barrier-to-autointegration factor proteome reveals chromatin-regulatory partnersbarrier-to-autointegration因子蛋白质组显示chromatin-regulatory伙伴.pdfVIP

Barrier-to-Autointegration Factor Proteome Reveals Chromatin-Regulatory Partners ´ 1 1 2 2 Rocıo Montes de Oca , Christopher J. Shoemaker , Marjan Gucek , Robert N. Cole , Katherine L. Wilson1* 1 Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2 Mass Spectrometry and Proteomics Facility, IBBS, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America 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 inc