The goal of our research is to determine the mechanisms by which the lamin isoform meshworks comprising the nuclear lamina (NL) organize chromatin in 3D and regulate gene expression. The experimental plan builds on recently developed techniques for determining lamin meshwork structures at nanoscale resolution using 3D-SIM combined with computational image analysis. This structural approach will be coordinated with studies of the biochemical/molecular interactions of each lamin isoform with chromatin using our recently developed HiLands model, which is the first to focus on the role of lamins in regulating chromatin organization. Throughout the proposed studies we will take advantage of our collection of single, double and triple lamin knockout mouse cells. Specifically we will elucidate the steps involved in the assembly of lamin isoform meshworks within the NL and determine how the assembly state of lamins impacts the 3D organization of chromatin and gene expression. These studies will be accompanied by a multi-pronged approach employing Hi-C, 4-C, epigenomics, transcriptomics, chromosome painting and FISH, the latter combined with 3D SIM- based immunostaining, to determine whether lamins regulate gene expression by maintaining 3D chromatin folding, enhancer-promoter interactions, and epigenetic modifications.
The major goal of this research project is to determine the roles of the nuclear lamin proteins in organizing and regulating the genome. The lamins are major architectural proteins of the nucleus of mammalian cells where they are involved in the regulation of many nuclear processes. Interest in the importance of the lamins in regulating nuclear functions has increased in recent years due to the large number of mutations in lamin genes causing many diseases ranging from premature aging to cardiomyopathies and muscular dystrophies. The studies proposed in this grant use high-resolution techniques to determine how the nuclear structures formed by the lamins regulate the organization of the genome in both healthy and diseased cells.
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