Mapping the micro-proteome of the nuclear lamina and lamina-associated domains

Abstract

The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

Off-the-shelf proximity biotinylation for interaction proteomics

“…GO-analyses were performed using Clusterprofiler v4.0.0 31 . For GO-based comparison of ProtA-Turbo Emerin with other published lamin-targeting strategies, we used data for nuclear lamina microdomain mapping (LAP2B-BioID 32 ), a two-component lamina BioID mapping strategy (2C-BioID 33 ), conventional Lamin A BioID 3 , and an antibody-HRP-based method for biotinylation by antibody recognition (BAR 17 ). Enriched proteins were defined according to the following criteria: LAP2B-BioID > 1.5-FC/ctrl; 2C-BioID > 1.5-FC/ctrl (-noAP21967); Lamin A BioID: proteins were downloaded from Table S1 ; BAR: all proteins of the LMNA Unbound sample that are >2 fold enriched over control, were not shared with the no antibody control and had at least 2 unique peptides.…”

In Silico and In Vivo Analysis of Amino Acid Substitutions That Cause Laminopathies

“…Given that the lamin meshwork forms a scaffold for the attachment of genomic DNA packaged into chromatin, it is possible that substitutions at R264 disrupt such contacts [19,[23][24][25]. To test for this, we analyzed the degree to which the fluorescent DAPI signal colocalized with the fluorescent signal from antibodies to LamC and lamDm 0 using the JACoP plugin for Fiji [68,69].…”

“…Support for this statement comes from studies showing that transient nuclear rupture and increased DNA damage are observed in skeletal muscle nuclei, which experience mechanical stress [21,22]. Lamins provide a scaffold for attachment of chromatin to the nuclear envelope at specific genomic locations called lamin-associated domains (LADs), which play a role in the orchestration of proper gene expression [19,[23][24][25]. LADs attract specific non-histone chromosomal proteins and are enriched for histone H3K9 tri-methylation, an epigenetic gene silencing mark [23].…”

“…Lamins provide a scaffold for attachment of chromatin to the nuclear envelope at specific genomic locations called lamin-associated domains (LADs), which play a role in the orchestration of proper gene expression [19,[23][24][25]. LADs attract specific non-histone chromosomal proteins and are enriched for histone H3K9 tri-methylation, an epigenetic gene silencing mark [23]. Lamins also interact with many other nuclear envelope proteins, including nuclear pore proteins [26].…”

“…This could explain the skeletal muscle and cardiac laminopathies, but perhaps not adipose tissue disorders. For tissues not under mechanical stress, the pathogenic mechanisms might relate to the function of lamins in genome organization, with abnormalities in the lamina causing altered gene expression [23,32]. Alternatively, tissue-specific defects could arise from cell type-specific interactions between lamins and nuclear envelope proteins [31].…”