Periodic Report Summary - BROCHROMITO (Mitosis with broken chromosomes)
My group is interested in deciphering the mechanisms that safeguard cells against aneuploidy. These mechanisms are of great interest as aneuploidy contributes to tumorigenesis. To gain insight into these mechanisms, we studie the behaviour of cells entering mitosis with damaged chromosomes. We use the endonuclease I-CreI to generate acentric chromosomes in Drosophila larval cells. While I-CreI expression produces acentric chromosomes in the majority of neuronal stem cells, remarkably, it has no effect on adult survival. Live studies reveal that acentric chromatids segregate efficiently to opposite poles. The acentric chromatid poleward movement is mediated through DNA tethers that connect the acentric fragment to its centric partner. These tethers are decorated with Polo kinase, a key mitotic regulator, the spindle checkpoint component BubR1 and two chromosomal passenger complex proteins, INCENP and Aurora-B but not the spindle checkpoint component Mad2 and the centromere histone variant Cid/CenpA. Reduced BubR1 or Polo function results in abnormal segregation of acentric chromatids, a decrease in acentric chromosome tethering and a great reduction in adult survival. Thus we propose that BubR1 and Polo facilitate the accurate segregation of acentric chromatids by maintaining the integrity of tethers that connect acentric chromosomes to their centric partners.
Our research project aims at deciphering the molecular mechanism by which the acentric chromosomes segregate accurately. We addressed the following questions:
1) Which double-strand break (DSB) repair machinery is involved in tether formation and proper acentric segregation?
2) Can we identify new components required for faithful acentric chromosome segregation?
3) How is BubR1 recruited to the tether?
4) What are the molecular pathways by which BubR1 acts on the tether?
1. Which DNA repair pathway is involved in tether formation?
We hypothesise that the presence of multiple DSB induced by I-CreI necessitates the long-term recruitment of the repair machinery. The cell may enter mitosis with repair intermediates that create the tether or promote its stability. The two major pathways that are known to repair DSBs are homologous recombination (HR) and non-homologous end-joining (NHEJ). The DNA Ligase IV (Lig IV) and Mus309 (RecQ Helicase homolog) are key components of the NHEJ and HR pathway respectively. We hypothesised that if either the NHEJ or the HR DNA repair mechanism is involved in maintaining the integrity of the tether, a lig IV or a mus309 mutant would be sensitive to I-CreI expression. Thus we tested the survival rate of the lig IV and mus309 null mutants upon I-CreI expression. We found that, unlike lig IV, mus309 mutant was highly sensitive to I-CreI expression. These results suggest that the RH but not the NHEJ DNA repair mechanism is involved in I-CreI-induced DSB repair and the formation of the tether.
2. Identifying new components involved in faithful acentric chromosome segregation
To identify new components involve in faithful acentric segregation, my group has started to study proteins that are known to play a role during mitosis. This study has led to the discovery of a completely new and unsuspected mechanism by which cells coordinate chromosome segregation with cell division, and, thus, ensure proper transmission of the genetic material into daughter cells. We have previously shown that I-CreI expression results in the production of acentric and centric chromosome fragments that remain attached by a DNA tether. In addition, we observed that this DNA tether transiently increases the length of the chromatid arms during anaphase. As a result, these abnormally long chromatids lag at the cleavage site during cytokinesis, increasing the risk of being trapped by the contractile ring. This would lead to the production of aneuploid cells. We made the striking observation that the cell adapts its length to the length of the trailing chromatid arms. This cell elongation is concomitant with the spreading of cortical myosin rings without compromising cytokinesis. The Rho Guanine-nucleotide exchange factor mediates this response. These studies reveal a novel chromatin to cortical myosin signalling pathway that ensures clearance of the chromatid arms from the cleavage plane prior to the completion of cytokinesis. This work has been published in The Journal of Cell Biology (2012, Vol. 199, No. 5).
