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.
With nearly 17,000 Americans on the waiting list for liver transplantation, the development of improved methods for the production of human hepatocytes is essential. By introducing defined cocktails of genes into cells obtained from a patient's skin, t is now possible to produce cells that resemble normal hepatocytes, but these transformed cells fail to activate particular regions of the genome that are crucial for liver function. The goal of his proposal is to identify the proteins that keep these regions of DNA dormant, and to determine which of these proteins should be depleted to enhance the efficiency and quality of existing hepatocyte conversion methods.
| Becker, Justin S; McCarthy, Ryan L; Sidoli, Simone et al. (2017) Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes. Mol Cell 68:1023-1037.e15 |
| Becker, Justin S; Nicetto, Dario; Zaret, Kenneth S (2016) H3K9me3-Dependent Heterochromatin: Barrier to Cell Fate Changes. Trends Genet 32:29-41 |
