preventing UFB formation, by contributing to the centromeric DNA decatenation process before the metaphase-anaphase transition. DNA catenation induced by the inhibition of Topo IIa has been shown to maintain sister chromatid cohesion in the absence of cohesin complexes [7,16]. We thus analyzed chromo- some spreads from BS cells and from GFP-BLM cells arrested in prometaphase (+colchicine), with (siPICH) or without (siCtrl) PICH knockdown (Figure 4A). For these experiments, Rad21, the cleavable subunit of cohesin, was depleted from all cell lines (siRad21) (Figure 4A, lower left panel). BLM deficiency and PICH knockdown were associated with an increase in centromeric cohesion. Indeed, we observed three distinct and different phenotypes: classical X-shaped chromosomes probably corre- sponding to cells not transfected with Rad21 siRNA (X-shapes), the expected fully disjoined chromatids resulting from cohesin depletion (complete disjunction), and a third, unusual phenotype of separated sister chromatids that were still physically linked, reflecting incomplete chromatid disjunction (incomplete disjunc- tion) (Figure 4A, upper left panels). Careful examination of these ‘‘separated but still paired’’ chromatids revealed that they were mostly linked via their centromeres (visualized as the major chromosomal constriction). This observation was confirmed by quantifying the intensity profiles of the centromeric region between the two chromatids of the ‘‘X-shape’’ and ‘‘incomplete disjunction’’ phenotypes (Figure 4A, upper right panel). We determined the frequency of each phenotype in the four cell lines and found that about 15% of the prometaphase cells presented the X-shaped phenotype. About 19% of siRad21-treated BS cells, siRad21+siPICH treated GFP-BLM cells, and siRad21+siPICH- treated BS cells presented completely disjoined chromatids, whereas about 66% of these cells, displayed an incomplete centromere disjunction phenotype (Figure 4A, lower right panel). By contrast, in GFP-BLM cells with Rad21 knockdown only, 34.2% of the cells displayed complete sister chromatid disjunction and 50.5% had separated sister centromeres that remained paired (as opposed to the 66% observed in the other cell lines). Similar results were obtained in Rad21-depleted HeLa cells, with a significantly higher frequency of cells presenting an ‘‘incomplete disjunction’’ phenotype and a significantly lower frequency of cells presenting a ‘‘complete disjunction’’ phenotype in cells depleted of BLM or PICH (Figure S4). Rad21 levels were initially similar in GFP-BLM cells and in HeLa cells and siRNA-mediated Rad21 downregulation was similarly effective in all cell lines. The differences in the percentage of cells with an incomplete disjunction phenotype therefore did not reflect differences in the efficacy of siRNA-mediated Rad21 downregulation (Figure S5). Finally, we performed the same chromosome spread analysis with GFP-BLM-I841T cells (inactive helicase domain), comparing the results with those for BS and GFP-BLM cells. Similar results were obtained with both Rad21-depleted GFP-BLM-I841T cells and Rad21-depleted BS cells, (Fig. 4B right panel), despite GFP-BLM- I841T and GFP-BLM cells having similar amounts of BLM protein (Figure 4B, left panel). Together, these results show that both BLM and PICH are involved in the complete disjunction of sister centromeres independently of the cohesin pathway, strongly suggesting that in the absence of BLM and/or PICH, some centromeric catenations are not processed, leading to an increase in centromeric non disjunction. The results for cells expressing BLM with an inactive helicase domain indicate that the helicase activity of BLM is involved in the complete disjunction of sister centromeres. Lastly, we observed no additive or synergistic effect of PICH and BLM deficiencies on the ‘‘incomplete disjunction’’ phenotype, BLM and PICH in Centromere Disjunction suggesting that these two proteins are involved in the same regulatory pathway. Centromeric UFBs are not Detectable in PICH-depleted Cells The findings reported above suggest that the higher frequency of centromeric UFBs in BLM-deficient cells [5], which we also confirmed, may be due to a lack of BLM processing activity of some centromeric DNA catenations. These results also suggest that the centromeric DNA catenations persisting in PICH- deficient cells probably result in an increase in centromere UFB frequency. We investigated the effect of PICH knockdown on the frequency of UFBs, by monitoring BLM. We found that PICH depletion resulted in a three-fold decrease in the number of cells presenting UFBs and in a five-fold decrease in the number of BLM-positive UFBs (Figure S6A and S6B). However, most of the BLM-positive UFBs detected were also positive for PICH staining, indicating that PICH downregulation was incomplete, with UFBs positive for both BLM and PICH probably forming in cells not efficiently transfected with PICH siRNA. Only a minor fraction of UFBs (5–6%) were positive for BLM staining and negative for PICH staining and the frequency of these UFBs was not affected by PICH downregulation (Figure S6B). The lack of detection of an increase in the frequency of BLM-positive UFBs in PICH-depleted anaphase cells can be accounted for by recent observations showing that PICH is required for BLM localization to anaphase threads [15]. Nevertheless, we cannot exclude the possibility that UFBs may be prematurely disrupted in the absence of PICH, as previously suggested [4], or that they may not form at all. We were unable to visualize UFBs in the absence of PICH, even by BrdU staining, probably due to the inherent limitations of this method [17]. However, as PICH downregulation is associated with an increase in the frequency of cells presenting an ‘‘incomplete disjunction’’ phenotype, UFB frequency is probably higher in these cells, although these structures are not detectable in the absence of PICH. BLM and PICH are Required for the Recruitment of Topo IIa to Centromeres Topo IIa is known to be responsible for the elimination of centromeric DNA catenation [16]. We investigated the possible role of BLM and PICH in centromeric decatenation, by assessing the recruitment of active Topo IIa to the pericentromeric region (satellite 3) and to the centromere of chromosomes X (a-satellite sequences DXZ1) and 17 (D17Z1). Topo IIa was trapped on DNA by 15 minutes of treatment with etoposide [18], and analyzed by chromatin immunoprecipitation (ChIP) coupled to semi-quantita- tive PCR (see Methods) in BS cells and GFP-BLM cells (Figure 5A) and in PICH-depleted GFP-BLM cells (siPICH) and their control cells (siCtrl) (Figure 5B). Immunoprecipitation of active Topo IIa in BS cells, PICH-depleted GFP-BLM cells and their respective control cells resulted in similar amplification of satellite 3 (Figure 5A and 5B, lower panels). We then used this as a control for normalization and found that the amounts of DXZ1 and D17Z1 amplified sequences were four and two times higher, respectively, in GFP-BLM control cells than in BS cells (Figure 5A, upper panel). DXZ1 and D17Z1 sequences bound to active Topo IIa were also twice as abundant in control cells as in PICH- depleted cells (Figure 5B, upper panel), whereas the total amount of Topo IIa was similar in all conditions (Figure 5C). We performed the same analysis with GFP-I841T cells, comparing the results obtained with those for BS and GFP-BLM cells, and obtained similar results for GFP-I841T cells and BS cells PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e33905