Most tissues of the eye are susceptible to developing fibrotic disease, blinding millions of people throughout the world. There are no effective approaches to prevent, slow or reverse this disease process. The cell type responsible for causing fibrotic disease is the myofibroblast. Understanding how a cell acquires an altered heritable phenotype to become a myofibroblast, leading to scarring associated with this pathological disease process, is a key question, likely to provide essential clues toward developing anti-fibrotic therapeutics. Changing transcriptional programming during reprogramming of a cell to a myofibroblast is not well understood. In many aspects, it may rely on changes in the epigenetic mechanisms of inheritance of chromatin structure during DNA replication. The mechanism of epigenetic inheritance during cell proliferation remains unknown, and we know even less about how epigenetic information and the corresponding transcriptional programs change during cell reprogramming. The gaps in our knowledge of these essential biological processes are based on the lack of direct experimental approaches that would allow examining the structure of chromatin and the state of transcription during and following DNA replication during cell proliferation and cell differentiation. Using newly developed experimental paradigms, we found that epigenetic marking during cell proliferation relies not on the transfer of modified histones to daughter strands, but rather on stable association of multiple histone-modifying proteins during DNA replication. Similar results were obtained in multiple lens models of cell reprogramming leading to fibrotic disease. Lens cells before injury and until the first day following surgery in the ex vivo chick model have chromatin that is characterized by a significant delay in the accumulation of the key repressive histone mark H3K27me3 following DNA replication. This signifies a de-condensed structure of nucleosomes. The same `open' post-replicative chromatin was also discovered in mouse and human lens cells during their induction to the myofibroblast phenotype, suggesting that this is a previously unknown pivotal property of all myofibroblast progenitor cells. Our data suggests that this `open' state of post-replicative chromatin is more amenable to binding of newly induced specific transcription factors (TFs) essential for cell reprogramming. Importantly, the state of `open' chromatin may be manipulated in order to change the ability of TFs to associate with their target sites on DNA to therapeutically target myofibroblast differentiation. We propose to further examine: 1) The epigenetic mechanisms involved in regulating cell reprogramming to a myofibroblast and 2) Whether epigenetic mechanisms can be manipulated to alter phenotypic outcome of cell reprogramming to a myofibroblast. We anticipate that the epigenetic molecular events and anti-fibrotic strategies discovered from the proposed studies will apply to treating fibrosis in the eye and most tissue types.
There are no effective approaches to prevent, slow or reverse the process of fibrotic disease, which affects almost every organ of the body, including most tissues in the eye to cause blindness in millions of people worldwide. We found a previously unknown, potentially pivotal epigenetic property of the cells from higher eukaryotes, including humans, that differentiate into a fibrotic disease causing cell type, called a myofibroblast. We propose to investigate how the newly discovered `open' state of chromatin following DNA replication may be manipulated in order to change the ability of essential transcription factors to associate with their target sites on DNA to therapeutically target myofibroblast differentiation.
