MLL5 maintains genomic integrity by regulating the stability of the chromosomal passenger complex through a functional interaction with Borealin

Abstract

Mixed lineage leukemia 5 (MLL5) is a versatile nuclear protein associated with many cellular events. We have shown previously that phosphorylation of MLL5 by Cdk1 is required for mitotic entry. In this paper, the function of MLL5 in mitotic regulation is further explored. SiRNA-mediated downregulation of MLL5 caused improper chromosome alignment at metaphase and resulted in failure of DNA segregation and cytokinesis. Mechanistic studies revealed that the chromosomal passenger complex (CPC), which plays a key role in chromosomal bi-orientation, was delocalized from the inner centromere region because of proteasome-mediated degradation in MLL5-depleted cells. Biochemical analyses further demonstrated that the central domain of MLL5 interacted with the C-terminus of Borealin, and the interaction is essential to maintain the stability of Borealin. Moreover, the mitotic defects in MLL5-depleted cells were rescued by overexpression of FLAG-MLL5, but not by a FLAG-MLL5 mutant that did not contain the central domain. Collectively, our results suggest that MLL5 functionally interacts with Borealin, facilitates the expression of CPC, and hence contributes to mitotic fidelity and genomic integrity.

Fig. 1.

Depletion of MLL5 by RNA interference causes genomic instability. (A) The average percentage of multinuclear cells in Negative Control-siRNA (NC-siRNA) and MLL5-siRNA-treated U2OS cells (P = 0.001, paired Student's t-test). Tubulin was stained green and nuclei were stained red. Arrowheads denotes multinuclear cells. Scale bars: 20 µm. (B) The cells were transfected with NC-siRNA, MLL5-siRNA or MLL5-siRNA-N every other day and harvested at day 7 for FISH. The percentage of cells exhibiting abnormal numbers of chromosomes 8 and 10 was scored (n>100 cells per sample). Scale bars: 5 µm. (C) The mitotic progression of U2OS cells expressing H2B-mCherry was monitored by live-cell imaging. Scale bars: 10 µm. (D) Cell cycle analyses showed that MLL5-depleted cells remained at 4N DNA content after releasing from nocodazole, and polyploid populations gradually increased. Noc, nocodazole.

Fig. 2.

MLL5-depleted cells fail to align chromosomes on metaplate because of impaired function of the kinetochore-MT attachment error-correcting machinery. (A) Metaphase-arrested cells were categorized into three groups: bipolar normal alignment, bipolar misalignment and multipolar misalignment. Experiments were repeated three times and the average percentage is presented. Scale bars: 5 µm. (B) The kinetochore-MT capture and attachment process were examined in a cold-induced MT depolymerization assay. Kinetochore fibers were stained with α-tubulin antibody and kinetochores were labeled with CREST. Scale bars: 5 µm. (C) The average inter-kinetochore distances (for sister kinetochores) were measured, as shown, in control cells (n = 70) and MLL5-depleted cells (n = 84). Scale bars: 5 µm. (D) A time-course study of the chromosome alignment process was conducted in NC- and MLL5-depleted cells after monastrol release. Cells were categorized into four groups: monopolar, bipolar misalignment, multipolar misalignment and bipolar normal alignment, and the percentages for each group at each time point after monastrol release are presented (n = 100 cells per sample). Scale bars: 5 µm.

Fig. 3.

CPC is downregulated in MLL5-depleted cells as a result of proteasome-mediated degradation. (A) CPC was delocalized in MLL5-depleted cells. Scale bars: 10 µm. (B) NC-siRNA- and MLL5-siRNA-siRNA-transfected cells were synchronized to S, G2 or M phase. The expression of Aurora-B, Survivin, Borealin and INCENP was analyzed by western blotting. Tubulin served as a loading control. (C) Blocking of the proteasome pathway by MG132 was able to rescue the expression of all CPC components.

Fig. 4.

MLL5 interacts with Borealin and maintains the stability of Borealin. (A) U2OS cells were transfected with CPC-siRNA or MLL5-siRNA and synchronized to M phase with nocodazole. The protein expression levels of Aurora-B, Survivin, Borealin, INCENP and MLL5 were analyzed by western blotting. Tubulin served as a loading control. (B) The stability of Borealin in NC-siRNA- and MLL5-siRNA-treated cells was compared by western blotting. CHX, cycloheximide. (C) A co-immunoprecipitation study revealed an association between MLL5 and Borealin. (D) A direct interaction between the central domain of MLL5 and the C-terminus of Borealin was established in in vitro binding assays. PS: N-terminal region containing PHD (plant homeodomain) and SET[Su(var)3-9, enhancer-of-zeste and trithorax] domain, aa 1-572; CD: central domain, aa 562-1150; CT: C-terminal domain, aa 1113-1858. (E) A GST pull-down assay demonstrated that the C-terminus of Borealin was required for the interaction with MLL5. The asterisk denotes the degradation of GST-Borealin and its mutants during induction. GST-Borealin-N: aa 1-120; GST-Borealin-C: aa 121-280. (F) The stabilities of HA-Borealin, HA–Borealin-C and HA–Borealin-N were compared in NC-siRNA- and MLL5-siRNA-treated cells. (G) Overexpression of FLAG–MLL5-CD significantly stabilized HA–Borealin-C in MLL5-depleted cells, but failed to rescue the degradation of HA–Borealin-N.

Fig. 5.

The expression of CPC and chromosome alignment in MLL5-siRNA-treated cells can be rescued by overexpression of FLAG-MLL5. (A) The lack of expression of CPC at G2 caused by MLL5 knockdown was rescued by overexpression of FLAG-MLL5 or HA-Borealin but not FLAG-MLL5ΔCD. The efficiency of the rescue was assessed and the percentages of cells expressing CPC are presented in the bar chart (n = 60 cells per sample). (B) The CPC localization and chromosome alignment defects observed in MLL5-siRNA-treated cells were rescued by FLAG-MLL5 and HA-Borealin overexpression. The rescue experiments were repeated three times, and the average percentages of cells (normal chromosome alignment, bipolar misalignment and multipolar misalignment) were determined (n = 100 cells per sample). Scale bars: 10 µm.