ABSTRACT 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.