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Investigating the differential structural organization and gene expression regulatory networks of lamin A Ig fold domain mutants of muscular dystrophy

S. Dutta,Prof Vikas Kumar,Madavan Vasudevan

Published 2024 in bioRxiv

ABSTRACT

In metazoan cells, lamins form a proteinaceous meshwork as a major structural component of the nucleus and also play a role in regulating essential cellular processes. Lamins, along with their interactors, act as determinants for chromatin organization throughout the nucleus. The major dominant missense mutations responsible for autosomal dominant forms of muscular dystrophies reside in the Ig fold domain of lamin A. However, how lamin A contributes to the distribution of heterochromatin and balances euchromatin, and how it relocates epigenetic marks to shape chromatin states, remains poorly defined, making it difficult to draw conclusions about the prognosis of lamin A-mediated muscular dystrophies.In the first part of this report, we identified the in-vitro organization of full-length lamin A proteins due to two well-documented Ig LMNA mutations, R453W and W514R, through biophysical and electron microscopy observations. We further demonstrated that both lamin A/C mutant cells predominantly expressed nucleoplasmic aggregates with reduced amounts at the nuclear envelope. Labeling specific markers of epigenetics, such as H3K9me3, H3K27me3, H3K36me3, and HP1α, allowed correlation of lamin A mutations with epigenetic mechanisms. Immunofluorescence and biochemical analyses indicated transcriptional upregulation. In addition to manipulating epigenetic mechanisms, our proteomic studies traced diverse expressions of transcription regulators, RNA synthesis and processing proteins, protein translation components, and posttranslational modifications. These data suggest severe perturbations in targeting other proteins to the nucleus.Our study also predicts specific structural configurations of lamin A determined by its interacting partners’ patterns. Significance This study investigates the effects of two severe mutations in the LMNA gene associated with muscular dystrophies: R453W and W514R. This research aims to provide new insights into the structural environment of full-length lamin A protein due to single-point mutations. Our study employs proteomics to map expression levels of regulators involved in epigenetic states, RNA synthesis and processing, and protein synthesis and processing. It assesses potential effects on binding affinity and transcription, as well as alterations in heterochromatin protein distribution and RNA polymerase II functionality due to mutations affecting lamin A. A systematic investigation of lamin A interactors is conducted to explore their associations with other proteins. Graphical Abstract Figure: Schematic diagram representing the results. Mutations in lamin A protein evicts the nucleosome from nuclear periphery and consequently promotes aberrant gene expression.

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