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: 9754140
• 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/9754140
• 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 肝移植是包括肝细胞癌在内的许多肝病的唯一治愈方法。 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 在胚胎发育过程中，随着细胞命运的选择和潜在的 分化成不同的血统是有限的。随着诱导多能性的发现，其中一个 体细胞通过异位表达的方式被重新编程为诱导多能干细胞(IPSC) 转录因子，人们发现有可能以以前 不可思议的事。除了重新编程到IPSC之外，还开发了多种方法来转换 分化：一种特性的细胞转化为另一种特性的分化，如诱导的 成纤维细胞转化为肝细胞。尽管存在这些方法，但已经有充分的文献证明，它们的细胞 产生在基因表达和功能上都不完全概括的靶细胞。精确单元格 重新编程或转换依赖于激活沉默的基因调控网络。我们发现 H3K9me3异染色质阻断重编程因子与靶点的结合并阻碍 在IPSC重编程和成纤维细胞直接转化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来实际提取这些顽固分子 异染色质区域，根据超声波抗性的标准，并确定其蛋白质 组合物；指定为抗超声波异染色质(SrHC)相关蛋白。这一过程 发现了H3K9me3和异染色质的已知调节因子以及以前没有发现的新蛋白质 已知在异染色质和细胞特性维持中起作用。我们对94个srHC的功能性siRNA筛选 相关蛋白鉴定了许多抑制异色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，我假设这些变化的表达 异染色质相关蛋白选择性修饰表基因组可以提高准确性和 成纤维细胞诱导的组氨酸肝素蛋白的功能。表观遗传修饰的HiHeps的功能评价 将通过免疫缺陷小鼠模型的rna-seq和系列移植研究来确定。 在那里可以进行肝脏人源化和肝功能评估。利用这种方法，我将确定 异染色质亚型如何形成和维持，以及如何选择性地破坏它们的稳定性 以促进对肝脏谱系的增强重编程。