To identify proteins that potentially associate with BAF we affinity-purified proteins from HeLa nuclear extracts using recombinant purified His-tagged human BAF (H6
BAF) dimers bound to Ni++
-agarose beads, with agarose beads alone as controls (see Methods
). Bound proteins were eluted sequentially with buffer containing 0.3 M NaCl to remove proteins of moderate affinity, then with 1 M NaCl to elute higher-affinity partners (). An aliquot (10%) of each eluate was resolved by SDS-PAGE and silver-stained for qualitative analysis. Reproducible patterns of BAF-associated bands were seen (; n
3). The remaining eluates (90%) from a single experiment were digested with trypsin in solution and all detectable constituent proteins were identified by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Data were analyzed using an in-house Mascot server and Scaffold software, resulting in over 70 significant, unique, and non-redundant protein “hits” (Table S1
). Proteins identified by Scaffold with at least 89% confidence in the 0.3 M or 1 M NaCl eluates from BAF-beads, and not detected in bead-only controls, are listed in . lists selected proteins that were detected with at least 89% confidence in a BAF-bead eluate(s) and also in a bead-only control(s). A few proteins appear in both tables based on peptides that fit each criterion. The full (100 megabyte) searchable Scaffold dataset is available upon request. We also scaled up sample preparation, and used isobaric tagging for relative and absolute quantitation (iTRAQ) reagents to independently identify and quantify proteins that were specifically enriched on BAF-beads, relative to bead-only controls. For iTRAQ analysis, the four samples were each labeled with a different iTRAQ reagent 
. This analysis yielded many proteins with significantly enriched binding to BAF-beads, relative to bead-only controls, expressed as enrichment ratios ().
Affinity purification of BAF-associated proteins from HeLa cell nuclear lysates.
Summary of all non-redundant proteins identified in the 0.3 M and 1 M NaCl elutions of HeLa nuclear lysate proteins bound to BAF beads only.
Summary of all non-redundant proteins identified in the 0.3 M and 1 M NaCl elutions of HeLa nuclear lysate proteins bound to BAF and control beads.
iTRAQ quantification of bound proteins eluted by 0.3 M or 1.0 M NaCl from BAF beads, relative to bead-only controls.
The iTRAQ analysis () yielded several abundant proteins known to bind BAF directly, including lamin A 
, core histones H3 and H4 
, plus one protein, histone H1.2, known to associate with BAF indirectly 
. All detected proteins were present in both the 0.3 and 1 M BAF-bead eluates (; 1 M/0.3 M column). Several proteins including H2A, H2B and Treacle were enriched 3–4 fold in the 1 M eluate, compared to the 0.3 M eluate (; 1 M/0.3 M column), suggesting relatively high affinity either for BAF or a co-purified partner (e.g., lamin A or histone H3 
). The iTRAQ results for the 0.3 M eluates showed that lamin A/C and all four core histones were enriched on BAF-beads (over control beads) by ~11-fold and 8.8-to-10.8-fold, respectively (; 0.3 M/control column). Similar enrichments for individual core histones implied similar stoichiometry and suggested they bound BAF as nucleosomes. This was consistent with evidence that BAF binds core histone H3 in vitro
and in vivo 
and associates with nucleosomes in vivo
(Montes de Oca, Andreassen & Wilson, in preparation). Alternatively, core histones might co-enrich on BAF-beads by binding other abundantly recovered proteins including lamins (via H2A/H2B; 
) or PARP1 
. Histone macro-H2A, which concentrates on the inactive X-chromosome 
, was enriched 13.5-fold (, 0.3/control column). Compared to controls, the iTRAQ analysis () also revealed significant enrichment for nucleophosmin (~16-fold), SET/I2PP2A (~21-fold), transcription factor NonO (~3-fold), Ser/Thr-protein phosphatase 1 (FB19; ~3-fold), Huntingtin (15-fold), RBBP4 and/or RBBP7 (~4-fold; see also ), FKBP-type peptidyl-prolyl cis-trans isomerase slyD (~50-fold), DNA-directed RNA polymerase beta (~43-fold) and Treacle (Treacher-Collins Syndrome protein; ~14-fold).
