Modifying regulators of heterochromatin to improve reprogramming to functional hepatocytes

修饰异染色质调节因子以改善功能性肝细胞的重编程

• 批准号: 10758720
• 负责人: Ryan Lehman McCarthy
• 金额: $ 8.72万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10758720
• 关键词: Modifying; regulators; heterochromatin; improve; reprogramming

PROJECT SUMMMARY/ABSTRACT Liver transplantation is the only curative treatment for many liver diseases, including hepatocellular carcinoma and biliary atresia. However, the supply of donor livers is insufficient to meet the growing need. Cell identity during embryogenesis is progressively established as cell fate choices are made and the potential to differentiate into alternative lineages is restricted. With the discovery of induced pluripotency, wherein a somatic cell is reprogrammed to an induced pluripotent stem cell (iPSC) through the ectopic expression of transcription factors, it was revealed that it was possible to manipulate cell identity in ways that were previously inconceivable. In addition to reprogramming to iPSC, multiple methods have been developed for trans- differentiation where cells of one identity are converted to another, such as the induced conversion of fibroblasts to hepatocytes. Although these methods exist, it has been well documented that the cells they produce incompletely recapitulate the target cell both in gene expression and functionality. Accurate cell reprogramming or conversion is dependent upon activating silent gene regulatory networks. We found that H3K9me3 heterochromatin blocks binding of the reprogramming factors to target sites and impedes the activation of the desired gene network both in reprogramming to iPSC and in direct conversion of fibroblasts to human induced hepatocytes (hiHeps). We developed methodology to physically extract these recalcitrant heterochromatin regions, based upon the criteria of sonication resistance, and determined their protein composition; designating as sonication resistant heterochromatin (srHC) associated proteins. This process found both well-known regulators of H3K9me3 and heterochromatin as well as novel proteins not previously known to have roles in heterochromatin and cell identity maintenance. Our functional siRNA screen of 94 srHC associated proteins identified many which repressed expression of heterochromatically silenced hepatocyte genes in hiHeps. Based upon our extensive preliminary data I hypothesize that altering expression of these heterochromatin associated proteins to selectively modify the epigenome can improve the accuracy and functionality of hiHeps induced from fibroblasts. Functional assessment of the epigenetically modified hiHeps will be determined through RNA-seq and serial transplantation studies in an immunodeficient mouse model where liver humanization can be performed and liver function assessed. Utilizing this approach I will identify how subtypes of heterochromatin form and are maintained, as well as how they can be selectively destabilized to facilitate enhanced reprogramming to the hepatic lineage.

项目总结/摘要 肝移植是治疗包括肝癌在内的许多肝脏疾病的唯一有效方法 和胆道闭锁然而，供体肝脏的供应不足以满足日益增长的需求。小区标识 在胚胎发生过程中，随着细胞命运的选择， 分化成不同的血统是受限制的。随着诱导的多能性的发现， 体细胞通过异位表达 转录因子，这表明它是可能的操纵细胞身份的方式，以前是 不可思议。除了重编程为iPSC之外，还开发了多种方法用于转基因。 分化，其中一种身份的细胞转化为另一种身份的细胞，如诱导的转化。 成纤维细胞转化为肝细胞。虽然这些方法存在，但已经有充分的文献证明， 在基因表达和功能上都不完全重现靶细胞。精确的细胞 重编程或转化依赖于激活沉默基因调控网络。我们发现 H3 K9 me 3异染色质阻断重编程因子与靶位点的结合，并阻碍重编程因子与靶位点的结合。 在重编程为iPSC和成纤维细胞直接转化为iPSC中激活所需的基因网络， 人诱导肝细胞（hiHeps）。我们开发了一种方法， 异染色质区域，基于抗超声标准，并确定其蛋白质 组合物;命名为抗超声异染色质（srHC）相关蛋白。这个过程 发现了H3 K9 me 3和异染色质的众所周知的调节因子以及以前没有的新蛋白质 已知在异染色质和细胞身份维持中起作用。我们对94 srHC的功能性siRNA筛选 相关蛋白鉴定出许多抑制异染色质沉默肝细胞表达的蛋白质 hiHep中的基因。基于我们广泛的初步数据，我假设改变这些基因的表达， 异染色质相关蛋白选择性修饰表观基因组可以提高准确性， 从成纤维细胞诱导的hiHep的功能性。表观遗传修饰的hiHeps的功能评估 将通过RNA-seq和免疫缺陷小鼠模型中的系列移植研究来确定 其中可以进行肝脏人源化并评估肝功能。利用这种方法，我将确定 异染色质的亚型如何形成和维持，以及它们如何被选择性地去稳定 以促进增强的肝谱系重编程。