BLAP75, an essential component of Bloom's syndrome protein complexes that maintain genome integrity

Abstract

Bloom's syndrome (BS) is a rare human genetic disorder characterized by dwarfism, immunodeficiency, genomic instability and cancer predisposition. We have previously purified three complexes containing BLM, the helicase mutated in this disease. Here we demonstrate that BLAP75, a novel protein containing a putative OB-fold nucleic acid binding domain, is an integral component of BLM complexes, and is essential for their stability in vivo. Consistent with a role in BLM-mediated processes, BLAP75 colocalizes with BLM in subnuclear foci in response to DNA damage, and its depletion impairs the recruitment of BLM to these foci. Depletion of BLAP75 by siRNA also results in deficient phosphorylation of BLM during mitosis, as well as defective cell proliferation. Moreover, cells depleted of BLAP75 display an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, BLAP75 is an essential component of the BLM-associated cellular machinery that maintains genome integrity.

Figure 1

BLAP75 is an integral component of BLM complexes. (A) Silver-stained and (B) Coomassie blue-stained SDS gels showing that the major polypeptides immunopurified by BLM and BLAP75 antibodies are identical, suggesting that these two proteins, together with the isolated polypeptides, are all components of BLM complexes. The major polypeptides visualized on Coomassie-stained gel (marked by arrows) from both preparations have been identified by MS. As controls, mock immunoprecipitation (IP) was carried out by using either a preimmune serum or protein A beads alone. The affinity-purified BLAP75 antibody was also shown as a control. The pattern of BLM-associated polypeptides reported here is somewhat different from the previous study (Meetei et al, 2003b), because preparation of the nuclear extracts used in these studies is different (see Materials and methods). It is noticed that the amount of antigen subunit (e.g., BLM in BLM IP) is lower compared to other components of the complex in (A). The reason could be that the antigen subunit binds directly to the polyclonal antibody beads, so that it is eluted less efficiently than other components of the complex under this condition. (C) Immunoblotting to show that BLAP75, BLM, Topo IIIα and RPA are present in complexes immunopurified by both BLM and BLAP75 antibodies, but absent in mock purifications using preimmune serum. The analysis was performed for the load, flow-through (FT) and the immunoisolated (IP) fractions. (D) Immunoblotting to show the Superose 6 gel filtration profiles of BLM and BLAP75 in nuclear extracts prepared from HeLa cells (upper two panels), a BS patient cell line (GM08505) (middle two panels) and the same patient cell line complemented by stably expressing GFP-BLM (bottom two panels). Notably, majority of BLAP75 in HeLa and GFP-BLM complemented cells fractionate in high-molecular-weight fractions (>670 kDa), but fractionate in low-molecular-weight fractions (about 440 kDa, fraction 28) in the BS cell line, suggesting that in the absence of BLM, the BLAP75 complex becomes smaller.

Figure 2

BLAP75 contains a putative OB-fold nucleic acid binding domain and is conserved in multiple eukaryotic species. (A) Sequence alignment of BLAP75 family of proteins from multiple eukaryotic species. The conserved N- and C-terminal regions are marked by brackets. The predicted β-sheets for the OB-fold nucleic acid binding domain are underlined. The abbreviations and accession numbers for different BLAP75 orthologs are as follows: human (humBLAP75; also called C9orf76 or FLJ12888; NP_079221), mouse (mouBLAP75; NP_083180.2), C. intestinalis (cioBLAP75; AK114784), D. rerio (danBLAP75; AAH45482.1) and C. elegans (celBLAP75; NP_741607.1). (B) Sequence alignment to show that part of the N-terminal domain of BLAP75 is also conserved in the Tudor domain 3 (Tdrd3) family of proteins. The abbreviations and accession numbers of these proteins are as follows: human (humTdrd3; also called FLJ21007; NP_110421.1), D. rerio (danTdrd3; AAH55609.1), Xenopus tropicalis (xenTdrd3; NP_989385.1), Drosophila (droTdrd3; NP_648724.1) and C. elegans (celTdrd3; NP_503358.1). A hypothetical protein of Schizosaccharomyces pombe (Yau3_schpo; NP_594146.1) was also found to contain the domain and was included in the alignment, even though it is not a member of the Tdrd3 family.

