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

Xianrong Wong,Karen L Reddy,Akhilesh Pandey,Anil K Madugundu,Victoria E Hoskins,Molly Gordon,Jevon A Cutler

doi:10.26508/lsa.202000774

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.

Highlights

DNA and proteins are highly organized within the eukaryotic cell nucleus
Establishment of BioID system to map the local proteomes at the nuclear periphery The BioID system was initially developed using the Lamin A protein as the bait allowing a robust interactome of this insoluble protein [25]
To expand on these efforts and to focus on an inner nuclear membrane (INM) protein implicated in lamina-associated domains (LADs) organization, we chose to identify vicinal proteins of the LAP2β in a BioID study

Summary

Introduction

DNA and proteins are highly organized within the eukaryotic cell nucleus. Sequestration of proteins into nuclear sub-domains and the higher order organization of chromatin itself have been implicated in the regulation of the genome [1, 2, 3, 4, 5, 6]. DamID (DNA Adenine Methyltransferase Identification), a genome-wide technique to identify nuclear lamina-proximal chromatin, has allowed the identification of lamina-associated domains (LADs) [9] These 100 kb to megabase (Mb) sized domains are enriched for genes that are transcriptionally silent and enriched in histone modifications indicative of facultative heterochromatin, such as histone H3 lysine 9 di/tri-methylation (H3K9me2/3) and histone H3 lysine 27 trimethylation (H3K27me3) [10, 11, 12, 13]. LADs represent a large fraction of the genome (30–50%, depending upon the cell type) and are highly correlated with the so-called heterochromatic “B-compartment,” as identified by chromatin conformation capture assays (HiC) (13, Preprint, Preprint) Given their importance to cellular function and identity, it is important to understand how these large domains of heterochromatin are regulated, maintained, and formed to understand global genome regulation and organization. An important element of understanding LAD organization and function is identifying which proteins are present at LADs and the nuclear lamina

Methods

Results

Conclusion