Barrier-to-Autointegration Factor Proteome Reveals Chromatin-Regulatory Partners

Rocío Montes De Oca,Katherine L Wilson,Christopher J Shoemaker,Marjan Gucek,Robert N Cole,Sebastian D Fugmann

doi:10.1371/journal.pone.0007050

Rocío Montes De Oca, Katherine L Wilson + Show 4 more

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https://doi.org/10.1371/journal.pone.0007050

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Journal: PloS one	Publication Date: Sep 16, 2009
Citations: 148	License type: CC BY 4.0

Affiliation: Johns Hopkins Medicine, Johns Hopkins University

Abstract

Nuclear lamin filaments and associated proteins form a nucleoskeletal (“lamina”) network required for transcription, replication, chromatin organization and epigenetic regulation in metazoans. Lamina defects cause human disease (“laminopathies”) and are linked to aging. Barrier-to-autointegration factor (BAF) is a mobile and essential component of the nuclear lamina that binds directly to histones, lamins and LEM-domain proteins, including the inner nuclear membrane protein emerin, and has roles in chromatin structure, mitosis and gene regulation. To understand BAF's mechanisms of action, BAF associated proteins were affinity-purified from HeLa cell nuclear lysates using BAF-conjugated beads, and identified by tandem mass spectrometry or independently identified and quantified using the iTRAQ method. We recovered A- and B-type lamins and core histones, all known to bind BAF directly, plus four human transcription factors (Requiem, NonO, p15, LEDGF), disease-linked proteins (e.g., Huntingtin, Treacle) and several proteins and enzymes that regulate chromatin. Association with endogenous BAF was independently validated by co-immunoprecipitation from HeLa cells for seven candidates including Requiem, poly(ADP-ribose) polymerase 1 (PARP1), retinoblastoma binding protein 4 (RBBP4), damage-specific DNA binding protein 1 (DDB1) and DDB2. Interestingly, endogenous BAF and emerin each associated with DDB2 and CUL4A in a UV- and time-dependent manner, suggesting BAF and emerin have dynamic roles in genome integrity and might help couple DNA damage responses to the nuclear lamina network. We conclude this proteome is a rich source of candidate partners for BAF and potentially also A- and B-type lamins, which may reveal how chromatin regulation and genome integrity are linked to nuclear structure.

Highlights

The nuclear envelope and lamin filament networks tether chromatin and influence chromatin organization and gene expression at several levels [1,2], through mechanisms that remain obscure
To identify proteins that potentially associate with Barrier-to-autointegration factor (BAF) we affinity-purified proteins from HeLa nuclear extracts using recombinant purified His-tagged human BAF (H6BAF) dimers bound to Ni++-agarose beads, with agarose beads alone as controls
It will be important in the future to verify the interaction between endogenous BAF and each endogenous partner

Summary

Introduction

The nuclear envelope and lamin filament networks tether chromatin and influence chromatin organization and gene expression at several levels [1,2], through mechanisms that remain obscure. A- and B-type lamins form separate networks of nuclear intermediate filaments, which are anchored to inner nuclear membrane (INM) proteins and ramify throughout the nuclear interior [3,4]. Lamins interact with chromatin and support most nuclear activities including replication, transcription and DNA damage repair [5,6,7,8]. Mutations in A-type lamins or other lamina components cause more than 15 human diseases (‘‘laminopathies’’) including Emery-Dreifuss muscular dystrophy and accelerated aging syndromes Cells expressing mutant lamins have compromised nuclear integrity. In HutchinsonGilford progeria cells the mutated lamin A precursor (‘‘progerin’’)

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