Additional candidates were independently identified by LC-MS/MS (–) including three transcription factors (Requiem, p15/SUB1/PC4 and LEDGF/p75 ) and a remarkable number of proteins involved in gene regulation or genome integrity including PARP1 (–), DDB1 and DDB2 () and Xeroderma pigmentosum complementation group C protein (XPC/XPCC; ). Also recovered were Mediator of DNA damage Checkpoint 1 protein (MDC1; which associates with phosphorylated H2AX [γH2AX] and Mre11 at sites of DNA damage 
; ), and five components of the nucleosome remodeling and histone deacetylase (NuRD) complex: unique components MTA2 and Mi2β/CHD4 
() plus HDAC1 or HDAC2 (unique peptides not recovered; ), and RBBP4 and RBBP7 (, ). Also identified were DPY-30-like protein (a component of Set1 histone methyltransferase complexes 
; ), G9a (an H3-K9 histone methyltransferase 
; ) and three isoforms of heterochromatin protein 1 (HP1α, HP1β, HP1γ; –), which specifically recognize and bind K9-methylated H3 
Independent validation of selected candidates
To evaluate the physiological relevance of this proteome, we chose 11 candidates to test independently for potential association with BAF in vivo
. Antibodies for many candidates were unavailable or unsuitable for immunoprecipitation, so we used an epitope-tag strategy. Each candidate was transiently expressed as a FLAG- or GFP-tagged protein in HeLa cells, then immunoprecipitated from whole cell lysates using antibodies against the epitope tag, resolved by SDS-PAGE and western blotted using antibodies specific for either the tag, endogenous BAF or other endogenous proteins (, , ). Compared to the positive and negative controls (FLAG-H3.1 and FLAG vector, respectively), BAF co-precipitated reproducibly with G9a and SET/I2PP2A (Montes de Oca, Andreassen & Wilson, in preparation) and five other candidates including Requiem () and PARP1 (), as detailed below. By contrast, four other candidates associated with BAF undetectably or weakly, under the same immunoprecipitation conditions: Mi2β and MTA2 (, lanes 1 and 2 vs
3 and 4; n
3), HDAC1 (data not shown) and DPY-30-like (, lane 6 vs
2, αBAF; n>4).
BAF associates with Requiem and PARP1 in vivo.
BAF associates efficiently with RBBP4, and weakly with RBBP7, in HeLa cells.
Dynamic associations of endogenous BAF and emerin with DDB2 and CUL4A in UV-treated cells.
BAF associates with Requiem in vivo
Requiem, with a Kruppel
-type zinc finger and four atypical zinc-fingers, is a pro-apoptotic transcription factor in myeloid cells homologous to the mouse Ubi-d4 protein 
. FLAG-Requiem reproducibly (n>4) and specifically co-immunoprecipitated both endogenous BAF and endogenous emerin from cells (, lane 2, αBAF and αEm). These results independently validated Requiem as BAF-associated in vivo
, and further suggested potential ternary interactions with emerin. Whether BAF or emerin (or both) bind Requiem directly was not tested.
BAF associates with PARP1 in HeLa and HEK293 cells
PARP1, represented by at least 31 distinct peptides, was the most significant protein identified by LC-MS/MS (–). PARP1 ADP-ribosylates many substrates including histones, regulates chromatin structure and has well-characterized roles in DNA damage responses 
. We therefore independently tested a potential association between FLAG-PARP1 and endogenous BAF in HeLa cells. Controls showed little or no background pelleting of endogenous BAF with FLAG vector alone (, lane 4, αBAF), and positive co-precipitation with FLAG-H3.1 (, lane 6, αBAF), as expected. Endogenous BAF co-precipitated reproducibly and at high levels with FLAG-PARP1 under both low stringency (150 mM NaCl, 0.15% TX-100; data not shown), and high-stringency conditions (300 mM NaCl, 0.3% TX-100; , lane 2, αBAF; n>4). A reciprocal experiment using a stable, tetracycline-inducible FLAG-BAF overexpressing cell line (HEK293, human embryonic kidney; 
) gave similar results: endogenous PARP1 co-immunoprecipitated with FLAG-BAF (data not shown). Further Western blotting of the FLAG-PARP1 immunoprecipitates confirmed the presence of lamins A and C (, lane 2, αLmnA/C; n>4) under high stringency conditions, as expected 
; and also revealed low levels of the nuclear membrane protein emerin (, lane 2, αEm; n>4), with higher signals under low stringency conditions (not shown). These results suggest BAF associates with PARP1 in two independent cell lines: HeLa and HEK293.
Selective association of BAF with RBBP4 in HeLa cells
RBBP4 (also known as Chromatin Assembly Factor-1 [CAF-1] p48 subunit or RbAp48) and RBBP7 (RbAp46) are homologous histone chaperones encoded by different genes. Both were specifically identified by LC-MS/MS () and at least one was identified by iTRAQ (). RBBP4 and RBBP7 are WD-repeat proteins that contribute, either together or separately, to several chromatin-modifying complexes including the NuRD complex 
and the CAF-1 complex, required for chromatin assembly after DNA replication and repair 
. We tested for potential interaction between endogenous BAF and either GFP-RBBP4 or GFP-RBBP7 in HeLa cells under normal conditions, or 1 hr after UV-irradiation, since RBBP4 and the CAF-1 complex mediate histone deposition during UV-damage repair. Controls confirmed little association of BAF with GFP alone (, lanes 1 and 2, αBAF) and positive association with GFP-H1.2 (, lanes 7 and 8, αBAF), as expected 
. Endogenous BAF co-precipitated robustly with GFP-RBBP4 in both undamaged and UV-treated cells (, lanes 3 and 4, respectively, αBAF; n
3). By contrast, near background levels of BAF co-precipitated with GFP-RBBP7 (, lanes 5 and 6, αBAF; n
3). Endogenous emerin co-precipitated very weakly with both GFP-RBBP4 and GFP-RBBP7 (, lanes 3–6, αEm; n
3). Control Western blots of whole cell lysates verified that UV-irradiation did not significantly alter the levels of endogenous BAF or emerin proteins, relative to loading control γ-tubulin (, inputs; n
3). BAF interaction with RBBP4 was further confirmed in reciprocal studies of stable HEK293 cells with tetracycline-inducible expression of FLAG-BAF (as above): FLAG-BAF co-immunoprecipitated endogenous RBBP4 (data not shown). Thus, despite 89.4% amino acid identity between RBBP4 and RBBP7 () 
, BAF associated robustly and selectively with RBBP4 in both untreated and UV-treated cells.
