Structure of post-replicative chromatin during cell reprogramming in fibrotic disease

项目摘要：眼睛的大部分组织都容易发展成纤维性疾病，致盲 全世界数以百万计的人。没有有效的方法来预防、减缓或逆转 这一疾病过程。导致纤维性疾病的细胞类型是肌成纤维细胞。 了解细胞如何获得改变的可遗传表型成为肌成纤维细胞，导致 疤痕形成与这一病理疾病过程有关，是一个关键问题，很可能提供必要的 开发抗纤维化疗法的线索。改变转录编程过程中 将细胞重新编程为肌成纤维细胞还不是很清楚。在许多方面，它可能依赖于 DNA复制过程中染色质结构遗传的表观遗传机制的变化。这个 细胞增殖过程中的表观遗传机制尚不清楚，我们知道的更少 关于表观遗传信息和相应的转录程序在细胞过程中如何变化 重新编程。我们对这些基本生物过程的认识上的差距是基于 缺乏直接的实验方法来检查染色质的结构 在细胞增殖和分化过程中DNA复制期间和之后的转录状态。 利用新开发的实验范式，我们发现细胞中的表观遗传标记 增殖不依赖于将修饰的组蛋白转移到子链上，而是依赖于稳定性 DNA复制过程中多个组蛋白修饰蛋白的联合作用。也得到了类似的结果 在导致纤维化疾病的细胞重编程的多个晶状体模型中。损伤前和损伤前的晶体细胞 在体外小鸡模型手术后的第一天，染色质的特征是 关键抑制性组蛋白标志H3K27me3在DNA后积累的显著延迟 复制。这意味着核小体的解聚结构。同样的‘开放’后复制 在小鼠和人类晶状体细胞诱导成肌成纤维细胞的过程中也发现了染色质。 表型，这表明这是以前未知的所有肌成纤维细胞前体细胞的关键特性 细胞。我们的数据表明，复制后染色质的这种开放状态更容易结合 新诱导的对细胞重新编程至关重要的特定转录因子(TF)。重要的是， 可以操纵染色质的状态以改变TF与其关联的能力 DNA上的靶点，用于治疗靶向肌成纤维细胞分化。我们建议进一步研究： 1)调控细胞重编程为肌成纤维细胞的表观遗传学机制；2) 表观遗传机制是否可以被操纵来改变细胞重编程的表型结果 成肌成纤维细胞。我们预计表观遗传学分子事件和抗纤维化策略 从拟议的研究中发现的将适用于治疗眼睛和大多数组织类型的纤维化。

Project Summary: Most tissues of the eye are susceptible to developing fibrotic disease, blinding millions of people throughout the world. There are no effective approaches to prevent, slow or reverse this disease process. The cell type responsible for causing fibrotic disease is the myofibroblast. Understanding how a cell acquires an altered heritable phenotype to become a myofibroblast, leading to scarring associated with this pathological disease process, is a key question, likely to provide essential clues toward developing anti-fibrotic therapeutics. Changing transcriptional programming during reprogramming of a cell to a myofibroblast is not well understood. In many aspects, it may rely on changes in the epigenetic mechanisms of inheritance of chromatin structure during DNA replication. The mechanism of epigenetic inheritance during cell proliferation remains unknown, and we know even less about how epigenetic information and the corresponding transcriptional programs change during cell reprogramming. The gaps in our knowledge of these essential biological processes are based on the lack of direct experimental approaches that would allow examining the structure of chromatin and the state of transcription during and following DNA replication during cell proliferation and cell differentiation. Using newly developed experimental paradigms, we found that epigenetic marking during cell proliferation relies not on the transfer of modified histones to daughter strands, but rather on stable association of multiple histone-modifying proteins during DNA replication. Similar results were obtained in multiple lens models of cell reprogramming leading to fibrotic disease. Lens cells before injury and until the first day following surgery in the ex vivo chick model have chromatin that is characterized by a significant delay in the accumulation of the key repressive histone mark H3K27me3 following DNA replication. This signifies a de-condensed structure of nucleosomes. The same `open' post-replicative chromatin was also discovered in mouse and human lens cells during their induction to the myofibroblast phenotype, suggesting that this is a previously unknown pivotal property of all myofibroblast progenitor cells. Our data suggests that this `open' state of post-replicative chromatin is more amenable to binding of newly induced specific transcription factors (TFs) essential for cell reprogramming. Importantly, the state of `open' chromatin may be manipulated in order to change the ability of TFs to associate with their target sites on DNA to therapeutically target myofibroblast differentiation. We propose to further examine: 1) The epigenetic mechanisms involved in regulating cell reprogramming to a myofibroblast and 2) Whether epigenetic mechanisms can be manipulated to alter phenotypic outcome of cell reprogramming to a myofibroblast. We anticipate that the epigenetic molecular events and anti-fibrotic strategies discovered from the proposed studies will apply to treating fibrosis in the eye and most tissue types.