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ENQUIRE PROJECT DETAILS BY GENERAL PUBLIC |
| Project Details |
| Funding Scheme : | General Research Fund | ||||||||||||||||
| Project Number : | 17126714 | ||||||||||||||||
| Project Title(English) : | Elucidating the function of the Bre1 E3 ubiquitin ligase in sister chromatid cohesion | ||||||||||||||||
| Project Title(Chinese) : | Bre1 E3泛素連接酶在姐妹染色單體粘連中的功能研究 | ||||||||||||||||
| Principal Investigator(English) : | Dr Yuen, Karen Wing Yee | ||||||||||||||||
| Principal Investigator(Chinese) : | |||||||||||||||||
| Department : | School of Biological Sciences | ||||||||||||||||
| Institution : | The University of Hong Kong | ||||||||||||||||
| E-mail Address : | kwyyuen@hku.hk | ||||||||||||||||
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| Co - Investigator(s) : |
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| Panel : | Biology & Medicine | ||||||||||||||||
| Subject Area : | Biological Sciences | ||||||||||||||||
| Exercise Year : | 2014 / 15 | ||||||||||||||||
| Fund Approved : | 931,950 | ||||||||||||||||
| Project Status : | Completed | ||||||||||||||||
| Completion Date : | 30-6-2018 | ||||||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
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| Realisation of objectives: | 1. Determine the execution time of Bre1 in cohesion during the cell cycle: To determine the cell-cycle stages at which Bre1 is important for sister chromatid cohesion, we exploited the Auxin-Inducible Degron (domain for inducing degradation) (AID) system to conditionally control the expression of Bre1 at specific cell-cycle stages and examined the cohesion phenotype in G2/M-arrested cells. We constructed an AID*−9Myc tag of Bre1 at the endogenous locus and expressed F-box Transport Inhibitor Response 1 (TIR1) from rice Oryza sativa in a strain containing the GFP–LacI:LacO system for cohesion assay. Auxin binds to AID* and OsTIR1, which targets AID*−9Myc-tagged Bre1 for polyubiquitination and proteasome-mediated degradation. We degraded Bre1 at specific cell-cycle stages. Once the arrest in G1, S or G2/M was achieved by α-F, HU or Noc, respectively, with or without auxin induction, the medium was washed and released into the next cell cycle. Cohesion phenotype in G2/M-arrested cells was assessed. Degradation of Bre1 in G1 only or in G1 and S phases showed similar proportions of cohesion defects (20.7% and 17.1%, respectively), which were comparable to the proportion of cells in which Bre1 was degraded in all cell-cycle stages (19.2%), suggesting that Bre1’s role in G1 phase contributes to sister chromatid cohesion. To our surprise, degradation of Bre1 in S phase only or in S and G2/M phases resulted in fewer cells with cohesion defects (12.4% and 10.2%, respectively), but still significantly more defective cells compared to control. The occurrence rate of the cohesion defect caused by degradation of Bre1 in G1 and S phases (17.1%) was significantly higher than that in cells in which Bre1 is degraded in S only (12.4%). Degradation of Bre1 in G2/M phase only resulted in an occurrence rate for cohesion defects similar to that in the the negative control, implying that Bre1 is not required in G2/M phase for cohesion. These findings demonstrated that Bre1’s role in sister chromatid cohesion is most prominent in G1 phase, but also in S phase, but Bre1 is not required in G2/M phase. 2 Elucidate the molecular function of Bre1 in sister chromatid cohesion: Our Bre1 degron mutant experiment suggested that Bre1 is important in G1 and S phases, and may be involved in cohesin loading in G1 or sister chromatid cohesion establishment in S phase. The enrichment of Scc1 at early origins or on chromatin was not affected by BRE1. Next, we elucidated that the association of Ctf4, Ctf18 and Eco1 with chromatin and at early origins in S phase was significantly reduced in bre1Δ cells compared to WT. Our results suggest that Bre1 facilitates the localization of these cohesion establishment factors to the chromatin, and specifically to the early origins. We hypothesized that Bre1 may signal to replication factors upstream of Ctf4 and Ctf18 to affect their localizations. ChIP-qPCR and chromatin spreads showed the occupancy of Psf2, Polα and Mcm10 at early replication origins and on chromatin in bre1Δ cells in S phase was significantly reduced. Bre1 plays a role in regulating the localization of an upstream replication factor, Mcm10, which is important for replication initiation at origins, replication fork progression, and recruitment of replication-coupled cohesion establishment factors. 