Figure 3

BLAP75 is required for BLM complex stability and mitotic phosphorylation of BLM. (A) Immunoblotting to show that suppression of BLAP75 expression by siRNA oligos reduces the levels of BLM and Topo IIIα in HeLa cells. As control, an siRNA oligo with scrambled sequence fails to reduce BLAP75 expression as well as the levels of BLM and Topo IIIα. Immunoblotting of β-actin is shown as loading control. (B) Immunoblotting to show that the level of BLAP75 is not significantly altered in cells depleted of BLM by siRNA, or in a BS patient cell line (BLM−/−) that lacks BLM. A lymphoblastoid cell line derived from a normal individual (WT) was used as a control. (C) Immunoblotting to show that the reduction of Topo IIIα and BLM protein levels in BLAP75-depleted HeLa cells is not due to increased apoptosis. HeLa cells were either untreated or treated with apoptosis-inducing drugs, TNFα and cycloheximide, as shown at the top of the figure. The presence of the specific cleavage product of PARP (ΔPARP) is a marker for cells undergoing apoptosis. Notably, the appearance of caspase 3 cleavage product of BLM (ΔBLM) correlates with apoptosis induction, but not BLAP75 depletion. In contrast, reduction in Topo IIIα protein level correlates with BLAP75 depletion, but not apoptosis induction. Polypeptides that crossreact with BLAP75 antibodies are marked by asterisks. (D) Flow cytometry histograms to show normal cell cycle progression and absence of apoptosis for BLAP75-depleted cells. The percentages of cells at different stages of cell cycles are shown within figures for HeLa cells growing under normal conditions (upper panel). Notably, the proportion of sub-G1 cells (marked by a bracket with <G1), which have less than 2N DNA content and include apoptotic cells, is very low under normal growth conditions (less than 1%), but is significantly increased when cells undergo apoptosis (bottom panel). (E) Immunoblotting to show that suppression of BLAP75 expression by siRNA reduces mitotic phosphorylation of BLM. Cells were treated without any drugs, or with mitotic inhibitors, Taxol and demecolcine, as indicated. The cells at different stages of the cell cycle were determined by flow cytometry analysis, and the percentages of cells at G2 and M phases are shown at the bottom. The hyper- and hypo phosphorylated BLM proteins are marked by a solid and an empty dot, respectively. A possible degradation product of BLM is marked by an asterisk.

Figure 4

BLAP75 is required for sustained cell proliferation. (A) Histogram to show that BLAP75 depletion reduces the total number of viable cells when compared to the cells treated with a control oligo. The viable cells are distinguished from dead ones by Trypan blue staining. (B) Immunoblotting to show that HeLa cells infected with BLAP75 siRNA in lentivirus vector have reduced expression of BLAP75, in comparison to same cells infected with a control siRNA using the same vector. The quantitated results (shown at the bottom) were obtained by scanning with a Typhoon Scanner and ImageQuant, and the normalization was performed using actin as a control. (C) Histogram to show that the number of colonies (which reflect sustained cell proliferation) is drastically reduced for cells treated with BLAP75 siRNA vector compared to those with the control vector. (D, E) Similar data as (B, C except that a different BLAP75 siRNA vector was used, and the experiment was performed in HCT116 cells.

Figure 5

BLAP75 colocalizes with BLM in nuclear foci in response to DNA damage and is required for BLM foci formation. (A) Images from indirect immunofluorescence analysis to show that BLAP75 in HeLa cells forms nuclear foci in response to several DNA damage reagents. DNA was visualized by costaining with Hoechst dye. (B) Representative immunofluorescence images to show that BLAP75 and BLM colocalize in nuclear foci in response to the DNA crosslinking drug MMC (50 ng/ml). These cells were also treated with a scrambled siRNA oligo to serve as a control for the next figure. (C) Representative images to show that HeLa cells treated with BLAP75 siRNA oligo display not only reduced BLAP75 staining but also reduced number of BLM foci. (D) Histogram of quantitative analysis of cells containing BLAP75 and BLM foci to illustrate that the percentage of cells containing BLM foci is drastically reduced in HeLa cells depleted of BLAP75 by siRNA oligo. A scrambled siRNA oligo was transfected in cells that were untreated with BLAP75 siRNA oligo to serve as a control (marked as ‘BLAP75 siRNA −'). The presence or absence of MMC is also indicated. Nuclei were tabulated in groups containing 0–5 and >5 foci per nucleus.

Figure 6

BLAP75-depleted cells display increased frequency of SCE similar to BLM-depleted cells. A histogram to show that depletion of BLAP75 by siRNA results in elevated frequency of SCE (as defined by the number of SCE per chromosome). HeLa cells treated with a scrambled oligo or a BLM siRNA oligo were used as negative and positive controls. SV40-transformed fibroblast cell lines from a normal individual (BLM+/+) and a BS patient (BLM−/−) are also shown for comparison. More than 600 chromosomes were scored for each group. Error bars represent standard deviation of each sample. Statistical analyses of the SCE data were performed using both one-factor ANOVA (analysis of variance) and the Student's two-tailed t-test. The results show that differences between control cells and those depleted of either BLM or BLAP75 are statistically significant, and the P-values (<0.001) were included in the figure.