Project Details
Funding Scheme : General Research Fund
Project Number : 785313
Project Title(English) : Deciphering centromeric chromatin assembly pathway and dynamics in holocentric Caenorhabditis elegans 
Project Title(Chinese) : 解讀秀麗隱桿線蟲中散漫著絲粒染色質的組裝途徑及動態 
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 
Tel :  
Co - Investigator(s) :
Panel : Biology & Medicine
Subject Area : Biological Sciences
Exercise Year : 2013 / 14
Fund Approved : 866,032
Project Status : Completed
Completion Date : 31-12-2016
Project Objectives :
Determine the role of RbAp48/LIN-53 in CENP-A/HCP-3 centromere assembly hierarchy in holocentric C. elegans
Elucidate the role of RbAp48/LIN-53 in centromeric chromatin remodeling and mitotic chromosome structure
Determine the dynamics of CENP-A/HCP-3 assembly over the cell cycles and the requirements for new CENP-A/HCP-3 loading
Abstract as per original application

Realisation of objectives: Objective 1: Determine the role of RbAp46/48LIN-53 in CENP-A/HCP-3 centromere assembly hierarchy in holocentric C. elegans 1. Determine the functional relationship between RbAp46/48/LIN-53 and M18BP1/KNL-2 in CENP-A/HCP-3 localization As mentioned in the midterm report, based on RNA interference and localization dependency experiments by live imaging and immunofluorescence, we found that CENP-A/HCP-3 centromeric localization is dependent on RbAp46/48LIN-53. In turn, the nuclear and centromeric localization of RbAp46/48/LIN-53 is dependent on CENP-A/HCP-3 and M18BP1/KNL-2, whereas M18BP1/KNL-2 localization is unaffected by RbAp46/48/LIN-53. Thus, we concluded that M18BP1/KNL-2 is upstream of RbAp46/48/LIN-53, and that RbAp46/48/LIN-53 and CENP-A/HCP-3 are interdependent of each other in the centromeric chromatin assembly process. 2. Determine if RbAp46/48/LIN-53 affects the mRNA and protein expression level and stability of CENP-A/HCP-3 and other canonical histones As mentioned in the midterm report, after RbAp46/48LIN-53 depletion, CENP-A/HCP-3 and H4 protein level dropped, but CENP-A/HCP-3 and H4 mRNA level remained unchanged. Our results suggest that RbAp46/48/LIN-53 recruits and stabilizes CENP-A/HCP-3-H4 nucleosomes to holocentromeres. On the other hand, H3 and H2B protein levels were reduced to a lesser degree, suggesting the effect of RbAp46/48/LIN-53 on CENP-A/HCP-3/ and H4 nucleosomes is specific, even though RbAp46/48/LIN-53 is also a H3-H4 chaperone. 3. Determine the chromosome missegregation frequency in RbAp46/48/LIN-53 depletion When RbAp46/48LIN-53was depleted, the metaphase plate could still be observed, yet 72% of one-cell embryos contain anaphase bridges and 41% undergo chromosome missegregation. Considering other cell stages in embryos, 24% of cells are aneuploid in at least one of the chromosomes. Objective 2: Elucidate the role of RbAp46/48LIN-53 in centromeric chromatin remodeling and mitotic chromosome structure 1. Determine if RbAp46/48LIN-53 affects histone acetylation and H3K27 methylation patterns by immunofluorescence To determine whether RbAp46/48/LIN-53 RNAi affects centromeric or global histone acetylation, we performed immunofluorescence on metaphase chromosomes. In wild-type, the poleward-facing holocentromeric region was not hypoacetylated, consistent with the lack of negative correlation between CENP-A/HCP-3 and H3/H4 acetylation from chromatin immunoprecipitation followed by DNA microarray (ChIP-chip) analysis in C. elegans (Liu et al., 2011, Gassmann et al., 2012). Global H3K9 and H4 acetylation (H3K9ac and H4K5/8/12/16ac) levels after RbAp46/48/LIN-53 RNAi were comparable to wild-type by immunofluorescence and western blot. These results suggest that RbAp46/48/LIN-53 does not regulate CENP-A/HCP-3 centromeric localization via histone acetylation in C. elegans. One of the RbAp46/48/LIN-53-containing complexes, polycomb repressor complex 2 (PRC2), has a role in generating the transcriptional repression mark, trimethylated H3 at K27 (H3K27me3), for gene silencing (Bender et al., 2004). This mark positively correlated (correlation index = 0.64) with CENP-A/HCP-3 based on ChIP-chip analysis in C. elegans (Gassmann et al., 2012), but whether a causal relationship exists between H3K27me3 and CENP-AHCP-3 localization is unknown. However, when we analyzed the H3K27me3 pattern on wild-type metaphase chromosomes by immunofluorescence, we