Unlocking heterochromatin to potentiate cell conversion to the hepatocyte fate

 描述(申请人提供)：本研究项目的目标是通过识别和克服阻止肝脏转录因子与其DNA靶标结合在异染色质中的障碍，增强细胞对肝细胞谱系的重新编程。随着大约17,000名美国人在等待肝脏移植的名单上，人们越来越需要从患者自己的组织中产生健康的肝细胞的方法，这种方法可以在不担心排斥的情况下进行细胞移植治疗。现在，通过几种肝转录因子的联合表达，可以将人皮肤成纤维细胞转化为肝细胞样细胞(人诱导肝细胞，或hiHeps)。然而，由此产生的细胞与正常肝细胞有显著的基因表达差异，导致功能缺陷，极大地限制了其临床应用。最近，我们的实验室发现，成纤维细胞中的大片异染色质通过阻止重编程因子在这些区域内与DNA中的同源位点结合，从而为诱导多能干细胞(IPS)的重编程提供了障碍。这些异染色区以组蛋白3赖氨酸9(H3K9me3)的三甲基化为标志，H3K9me3甲基转移酶的敲除减轻了因子结合的障碍，提高了iPS重新编程的效率。我们现在发现，这些大的H3K9me3结构域，随着细胞类型的不同，同样会阻碍hiHep重新编程：在肝细胞中表达的在成纤维细胞H3K9me3结构域中受到抑制的基因，包括肝细胞转录因子和对肝功能重要的基因，在hiHep细胞中与正常肝细胞相比一直受到抑制。然而，定位于这些异染色质结构域的蛋白质组分介导了因子结合的障碍，进而导致重新编程的低效，人们对此知之甚少。该方案的目的1旨在利用我们开发的一种新的方法，在成纤维细胞中发现大的H3K9me3结构域的组成蛋白，以富集异染色质，并通过质谱分析鉴定共纯化的蛋白。这一方法的初步数据揭示了许多已知的异染色质蛋白和阻遏物，以及对RNA结合蛋白的意外浓缩。这项建议的目的2试图确定蛋白质组学或文献所确定的哪些异染色质蛋白具有限制重编程到肝细胞谱系的功能。这些蛋白质将在hiHep重新编程过程的早期被siRNA系统地耗尽，以便确定促进成纤维细胞异染色质中更有效的细胞转化和基因重新激活的条件。然后，通过siRNA治疗增强重编程的细胞将在移植到小鼠肝衰竭模型后，测试它们在体内恢复肝功能的能力。因此，这里提出的研究将揭示人类异染色质的成分，这些成分阻碍了细胞身份向成熟肝细胞命运的转变。

 DESCRIPTION (provided by applicant): The goal of this research project is to enhance cell reprogramming to the hepatocyte lineage, by identifying and overcoming the barriers that prevent hepatic transcription factors from binding their DNA targets in heterochromatin. With some 17,000 Americans on the liver transplant waiting list, there is increasing need for methods to generate healthy hepatocytes from a patient's own tissue, which could allow cell transplantation therapy without concern for rejection. It is now possible to convert human skin fibroblasts into hepatocyte-like cells (human induced hepatocytes, or hiHeps) by combined expression of several liver transcription factors. However, the resulting cells have substantial gene expression differences from normal hepatocytes, resulting in functional deficiencies that greatly limit their clinical utility. Recently, our lab showed that large regions of heterochromati in fibroblasts present a barrier to reprogramming to induced pluripotent stem (iPS) cells, by preventing the reprogramming factors from binding their cognate sites in DNA within these domains. These heterochromatic regions are marked by trimethylation of histone 3 lysine 9 (H3K9me3), and knockdown of H3K9me3 methyltransferases relieves the impediment to factor binding and enhances the efficiency of iPS reprogramming. We now find that these large H3K9me3 domains, which vary by cell type, similarly impede hiHep reprogramming: genes expressed in liver cells that are repressed in fibroblast H3K9me3 domains, including hepatocyte transcription factors and genes important for liver function, are consistently repressed in hiHep cells relative to normal hepatocytes. However, the protein components localized to these heterochromatin domains that mediate the impediment to factor binding, and in turn the inefficiency of reprogramming, are poorly understood. Aim 1 of this proposal seeks to discover the constituent proteins of large H3K9me3 domains in fibroblasts, using a novel method we have developed to enrich heterochromatin and identify the co-purifying proteins by mass spectrometry. Preliminary data from this approach reveals many known heterochromatin proteins and repressors, as well as an unexpected enrichment for RNA-binding proteins. Aim 2 of this proposal seeks to determine which heterochromatin proteins, as identified by proteomics or the literature, function to restrict reprogramming to the hepatocyte lineage. These proteins wil be systematically depleted by siRNA early in the hiHep reprogramming process in order to identify conditions that promote more efficient cell conversion and reactivation of genes in fibroblast heterochromatin. Cells whose reprogramming is enhanced by siRNA treatment will then be tested for their ability to restore liver function in vivo after transplantation into a murne liver failure model. The research proposed here will thus reveal the components of human heterochromatin that impede conversion of cell identity to the mature hepatocyte fate.