Investigating imitation SWI chromatin remodeling complexes in mammalian tissue regeneration

项目摘要 一项观察表明，在哺乳动物中，涡虫、蝾螈和斑马鱼的强大再生能力在很大程度上丧失了。 这对人类健康有着切实的影响。受损的组织再生可能导致疾病 如肝硬化或炎症性肠病（IBD）。肝脏疾病环境中的有限再生可以 也排除了对癌症进行大的外科切除的能力。相对缺乏再生能力 部分原因可能是发现新的靶点很困难，并且依赖于体内系统， 不能在培养皿中复制。为了确定可以靶向增强再生的新途径， 我们建立了体内CRISPR-Cas9筛选平台来评估小鼠肝脏中的基因。使用此 高通量系统，我们评估了152个基因编码的表观遗传机器，如组蛋白， 阅读器、书写器和橡皮擦[2]。这鉴定了两个仿SWI/SNF（ISWI）染色质重塑复合物 由Baz 2a和Baz 2b编码的亚基，这些基因以前不知道调节再生。的 BAZ 2蛋白与SMARCA 5酶之间的相互作用定义了核仁重塑复合物 （NoRC），ISWI复合物的五种亚型之一，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，Baz 2a或Baz 2b的体内Cas9缺失可以抑制Baz 2a或Baz 2b的表达。 Baz 2b在肝细胞再生模型中增加肝再生。类似地，BAZ 2A的化学抑制 和BAZ 2B使用特异性溴结构域抑制剂GSK 2801导致肝再生增加[4]。 此外，我们还发现GSK 2801可以促进结肠炎小鼠模型的肠道恢复。这些 数据表明，含有BAZ 2的ISWI/NoRC染色质重塑复合物对于器官的生长是重要的。 再生和多种疾病的有前途的治疗靶点。我们的中心假设是， SWI组分BAZ 2A和BAZ 2B通过限制蛋白质的增加而限制有效的组织再生 通过抑制rRNA转录合成。我们将通过验证这一假设的不同方面来测试 Baz 2a和Baz 2b基因作为真正的再生调节因子（Aim 1），通过了解全球表观遗传学 活性的BAZ 2含有复合物（目的2），并通过确定是否增加蛋白质合成的关键 BAZ 2抑制促进再生的机制（目的3）。该项目将定义再生 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 用于增强组织再生的BAZ 2蛋白的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.