did not observe an enrichment of this modification at centromeres at this resolution. Depletion of CENP-A/HCP-3did not affect the level of chromosomal H3K27me3, but RbAp46/48/LIN-53 depletion reduced H3K27me3 level on metaphase chromosomes to 11% compared to wild-type. To test whether RbAp46/48LIN-53 depletion affects CENP-A/HCP-3 localization through modulating H3K27me3 level, we depleted histone methyltransferase EZH2/MES-2 in PRC2 complex, which reduced chromosomal H3K27me3 level to 17% of wild-type. However, EZH2/MES-2 depletion did not significantly reduce centromeric GFP::CENP-A/HCP-3 level. This suggests that RbAp46/48/LIN-53 does not recruit CENP-A/HCP-3 to the centromere through PRC2 complex. We also systematically depleted the core enzymatic component or the largest subunit of the other six RbAp46/48/LIN-53-containing complexes. None of these perturbations led to a decrease in centromeric GFP::CENP-AHCP-3 level. These results suggest that RbAp46/48LIN-53’s role in centromeric CENP-AHCP-3 deposition may be independent of its roles as part of these known chromatin-modifying complexes. 2. Determine if RbAp46/48LIN-53 affects chromosome condensation in mitosis As mentioned in the midterm report, chromosomes afterRbAp46/48/LIN-53 depletion displayed a condensation kinetics pattern that is comparable to that after mock treatment. Also, the metaphase plate width in RbAp46/48LIN-53 depletion was similar to that in mock treatment. These results suggested chromosome condensation was not impaired in RbAp46/48LIN-53 depletion. Objective 3: Determine the dynamics of CENP-A/HCP-3 assembly over the cell cycles and the requirements for new CENP-A/HCP-3 loading 1. Validate that CENP-A/HCP-3 on chromatin is newly assembled during every cell cycle in C. elegans embryos: Following up on the mid-term report, both N- or C-terminal Dendra::CENP-A/HCP-3 transgenic strains do not give green fluorescence signal before photoconversion. After photoconversion trial, there is also no red fluorescence signal. However, we confirmed that most (93.7%) of CENP-A/HCP-3 on chromatin is turned over in the next cell cycle based on GFP::CENP-A/HCP-3, consistent with our previous finding in Gassmann et al., 2012. 2. Elucidate the requirements for new CENP-AHCP-3 loading during the cell cycle in C. elegans: To test if spindle checkpoint activation affects CENP-A/HCP-3 turnover, we depleted ZYG-1, which is required for centriole duplication and results in monopolar spindle in the second mitosis. In ZYG-1 depleted embryos, as in WT, the CENP-A/HCP-3 turned over completely (98.2%) within the anaphase of the first division and the metaphase of the second mitosis (during which the spindle checkpoint is activated). However, the variable cell shapes in the third mitosis prevent us from analyzing the turnover in the next cell division. This result suggests that spindle checkpoint is not required for HCP-3 turnover, or alternatively, HCP-3 was exchanged between the time when we photobleached (anaphase) and checkpoint activation (next metaphase). We permeabilized the embryos by PERM-1 RNAi, and treated GFP::CENP-A/HCP-3 embryos with latrunculin A, nocodazole and clasto-lactacystin-beta-lactone separately. Latrunculin A inhibits actin polymerization, hence it prevents furrow ingression when added before the event. Nocodazole inhibits microtubules polymerization, hence it blocks cytokinesis when added after anaphase onset. As Latrunculin A-treated and nocodazole-treated embryos simply arrested before the next metaphase or occasionally have their two daughter nuclei fused back, it is not feasible to measure the ratio between the bleached sister versus the unbleached sister in the next cell cycle. Clasto-lactacystin-beta-lactone is a specific 26S proteasome inhibitor that can prevent metaphase to anaphase progression when added before nuclear envelope breakdown (NEBD). Clasto-lactacystin-beta-lactone treated embryos are expected to arrest in metaphases of the second mitosis. Most (71.2%) CENP-A/HCP-3 on the chromatin is turned over in the next cell cycle, which is not significantly different from the (perm-1 RNAi) control embryos (75.5%). In summary, we have found that perturbed centrosome duplication or reduced proteasome activity in mitosis do not affect HCP-3 turnover from our photobleaching experiment.
