LB: Creating a nuclear envelope targeting assay to study chromatin localization at the nuclear envelope and how emerin EDMD1 mutants affect localization


Pre-Clinical Research

Poster Number: T434


James Holaska, PhD, Cooper Medical School of Rowan University, Nicholas Marano, Cooper Medical School of Rowan University

Emery-Dreifuss muscular dystrophy (EDMD) is a rare, inherited muscular disorder characterized by early-onset contractures, progressive muscle weakness, and cardiac involvement. It affects both skeletal and cardiac muscles and can lead to significant disability and life-threatening cardiac complications. EDMD1 is caused by mutations in the EMD gene, which encodes the protein emerin. Emerin is a protein of the inner nuclear membrane and plays a critical role in maintaining the integrity and function of the nucleus in muscle cells. Beyond structural support, emerin is involved in organizing chromatin and regulating gene expression. It participates in tethering chromatin to the nuclear envelope to organize the spatial arrangement of genes within the nucleus. Mutations in the EMD gene disrupt emerin’s normal functions, but the exact pathways through which emerin mutations lead to muscle pathology are not fully understood. Based upon emerin’s demonstrated role in organizing repressive genomic loci to the nuclear envelope, we hypothesize that emerin dysfunction seen in EDMD1 alters the transcriptional reprogramming during satellite cell differentiation by failure to properly reorganize the genome resulting in impaired muscle regeneration. Thus, it is incumbent on us to understand the molecular mechanisms underlying this reorganization and how it goes awry in EDMD1. Chromatin at the nuclear periphery is enriched for repressive marks H3K9me2 and H3K27me3 while also exhibiting a lack of histone acetylation marks. The mechanisms responsible for deposition of these marks on chromatin at the nuclear periphery or recruitment of chromatin containing these marks to the nuclear periphery remains to be elucidated. To establish this mechanism, we developed a nuclear envelope targeting (NET) assay using histone tail peptide sequences with repressive modifications, including H3K9me2 or H3K27me3. These peptides were conjugated to recombinant GST-GFP containing a nuclear localization signal (GST-GFP-NLS). Here, we show nuclear envelope targeting is dependent upon cytosol, temperature, and the presence of an energy-regenerating system. We anticipate the NET assay will be a powerful tool to identify the major players in chromatin targeting to the nuclear envelope and to investigate the mechanism(s) regulating the localization of repressive chromatin to the nuclear envelope.