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.
Many patients with chronic liver disease are unable to receive life saving liver transplants due to an insufficient supply of donor livers, and those patients who do receive transplants are subjected to a lifetime of immunosuppresants and associated, often severe, side effects. Although remarkable progress has been made in the field of regenerative medicine enabling the conversion of human skin fibroblast cells into induced hepatocyte cells, the resulting cells fail to fully recapitulate a true hepatocyte and lack key functionality due to the failure to activate expression of many crucial genes which are maintained in a repressed state by heterochromatin. The aim of this research proposal is to identify heterochromatin regulators repressing these key hepatic genes to learn how heterochromatin enforces cell identity and how it can be altered to improve the function of induced hepatocytes.