3. How is BubR1 recruited to the tether?
To identify the sequence responsible for BubR1 localisation on the tether, we have cloned several truncated versions of the protein tagged with GFP. We have identified four domains in Drosophila BubR1. The first domain called KEN, contains the well conserved KEN box imporant for BubR1 interaction with Cdc20, a subunit of the E3 ubiquitin ligase APC/C, and BubR1 function as the spindle checkpoint. The second domain is called the Bub3-binding domain (Bub3-BD also called the GLebs motif) and was shown to be important for BubR1 interaction with Bub3 and its localisation to the kinetochore. The thrid domain, called domain 3, has an unknown function and the fourth domain is required for its kinase activity. We produced transgenic flies expressing truncated BubR1 versions lacking one or more of these four domains. We found that Bub3-BD is required and sufficient for BubR1 localisation on the tether as well as the kinetochore. More importantly, we found that the glutamate 481 in the Bub3-BD domain is essential for BubR1 localisation on both the kinetochore and the tether. Since E481 is essential for BubR1 interaction with Bub3 and its localisation on the kinetochore, we investigated whether Bub3 localises to the tether. We found that Bub3, like BubR1, accumulates on the tether throughout mitosis. Moreover, BubR1 was not required for Bub3 localisation to the tether suggesting that Bub3 acts upstream of BubR1 on the tether. Studies in mammals have shown that the interaction of BubR1 and Bub3 with KNL1, another kinetochore protein, promotes their recruitment to the kinetochore. Therefore, we analysed the localisation of Spc105, the Drosophila homolog of KNL1, to the tether. While Spc105 strongly accumulates on the kinetochore throughout mitosis, no signal was detected on the tether. All together these results suggest that the molecular pathway regulating BubR1 and Bub3 recruitment to the tether is not identical to that of BubR1 and Bub3 localisation to the kinetochore.
4. What are the molecular pathways by which BubR1 acts on the tether?
We have previously found that the BubR1 KEN box is important for BubR1 function on the tether. Since the KEN box is important for BubR1 interaction with Cdc20, we speculate that some APC/C substrates are associated with the tether and are protected from degradation by BubR1. BubR1 remains strongly associated with the tether well into anaphase at the time the APC/C activity is high. It may be that BubR1 efficiently inhibits the APC/C activity locally on the tether by sequestering its regulatory subunit Cdc20, hence preserving tether integrity throughout mitosis. To test this hypothesis we analyzed the localisation of Cdc20 during mitosis after I-CreI expression. As previously published, we observed that Cdc20 is sequestered at the kinetochore during prometaphase and is released from the kinetochore at the transition from metaphase to anaphase, resulting in APC/C activation. However, we found that Cdc20 remains strongly localised on the tether that connects the two broken chromosome fragments throughout mitosis. This result suggests that Cdc20 is sequestered on the tether probably via its interaction with BubR1.
In conclusion, our work has revealed two novel pathways that ensure proper segregation of chromosomes. The first pathway involves the BubR1 and Bub3-dependent formation of DNA tether connecting two chromosome fragments. The second pathway involves the Rho-GEF-dependent cell elongation during segregation of abnormally long chromosomes.
Our research project aims at deciphering the molecular mechanism by which the acentric chromosomes segregate accurately. We addressed the following questions:
1) Which double-strand break (DSB) repair machinery is involved in tether formation and proper acentric segregation?
2) Can we identify new components required for faithful acentric chromosome segregation?
3) How is BubR1 recruited to the tether?
4) What are the molecular pathways by which BubR1 acts on the tether?
1. Which DNA repair pathway is involved in tether formation?
We hypothesise that the presence of multiple DSB induced by I-CreI necessitates the long-term recruitment of the repair machinery. The cell may enter mitosis with repair intermediates that create the tether or promote its stability. The two major pathways that are known to repair DSBs are homologous recombination (HR) and non-homologous end-joining (NHEJ). The DNA Ligase IV (Lig IV) and Mus309 (RecQ Helicase homolog) are key components of the NHEJ and HR pathway respectively. We hypothesised that if either the NHEJ or the HR DNA repair mechanism is involved in maintaining the integrity of the tether, a lig IV or a mus309 mutant would be sensitive to I-CreI expression. Thus we tested the survival rate of the lig IV and mus309 null mutants upon I-CreI expression. We found that, unlike lig IV, mus309 mutant was highly sensitive to I-CreI expression. These results suggest that the RH but not the NHEJ DNA repair mechanism is involved in I-CreI-induced DSB repair and the formation of the tether.