3. Decipher the biochemical action of Bre1 in sister chromatid cohesion: Ctf18 and Eco1 are both required for the acetylation of Smc3 during S phase to generate a stably chromosome-bound cohesin pool for enduring sister chromatid cohesion. In the absence of Bre1, Smc3 acetylation was significantly diminished, whereas the protein levels of Smc3 in WT and bre1Δ cells were similar. E3 ubiquitin ligase Bre1 is known to function with E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate histone H2B at K123 (H2Bub1) through the conserved RING domain of Bre1. We performed the cohesion assay in G2/M-arrested cells by adding nocodazole for 3 hr. The cohesion defect in rad6Δ (19.6% ), bre1-RINGΔ (18.0%), which lacks E3 activity and cannot ubiquitinate H2B, and htb1-K123R htb2Δ (17.9%) occured at frequencies comparable to that in bre1Δ in nocodazole (18.8%). These results suggest that Rad6 and Bre1-catalyzed H2Bub1 accounts for Bre1’s function in sister chromatid cohesion. By contrast, deletion of the other known substrate of Bre1, swd2Δ (5.6%), showed only a WT level of cohesion defect. | ||||||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | |||||||||||||||||
| Major findings and research outcome: | Bre1, a conserved E3 ubiquitin ligase in Saccharomyces cerevisiae, together with its interacting partner Lge1, are responsible for histone H2B monoubiquitination, which regulates transcription, DNA replication, and DNA damage response and repair, ensuring the structural integrity of the genome. Deletion of BRE1 or LGE1also results in whole chromosome instability. We discovered a novel role for Bre1, Lge1 and H2Bub1 in chromosome segregation and sister chromatid cohesion. Bre1’s function in G1 and S phases contributes to cohesion establishment, but it is not required for cohesion maintenance in G2 phase. Bre1 is dispensable for the loading of cohesin complex to chromatin in G1, but regulates the localization of replication factor Mcm10 and cohesion establishment factors Ctf4, Ctf18 and Eco1 to early replication origins in G1 and S phases, and promotes cohesin subunit Smc3 acetylation for cohesion stabilization. H2Bub1 epigenetically marks the origins, potentially signaling the coupling of DNA replication and cohesion establishment. | ||||||||||||||||
| Potential for further development of the research and the proposed course of action: |
In the future it will be worthwhile to investigate whether genetic mutations that prevent sister chromatids adhering to each other is a major cause of the chromosome abnormalities seen in cancer cells. This knowledge may be useful for diagnosing cancers. Drugs that prevent the activity of Bre1 and other proteins involved in holding together sister chromatids could also be developed as potential cancer treatments that kill cancer cells by causing instability in their number of chromosomes. | ||||||||||||||||
| Layman's Summary of Completion Report: | Most of the DNA in a cell is stored in structures called chromosomes. During every cell cycle, each cell needs to replicate its chromosomes, hold the two chromosome copies (also known as “sister chromatids”) together before cell division, and distribute them equally to the two new cells. Each step must be executed accurately otherwise the new cells will have extra or missing chromosomes – a condition that is seen in many cancer cells and that can cause embryos to die. Since these processes are so essential to life, they are highly similar in a range of species, from single-celled organisms such as yeast to multicellular organisms like humans. However, it was not clear when and how sister chromatids first join together, or how this process is linked to DNA replication. The DNA in the sister chromatids is wrapped around proteins called histones to form a structure known as chromatin. An enzyme called Bre1 plays roles in gene transcription and DNA replication and repair by adding ubiquitin molecules to a histone called H2B. By using genetic, molecular and cell biological approaches to study baker and brewer yeast cells, Zhang et al. show that the activity of Bre1 helps to hold sister chromatids together. Specifically, Bre1 recruits proteins to the chromatin before and during DNA replication, which help to initiate replication and to establish cohesion between the sister chromatids. The ubiquitin molecule attached to H2B by Bre1 is also essential for establishing cohesion, acting as a mark that helps to link the two processes. | ||||||||||||||||
| Research Output | |||||||||||||||||
| Peer-reviewed journal publication(s) arising directly from this research project : (* denotes the corresponding author) |
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| Recognized international conference(s) in which paper(s) related to this research project was/were delivered : |
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| Other impact (e.g. award of patents or prizes, collaboration with other research institutions, technology transfer, etc.): |
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| SCREEN ID: SCRRM00542 |