BAF and emerin interactions with DDB2 and CUL4 in UV-irradiated HeLa cells
Three proteomic candidates (DDB1, DDB2, XPC; ) are involved in DNA damage repair. DDB1 and DDB2 are key components of the UV-DNA-damage-binding protein complex (UV-DDB) 
, which recognizes DNA-distorting lesions and mediates nucleotide-excision repair, in part by recruiting XPC-RAD23B heterodimers 
. In addition, DDB1 and DDB2 also associate with Cullin 4 (CUL4)-containing E3 ubiquitin ligase complexes. One such complex, CUL4-DDB-ROC1, is recruited rapidly (5–10 min) by DDB1-DDB2 to sites of UV-induced DNA damage, where it ubiquitylates H3 and H4 causing nucleosome eviction and exposure of damaged DNA 
. The CUL4-DDB-ROC1 complex then promotes repair by recruiting repair proteins XPC and its partner RAD23B to damage sites 
We found that endogenous DDB1 co-immunoprecipitated with FLAG-BAF in tetracycline-induced HEK293
BAF cells (data not shown), independently validating its association with BAF in vivo
. Given the above-described roles for DDB1-DDB2 and XPC-RAD23B, and recognizing that CUL4 was not identified in our (unirradiated) HeLa cell proteome, we hypothesized that BAF might be involved in cellular responses to UV-damage. To test this idea we expressed either FLAG-CUL4A, FLAG-DDB2, FLAG-H3.1 (positive control) or FLAG vector alone (negative control) transiently (~24 hrs) in HeLa cells. Cells were then either treated with UV light (20 J/m2
) or left untreated as controls, and allowed to recover for the indicated time. Whole cell lysates were then prepared and either Western blotted directly (, inputs) or after immunoprecipitation with FLAG-conjugated beads (, pellets). To assess whether cells were effectively damaged by UV-treatment, whole cell lysates were Western blotted with antibodies against endogenous PARP1 to assay production of its characteristic apoptosis-induced cleavage product 
. PARP1 cleavage was weak or undetectable in untreated cells (, “—”; arrowhead), and detectable but faint 60 min after UV-treatment (, lanes 2, 4, 9, 12, 14, 19; arrowhead; n
3). PARP1 cleavage was obvious 120 min after UV-treatment (, lanes 10 and 20; arrowhead), suggesting cells had entered apoptosis. FLAG-pulldowns from untreated cells or from each timepoint after UV-irradiation, were probed with antibodies specific for either FLAG, endogenous DDB1, endogenous emerin or endogenous BAF (). Endogenous DDB1 co-precipitated with FLAG-DDB2 at all timepoints (, lanes 5–10, αDDB1; n
3) as expected, since these proteins form heterodimers 
. Validating our proteomic results, endogenous BAF co-precipitated with FLAG-DDB2 (, lanes 5–10, αBAF; short and long exposures both shown; n
3) above the FLAG vector control background (, lanes 1–2, αBAF; n
3), in both UV-treated and untreated cells. Endogenous emerin also co-precipitated efficiently with FLAG-DDB2 (, lanes 5–10, αEm; n
3) in both UV-damaged and undamaged cells. In addition, emerin also co-precipitated with FLAG-CUL4A in undamaged cells, and this association transiently decreased, then increased after UV-treatment (, lanes 15–20, αEm; n
3). A similar trend was seen for the endogenous BAF association with FLAG-CUL4A (, lanes 15–20, αBAF; n
3). Despite experiment-to-experiment variation in the exact timing of highest association between BAF or emerin and FLAG-CUL4A, endogenous BAF and emerin consistently “co-peaked” in their interaction with FLAG-CUL4A after UV-treatment. These results validated CUL4A, DDB2 and DDB1 as partners for BAF in vivo
, and suggested their association with BAF is differentially regulated in response to UV-damage. Furthermore, we discovered robust and potentially UV-regulated associations of the nuclear membrane protein emerin with both DDB2 and CUL4A. These results suggest novel roles for both BAF and emerin in genome integrity.