2. Identifying new components involved in faithful acentric chromosome segregation
To identify new components involve in faithful acentric segregation, my group has started to study proteins that are known to play a role during mitosis. This study has led to the discovery of a completely new and unsuspected mechanism by which cells coordinate chromosome segregation with cell division, and, thus, ensure proper transmission of the genetic material into daughter cells. We have previously shown that I-CreI expression results in the production of acentric and centric chromosome fragments that remain attached by a DNA tether. In addition, we observed that this DNA tether transiently increases the length of the chromatid arms during anaphase. As a result, these abnormally long chromatids lag at the cleavage site during cytokinesis, increasing the risk of being trapped by the contractile ring. This would lead to the production of aneuploid cells. We made the striking observation that the cell adapts its length to the length of the trailing chromatid arms. This cell elongation is concomitant with the spreading of cortical myosin rings without compromising cytokinesis. The Rho Guanine-nucleotide exchange factor mediates this response. These studies reveal a novel chromatin to cortical myosin signalling pathway that ensures clearance of the chromatid arms from the cleavage plane prior to the completion of cytokinesis. This work has been published in The Journal of Cell Biology (2012, Vol. 199, No. 5).
3. How is BubR1 recruited to the tether?
To identify the sequence responsible for BubR1 localisation on the tether, we have cloned several truncated versions of the protein tagged with GFP. We have identified four domains in Drosophila BubR1. The first domain called KEN, contains the well conserved KEN box imporant for BubR1 interaction with Cdc20, a subunit of the E3 ubiquitin ligase APC/C, and BubR1 function as the spindle checkpoint. The second domain is called the Bub3-binding domain (Bub3-BD also called the GLebs motif) and was shown to be important for BubR1 interaction with Bub3 and its localisation to the kinetochore. The thrid domain, called domain 3, has an unknown function and the fourth domain is required for its kinase activity. We produced transgenic flies expressing truncated BubR1 versions lacking one or more of these four domains. We found that Bub3-BD is required and sufficient for BubR1 localisation on the tether as well as the kinetochore. More importantly, we found that the glutamate 481 in the Bub3-BD domain is essential for BubR1 localisation on both the kinetochore and the tether. Since E481 is essential for BubR1 interaction with Bub3 and its localisation on the kinetochore, we investigated whether Bub3 localises to the tether. We found that Bub3, like BubR1, accumulates on the tether throughout mitosis. Moreover, BubR1 was not required for Bub3 localisation to the tether suggesting that Bub3 acts upstream of BubR1 on the tether. Studies in mammals have shown that the interaction of BubR1 and Bub3 with KNL1, another kinetochore protein, promotes their recruitment to the kinetochore. Therefore, we analysed the localisation of Spc105, the Drosophila homolog of KNL1, to the tether. While Spc105 strongly accumulates on the kinetochore throughout mitosis, no signal was detected on the tether. All together these results suggest that the molecular pathway regulating BubR1 and Bub3 recruitment to the tether is not identical to that of BubR1 and Bub3 localisation to the kinetochore.
4. What are the molecular pathways by which BubR1 acts on the tether?
We have previously found that the BubR1 KEN box is important for BubR1 function on the tether. Since the KEN box is important for BubR1 interaction with Cdc20, we speculate that some APC/C substrates are associated with the tether and are protected from degradation by BubR1. BubR1 remains strongly associated with the tether well into anaphase at the time the APC/C activity is high. It may be that BubR1 efficiently inhibits the APC/C activity locally on the tether by sequestering its regulatory subunit Cdc20, hence preserving tether integrity throughout mitosis. To test this hypothesis we analyzed the localisation of Cdc20 during mitosis after I-CreI expression. As previously published, we observed that Cdc20 is sequestered at the kinetochore during prometaphase and is released from the kinetochore at the transition from metaphase to anaphase, resulting in APC/C activation. However, we found that Cdc20 remains strongly localised on the tether that connects the two broken chromosome fragments throughout mitosis. This result suggests that Cdc20 is sequestered on the tether probably via its interaction with BubR1.
In conclusion, our work has revealed two novel pathways that ensure proper segregation of chromosomes. The first pathway involves the BubR1 and Bub3-dependent formation of DNA tether connecting two chromosome fragments. The second pathway involves the Rho-GEF-dependent cell elongation during segregation of abnormally long chromosomes.