Barrier-to-autointegration factor proteome reveals chromatin-regulatory partners.
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.
Montes de Oca
Rocío
R
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Shoemaker
Christopher J
CJ
Gucek
Marjan
M
Cole
Robert N
RN
Wilson
Katherine L
KL
D000595:Amino Acid Sequence; D002843:Chromatin / Q000737:chemistry*; D044842:Cullin Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D034881:Nuclear Lamina / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000737:chemistry*; D056505:Retinoblastoma-Binding Protein 4 / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C501959:BANF1 protein, human; C114234:CUL4A protein, human; D002843:Chromatin; D044842:Cullin Proteins; C091435:DDB1 protein, human; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D006657:Histones; D008565:Membrane Proteins; D009687:Nuclear Proteins; D020543:Proteome; C082637:RBBP4 protein, human; D056505:Retinoblastoma-Binding Protein 4; C092710:emerin; D011065:Poly(ADP-ribose) Polymerases