Saturday, May 4, 2024
5/4/2024
PROJECT SUMMARY: 5-methylcytosine oxidation in development and disease DNA methylation at gene promoters and enhancers is usually an indication that genes are silenced, whereas loss of methylation through the TET-mediated 5-methylcytosine (5mC) oxidation pathway can lead to gene activation. TET proteins and the 5mC oxidation pathway are known, pivotal actors in embryonic development and disease, yet the mechanisms by which TET-mediated 5mC oxidation is regulated are completely unknown. We recently discovered that the protein SMCHD1 is a potential negative regulator of TET activity. In humans, SMCHD1 depletion activates the DUX4 gene(s) that cause(s) cell toxicity, muscle degeneration, and facioscapulohumeral muscular dystrophy (FSHD). We hypothesize that one key function of SMCHD1 is to control 5mC oxidase activities and that lack of SMCHD1 leads to an over-activation of TET-mediated epigenetic function, and the activation of DUX4. The objective of this project is to test this hypothesis and obtain a fundamental understanding of how SMCHD1 interacts with and controls TET-mediated epigenetic function in normal embryonic development and disease. In Aim 1, we will use mouse embryonic stem cells and human induced pluripotent stem cells to determine the basic mechanisms of SMCHD1-regulated TET function in normal development. We will employ SMCHD1-knockout cell and mouse lines and determine how TET protein distribution in the genome is altered; which loci are affected; if the distribution of oxidized 5-methylcytosine bases (5hmC) is specific to SMCHD1 binding sites; which regions of the (zygotic) genome are activated upon SMCHD1 loss; and if there are shared SMCHD1-mediated regulatory pathways between mouse and human pluripotent cells. The results of these experiments will establish the negative regulatory function of SMCHD1 in normal development and set the stage for disease-specific studies in Aim 2. In Aim 2, we will inactivate SMCHD1 in human muscle cells and determine the epigenetic role of SMCHD1 in cell differentiation and disease by determining gene expression changes, chromatin structure, and genome-wide DNA methylation status. We expect that SMCHD1-mediated TET inhibition and regulation of gene expression will be similar in human and mouse cells, and that depleting TET activity in SMCHD1 knockout cells will at least partially reverse the effects of SMCHD1 loss-of-function phenotypes. In Aim 3, we will perform biochemical studies of SMCHD1-TET protein-protein interactions to identify key binding domains, and determine if TET inhibitors can silence DUX4 expression in cell lines lacking functional SMCHD1, and in muscle cells derived from FSHD patients. If successful, these data will provide proof of principle that TET inhibitors may have therapeutic benefit for FSHD patients. Together, the results of this project will generate the first mechanistic, regulatory model of the TET- mediated 5mC oxidation pathway in mouse and man, and the role of the novel TET-SMCHD1 axis in normal development and disease.
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