设 没有大部分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组蛋白 使用新的 CUT&Tag 技术对野生型和 PRC2 突变体再生鳍进行修饰模式。我们将 使用 RNA-Seq 将 PRC2 依赖性转录组与改变的染色质景观相关联。在目标#2中， 我们将通过在实验过程中瞬时表达 Ezh2 来实验性地绕过 ezh1/ezh2 突变体的致死性 使用 mRNA 注射和诱导转基因方法进行胚胎发育。我们将表征 再生缺陷，如果有的话，在衍生的空 PRC2 成人中确定关键的 H3K27me 控制的监管 鳍再生网络。综合成果将为更大的项目研究提供中心前提 器官再生的染色质动力学和细胞转变。更广泛的影响包括使用指南 基于染色质的扰动物可增强再生医学并作为儿科神经胶质瘤的治疗方法， 其他癌症以及由 PRC2/H3K27me 破坏引起的先天性缺陷。