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.

项目总结 成年斑马鱼迅速再生切断的鳍，包括复杂的骨骼，恢复到它们原来的大小和 形式。来自损伤诱导的去分化成熟细胞的谱系受限的祖细胞增殖 并重新分化以恢复丢失的鳍组织。因此，了解对单元状态转换的控制 在分化状态和祖细胞状态之间，保持细胞身份，是理解强健的核心 附肢再生。基因表达程序的深刻变化推动去分化和重新分化 差异化状态转换。染色质景观被认为是稳定祖细胞和 不同的状态程序和因此受调控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上保持了类似网格的位置一致性，从而引导 再生生长的鳍大小-恢复量。多梳抑制复合体2(PRC2)抑制 Ezh1/Ezh2催化组蛋白H3(H3K27me)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，以及 维护小区命运和位置标识，包括Hox代码。我们早些时候将H3K27Me的移除 上调的组蛋白去甲基酶标记与启动FIN相关的广泛基因激活 再生。最近，我们针对ezh1和eZH2产生了成年存活的PrC2突变斑马鱼。令人惊讶的是， 尽管全球H3K27me2/3水平大幅下降，但多轮鳍再生仍正常发生 伴随着激活相关的H3K27乙酰化升高。因此，H3K27Me的大部分不是 在鳍片再生期间调节状态转换或维持单元身份所需。这些 结果在令人信服的脊椎动物再生背景下挑战PRC2/H3K27me3教条。我们将继续追查 区分几种解释鳍再生过程的假说的探索性研究 没有大多数这种主要的抑制性组蛋白修饰。对于目标1，我们将描述全基因组的组蛋白 使用新的切割和标签技术的野生型和突变型再生鳍的修改模式。我们会 使用RNA-Seq将依赖于PrC2的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Ezh2来绕过我们的ezh1/ezh2突变体的致命性 使用mRNA注射和诱导性转基因方法进行胚胎发育。我们将描述 衍生缺失的PRC2成体的再生缺陷，如果有的话，与定义关键的H3K27me控制的调控有关 鳍片再生网络。综合结果将为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变。更广泛的影响包括关于使用 以染色质为基础的微扰剂，以增强再生医学并作为儿童胶质瘤的治疗药物， 其他癌症，以及PrC2/H3K27me基因突变引起的先天性缺陷。