BLM and PICH in Centromere Disjunction together with the mean number of GFP-BLM-positive centromeres per cell (right panel). Bars indicate the standard deviation (SD). (C) Localization of GFP-BLM (green) to centromeres (red) in prophase cells. The nucleus and centromeres were visualized as in A. Scale bar = 5 mm. (D) Localization of GFP-BLM (green) with an inactive helicase domain (GFP-I841T) or with both the helicase and DNA-binding domains inactivated (GFP-G891E). Nuclei and centromeres were visualized as in A. Scale bar = 5 mm. doi:10.1371/journal.pone.0033905.g001 isomerase IIa (Topo IIa) activity and occurs after the onset of anaphase, after the disappearance of cohesin [6,7]. Thus, sister centromeres are held together through double-stranded DNA catenations until the end of anaphase, accounting for the strong induction of centromeric UFBs by catalytic inhibitors of Topo II [4,5]. The frequency of PICH-positive UFBs is also higher in BLM-deficient cells than in control cells, suggesting the involve- ment of BLM in their resolution [5]. This point is of particular interest because BLM deficiency causes Bloom’s syndrome, an autosomal recessive disease displaying one of the strongest known correlations between chromosomal instability and an increase in the risk of cancer at an early age [8]. The hallmark of BLM- deficient cells is a high frequency of sister chromatid exchanges (SCEs) [8]. Thus, BLM plays a crucial role in preventing genetic instability and cancer. In normal cells, BLM is detected only on UFBs in anaphase, whereas PICH staining is detected as early as metaphase [5]. PICH is required for the localization of BLM to anaphase UFBs, and BLM is required for the chromatin remodeling function of PICH in vivo, the two proteins cooperating to limit histone incorporation into UFBs and to promote their resolution [9]. We hypothesized that the functional relationship between PICH and BLM might also be of major importance before anaphase in the maintenance of centromere integrity. We report here the localization of BLM to the centromere of chromosomes from G2 phase to mitosis. We show that PICH and BLM deficiencies are associated with changes in centromere structure, with an increase in centromeric non disjunction in cohesin-depleted cells and a defect in the recruitment of active Topo IIa to centromeres. Our results reveal the existence of a new centromeric mechanism involving cooperation between PICH and BLM, likely to render some centromeric catenates accessible to Topo IIa before anaphase onset, thereby facilitating correct centromeric disjunction and preventing the formation of super- numerary UFBs. Results BLM is a Centromeric Protein PICH is a centromeric protein [4]. As BLM, like PICH, localized with centromeric UFBs [5], we first investigated whether BLM also localized to centromeres. We analyzed the subcellular distribution of stably expressed GFP-tagged BLM protein in asynchronous GM08505 BS cells (GFP-BLM cells) [10]. We detected GFP-BLM in PML bodies, as expected [11] (Figure S1), but in some cells, BLM foci were smaller, double-dotted and no longer colocalized within PML bodies. Using CREST serum as a centromeric marker, we showed, by cyclin B1 staining [12], that 73% of cells with GFP-BLM-positive centromeres were in G2- phase or early mitosis (Figure 1A and 1B, left panel). These cells included a mean of 27 GFP-BLM-positive pairs of centromeres (7 to 59 pairs). By contrast, only 10 GFP-BLM-positive centromeres (1 to 24 pairs) were detected in late S-phase cells (most centromeres being paired), negative for cyclin B1 staining (Figure 1B, right panel). All the centromeres in prophase cells displayed positive staining for GFP-BLM (Figure 1C). These results suggest that BLM is progressively loaded to the centromeres from late S phase until mitosis. We then investigated whether the helicase and/or DNA binding activities of BLM were required for centromeric localization, using GM08505 BS cells stably trans- fected with a construct encoding either the BLM protein with an inactive helicase domain (GFP-I841T) or with both the helicase and DNA binding domains inactivated (GFP-G891E) [13]. We found that DNA-binding activity was required for the centromeric localization of BLM, whereas helicase activity was not. Indeed, GFP-G891E displayed a diffuse distribution in BS cells and was not detectable at centromeres, suggesting that BLM may interact with centromeric DNA (Figure 1D). The centromeric localization of endogenous BLM was also investigated in immunofluorescence experiments with two differ- ent anti-BLM antibodies, on mitotic chromosome spreads from HeLa S3 cells at prometaphase/metaphase stage, downregulated for BLM (siBLM) or without BLM downregulation (siCtrl). In HeLa siCtrl cells, we detected endogenous BLM at centromeres, with CREST serum used as a centromeric marker. The siRNA- mediated depletion of BLM (siBLM) resulted in the absence of the BLM signal at centromeres, confirming the specificity of the anti- BLM antibodies (Figure 2A). Closer examination of the centro- meres of each chromosome showed the BLM signal to be present on about 84% of centromeres in HeLa siCtrl cells and on 1.7% of centromeres from HeLa siBLM cells (Figure 2B). Thus, BLM is clearly a centromeric protein. BLM and PICH Deficiencies are Associated with Changes in Centromeric DNA Structure We investigated the possible effects of BLM deficiency on centromere structure. We used a FISH (fluorescence in situ hybridization)-based assay to investigate the centromeric DNA organization of one chromosome and its volume (chromosome 8, CEN-8 probe [14]) in GM08505 BLM-deficient cells stably expressing a GFP vector (BS cells) and in GFP-BLM cells during prometaphase and metaphase (Figure 3A). We found that CEN-8 signal volume in BS cells was less than half that in control cells, indicating that BLM is involved in centromeric DNA structure (Figure 3B, left panel). For confirmation of these findings, we analyzed the effects of BLM deficiency on the structural morphology of the centromeric DNA in BS and GFP-BLM metaphase cells by electron microscopy (EM) (Figure 3C). Centromeric chromatin was identified adjacent to the typical kinetochore structure. Immunofluorescence analysis of several kinetochore/centromere markers confirmed that BLM deficiency had no major effect on kinetochore structure, as previously shown for PICH-deficient cells [4] (Figure S2). However, the centromeric chromatin was clearly more dense in BS cells than in GFP-BLM cells (Figure 3C), confirming an effect on centromeric DNA organization of the absence of BLM. We next performed FISH experiments in GFP-BLM cells downregulated for PICH (siPICH) or without PICH downregulation (siCtrl). We found that CEN-8 signal volume in PICH-downregulated cells was about half that in control cells, indicating that PICH is also involved in centromeric DNA structure before anaphase onset (Figure 3B, right panel). These results are not the consequence of a coregulation of PICH and BLM genes, because the deficiency of one protein did not affect the expression of the other (Figure 4A, lower left panel). Finally, CEN-8 signal volume in HeLa cells depleted of BLM, PICH or both proteins was significantly lower than that in control cells (Figure S3). There was therefore no additive or synergistic PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e33905