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: 10214599
• 负责人: Ryan Lehman McCarthy
• 金额: $ 10.53万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214599
• 关键词: Albumins; Biliary; Biliary Atresia; Binding; Binding Proteins; Biological Assay; Cells; Chromatin; Complex; Cytochromes; Data; Ectopic Expression; Elements; Embryonic Development; Epigenetic Process; Exhibits; Failure; Fibroblasts; Gene Expression; Generations; Genes; Goals; Hepatic; Hepatocyte; Heterochromatin; Histologic; Histones; Human; Learning; Life; Liver; Liver diseases; Maintenance; Methodology; Methods; Modification; Mus; Patients; Primary carcinoma of the liver cells; Process; Proteins; Proteomics; RNA-Binding Proteins; Regenerative Medicine; Regulator Genes; Research Proposals; Resistance; Role; Savings; Series; Site; Skin; Small Interfering RNA; Somatic Cell; Sonication; Transplantation; base; cell type; chronic liver disease; combinatorial; curative treatments; epigenome; experimental study; gene function; gene repression; immunodeficient mouse model; improved; improved functioning; in vitro Assay; in vivo; induced pluripotent stem cell; knock-down; liver function; liver transplantation; novel; pluripotency; side effect; transcription factor; transcriptome sequencing; transdifferentiation; transplant model

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.

项目摘要/摘要 肝移植是许多肝脏疾病的唯一治疗方法，包括肝细胞癌 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。细胞身份 在胚胎发生过程中，随着细胞命运的选择和潜在的选择，逐渐建立起来 分化成替代谱系受到限制。随着诱导多能性的发现，其中 体细胞通过异位表达被重编程为诱导多能干细胞（iPSC） 转录因子，人们发现可以通过以前的方式操纵细胞身份 不可思议。除了对 iPSC 进行重编程之外，还开发了多种方法用于反式 分化，其中一种身份的细胞转化为另一种身份，例如诱导转化 成纤维细胞至肝细胞。尽管存在这些方法，但已有充分证据表明，它们所用的细胞 产生不完全重现靶细胞的基因表达和功能。准确的细胞 重编程或转换取决于激活沉默基因调控网络。我们发现 H3K9me3 异染色质阻断重编程因子与靶位点的结合并阻碍 在重编程为 iPSC 和将成纤维细胞直接转化为 iPSC 时，激活所需的基因网络 人类诱导肝细胞（hiHeps）。我们开发了物理提取这些顽固分子的方法 异染色质区域，基于超声抗性标准，并确定其蛋白质 作品;指定为超声抗性异染色质 (srHC) 相关蛋白。这个过程 发现了众所周知的 H3K9me3 和异染色质调节因子以及以前未发现的新型蛋白质 已知在异染色质和细胞身份维持中发挥作用。我们对 94 srHC 的功能 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 hiHeps 中的基因。根据我们广泛的初步数据，我假设改变这些的表达 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和 成纤维细胞诱导的 hiHeps 的功能。表观遗传修饰 hiHeps 的功能评估 将通过免疫缺陷小鼠模型中的 RNA-seq 和连续移植研究来确定 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 异染色质亚型如何形成和维持，以及如何选择性地使其不稳定 促进肝谱系的增强重编程。