Summary of objectives addressed:
Objectives Addressed Percentage achieved
1.Determine the role of RbAp48/LIN-53 in CENP-A/HCP-3 centromere assembly hierarchy in holocentric C. elegansYes100%
2.Elucidate the role of RbAp48/LIN-53 in centromeric chromatin remodeling and mitotic chromosome structureYes100%
3.Determine the dynamics of CENP-A/HCP-3 assembly over the cell cycles and the requirements for new CENP-A/HCP-3 loadingYes95%
Research Outcome
Major findings and research outcome: The major findings in this project has been published in: Lee BCH, Lin Z and Yuen KWY*. RbAp46/48LIN-53 is Required for Holocentromere Assembly in Caenorhabditis elegans. Cell Reports. 2016. 14:1819-1828. DOI:http://dx.doi.org/10.1016/j.celrep.2016.01.065 (as in Part C 8) • 2016 Impact factor: 8.282; 5-Year Impact Factor: 8.728; • SJR 8.588; SJR ranking 101/29715= top 0.33%; 32/1848 = top 1.73% (Biochemistry, Genetics and Molecular Biology); • Number of citations: 2; • Precise role of contribution: Corresponding author; the other 2 authors are my PhD students • The paper has been awarded 2016 Second Research Output Prize, Strategic Research Theme of Development and Reproduction, University of Hong Kong, HK$20,000 (as in Part C 11) Summary: Centromeres, the specialized chromosomal regions for recruiting kinetochores and directing chromosome segregation, are epigenetically marked by a centromeric histone H3 variant, CENP-A. To maintain centromere identity through cell cycles, CENP-A diluted during DNA replication is replenished. The licensing factor M18BP1/KNL-2 is known to recruit CENP-A to holocentromeres. Here, we show that RbAp46/48/LIN-53, a conserved histone chaperone, is required for CENP-A/HCP-3 localization in holocentric Caenorhabditis elegans. Indeed, RbAp46/48/LIN-53 and CENP-A/HCP-3 localizations are interdependent. RbAp46/48/LIN-53 localizes to the centromere during metaphase in a CENP-A/HCP-3- and M18BP1/KNL-2-dependent manner, suggesting CENP-A/HCP-3 loading may occur before anaphase. RbAp46/48/LIN-53 does not function at the centromere through histone acetylation, H3K27 trimethylation, or its known chromatin-modifying complexes. RbAp46/48/LIN-53 may function independently to escort CENP-A/HCP-3 for holocentromere assembly but is dispensable for other kinetochore protein recruitment. Nonetheless, depletion of RbAp46/48/LIN-53 leads to anaphase bridges and chromosome missegregation. This study unravels the holocentromere assembly hierarchy and its conservation with monocentromeres.
Potential for further development of the research
and the proposed course of action:
Based on the results from this 2012 GRF, I have successfully applied for a continued GRF project in 2017 (17126717), HK$1,221,692, Duration: 3 years, Score 5/5. “Holocentric but not everywhere on the chromosome: How and where does histone chaperone RbAp46/48/LIN-53 assemble Centromeric Protein A (CENP-A/HCP-3) in Caenorhabditis elegans?”.
Layman's Summary of
Completion Report:
In Brief: Histone H3 variant CENP-A epigenetically marks all functional centromeres, but how it is assembled on holocentromeres is unknown. We show that the histone chaperone RbAp46/48/LIN-53 is required for CENP-A/HCP-3 localization and accurate chromosome segregation in holocentric C. elegans. RbAp46/48/LIN-53 may function to escort CENP-A/HCP-3 for holocentromere assembly. Highlights: • RbAp46/48/LIN-53 is crucial for chromosome segregation in holocentric C. elegans • RbAp46/48/LIN-53 localizes to centromeres in metaphase and disappears in anaphase • RbAp46/48LIN-53 localizes and stabilizes CENP-AHCP-3 for holocentromere assembly • RbAp46/48LIN-53’s centromeric role is not due to histone acetylation or methylation
Research Output
Peer-reviewed journal publication(s)
arising directly from this research project :
(* denotes the corresponding author)
Year of
Author(s) Title and Journal/Book
2016 Lee BCH, Lin Z and Yuen KWY*  RbAp46/48LIN-53 is Required for Holocentromere Assembly in Caenorhabditis elegans. Cell Reports. 2016. 14:1819-1828. DOI: http://dx.doi.org/10.1016/j.celrep.2016.01.065 
Recognized international conference(s)
in which paper(s) related to this research
project was/were delivered :
Month/Year/City Title Conference Name
Waltham Deciphering the Role of RbAp46/48/LIN-53 in Centromere Function in Caenorhabditis elegans  Gordon Research Conference on Centromere Biology 
Other impact
(e.g. award of patents or prizes,
collaboration with other research institutions,
technology transfer, etc.):