Investigating imitation SWI chromatin remodeling complexes in mammalian tissue regeneration

项目总结 一项观察发现，在哺乳动物中，响尾藻、蝾螈和斑马鱼的强大的再生能力基本上消失了 这对人类健康有切实的影响。受损的组织再生可能导致疾病 如肝硬变或炎症性肠病(IBD)。肝病环境中有限的再生可能 也排除了为癌症进行大手术切除的能力。相对缺乏再生性 治疗的部分原因可能是因为发现新的靶点是困难的，而且依赖于体内系统 不能在培养皿中复制。为了确定可以作为促进再生的靶点的新途径， 我们建立了一个体内CRISPR-Cas9筛选平台来评估小鼠肝脏中的基因。使用这个 高通量系统，我们评估了152个基因编码组蛋白等表观遗传机制的影响 读者、作家和橡皮擦[2]。这鉴定了两个模拟的SWI/SNF(ISWI)染色质重塑复合体 由Baz2a和Baz2b编码的亚基，这是以前未知的调控再生的基因。这个 BAZ2蛋白与SMARCA5酶的相互作用定义了核仁重塑复合体 (NORC)，ISWI复合体的五种亚型之一，它使用ATP水解来重塑核小体，特别是 核糖体DNA(RDNA)基因座[3]。我们的验证研究表明，在体内Cas9缺失Baz2a或 在肝细胞再繁殖模型中，Baz2b促进了肝再生。同样，BAZ2A的化学抑制作用 而使用特定溴域抑制剂GSK2801的BAZ2B导致肝脏再生增加[4]。 此外，我们还发现GSK2801可以促进结肠炎小鼠模型的肠道恢复。这些 数据表明，含有ISWI/NORC染色质重塑复合体的BAZ2对器官具有重要意义 多种疾病的再生和有前景的治疗靶点。我们的中心假设是，模仿 SWI成分BAZ2A和BAZ2B通过限制蛋白质的增加来限制有效的组织再生 通过抑制rRNA转录来合成。我们将通过验证来测试这个假设的不同方面 Baz2a和Baz2b基因作为真正的再生调节因子(目标1)，通过了解全球表观遗传学 含BAZ2的络合物的活性(目标2)，并通过确定增加蛋白质合成是否是关键 抑制BAZ2促进再生的机制(目标3)。该项目将定义可再生的 与ISWI染色质重塑相关的表型和分子机制。我们的研究将有助于 促进组织再生的BAZ2蛋白小分子抑制剂的临床药物开发 在肝脏和肠道中。

PROJECT SUMMARY The potent regenerative capabilities of planaria, newts, and zebrafish are largely lost in mammals, an observation that has a tangible impact on human health. Compromised tissue regeneration likely contributes to disorders such as liver cirrhosis or inflammatory bowel diseases (IBD). Limited regeneration in liver disease settings can also preclude the ability to perform large surgical resections for cancer. The relative lack of regenerative therapies could in part be because discovering new targets is difficult and is dependent on in vivo systems that cannot be replicated in the culture dish. To identify new pathways that can be targeted to enhance regeneration, we established an in vivo CRISPR-Cas9 screening platform to evaluate genes in the mouse liver. Using this high-throughput system, we assessed the impact of 152 genes encoding epigenetic machines such as histone readers, writers, and erasers [2]. This identified two imitation SWI/SNF (ISWI) chromatin remodeling complex subunits encoded by Baz2a and Baz2b, genes that were not previously known to regulate regeneration. The interaction between BAZ2 proteins with the SMARCA5 enzyme defines the nucleolar remodeling complex (NoRC), one of five subtypes of ISWI complexes that uses ATP hydrolysis to remodel nucleosomes, particularly at ribosomal DNA (rDNA) loci [3]. Our validation studies showed that in vivo Cas9 deletion of either Baz2a or Baz2b increased liver regeneration in a hepatocyte repopulation model. Similarly, chemical inhibition of BAZ2A and BAZ2B using the specific bromodomain inhibitor GSK2801 resulted in increased liver regeneration [4]. Moreover, we also found that GSK2801 could promote intestinal recovery in a mouse model of colitis. These data suggest that BAZ2 containing ISWI/NoRC chromatin remodeling complexes are important for organ regeneration and promising therapeutic targets for multiple diseases. Our central hypothesis is that the imitation SWI components BAZ2A and BAZ2B limit efficient tissue regeneration by restricting increases in protein synthesis via suppression of rRNA transcription. We will test different aspects of this hypothesis by validating Baz2a and Baz2b genes as bona fide regeneration regulators (Aim 1), by understanding the global epigenetic activities of BAZ2 containing complexes (Aim 2), and by determining if increased protein synthesis is a key mechanism by which BAZ2 inhibition promotes regeneration (Aim 3). This project will define the regenerative phenotypes and molecular mechanisms associated with ISWI chromatin remodeling. Our studies will facilitate clinical drug development of small molecule inhibitors of BAZ2 proteins for use in enhancing tissue regeneration in the liver and intestine.