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Project Details
Funding Scheme : General Research Fund
Project Number : 17126717
Project Title(English) : 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? 
Project Title(Chinese) : 染色體上的全著絲粒結構並非無處不在——在秀麗隱桿線蟲中組蛋白伴侶RbAp46/48/LIN-53如何及於何處組裝著絲粒蛋白A (CENP-A/HCP-3)? 
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) :
Prof Li, Xiang David
Prof Zhao, Zhongying
Panel : Biology & Medicine
Subject Area : Biological Sciences
Exercise Year : 2017 / 18
Fund Approved : 1,221,692
Project Status : Completed
Completion Date : 31-12-2020
Project Objectives :
Determine how histone chaperone LIN-53 assembles HCP-3 to holocentromeric chromatin in vivo
Elucidate the physical interaction between LIN-53 and HCP-3 in vitro
Decipher the effect of LIN-53 and transcription on the genome-wide localization of holocentromeres by DNA Adenine Methyltransferase Identification (DamID)
Abstract as per original application
(English/Chinese):

Realisation of objectives: Objective 1: Determine how histone chaperone LIN-53 assembles HCP-3 to holocentromeric chromatin in vivo CENP-AHCP-3 could not be detected in the soluble nucleoplasm fractions. LIN-53 can be co-purified with chromatin-bound CENP-AHCP-3 for immunoprecipitation, and such interaction was confirmed reciprocally. LIN-53 can also be co-purified with KNL-2. In consistent with previous studies, the above results show that LIN-53 may concomitantly target CENP-AHCP-3 to the centromere and interact with KNL-2 at the licensed centromeric chromatin. In C. elegans, injecting foreign DNA, even devoid of C. elegans sequences, into its gonad could form episomal extra-chromosomal arrays, also known as artificial chromosomes (ACs), in the embryonic cells. We used the ACs system to study how RbAp46/48LIN-53 assembles CENP-AHCP-3 to the AC chromatin in vivo. We found that RbAp46/48LIN-53 deposits CENP-AHCP-3 on endogenous chromosomes, but HAT-1 depletion has no effect on CENP-AHCP-3 on endogenous chromosomes. Mis18BP1KNL-2 and CENP-AHCP-3 are interdependent for each other’s localization on endogenous chromosomes of C. elegans. On endogenous chromosomes, RbAp46/48lin-53 depletion only reduced CENP-AHCP-3 level but did not affect Mis18BP1KNL-2 level. We monitored the M18BP1KNL-2 signal on the chromatin of nascent ACs in lin-53 RNAi-treated embryos. Surprisingly, at de novo centromeres on nascent ACs, RbAp46/48LIN-53 depletion also leads to the loss of initial Mis18BP1KNL-2 deposition, which suggests that while Mis18BP1KNL-2 could be a self-directing factor for centromere maintenance in the existing centromeres, it is downstream of RbAp46/48LIN-53 in de novo centromere establishment. Objective 2: Elucidate the physical interaction between LIN-53 and HCP-3 in vitro Proteins containing 6xHis fusion tags were captured and immobilized to Ni-NTA agarose beads. Upon incubation with histone H3 and H4, it was found that 6xHis-SUMO tagged LIN-53 could successfully pull-down H3 and H4 individually. Besides, LIN-53 was also pulled-down by purified 6xHis-tagged CENP-A protein immobilized on beads. Together, these findings have demonstrated that LIN-53 can physically interact with histone H3, H4 and CENP-AHCP-3 individually. Interestingly, the binding of CENP-AHCP-3 can be out competed by titrating in H3. Purified C. elegans histone H3 and H4 were mixed in equimolar to reconstitute (H3-H4)2 tetramers, using adapted protocol from the reconstitution of Xenopus laevis histones subunits (Dyer et al., 2003; Luger et al., 1999). To assess the oligomeric state and purity of the reconstituted (H3-H4)2 tetramers, the sample was purified over a size-exclusion chromatography column. The chromatography trace showed that C. elegans histone H3 and H4 can be successfully reconstituted to tetrameric (H3-H4)2 form with high homogeneity. It is found that (H3-H4)2 tetramers, like histone H3 and H4, can be pulled-down by 6xHis-SUMO tagged LIN-53. It is found that (H3-H4)2 tetramers could be pulled-down by 6xHis-SUMO tagged LIN-53 under [NaCl] concentrations ranging from 150 mM to 1000 mM. However, (H3-H4)2 tetramers showed salt-dependent decrease in binding to 6xHis-SUMO tagged LIN-53. The quantification result showed that over 60% of (H3-H4)2 tetramers would be retained to bind LIN-53 under high ionic concentration (1000 mM NaCl) when compared to binding under physiological salt concentration at 150 mM NaCl. Objective 3: Decipher the effect of LIN-53 and transcription on the genome-wide localization of holocentromeres by DNA Adenine Methyltransferase Identification (DamID) i. Creating C. elegans DamID strains and validation of DamID fusion proteins by immunofluorescence (IF) After the construction of Dam::CENP-A transgenic strain and verification of expression by immunofluorescence and Western as stated in the mid-term report, we continue in the DamID sample collection and analysis. ii. Low input DamID Methodology 10 worms or a single worm was put directly into a PCR tube containing 3 μl lysis buffer. Subsequent sample processing involves a few steps: DpnI digestion, which is highly specific for Dam-methylated GmATC motifs, followed by a specific double strand adaptor ligation and then a PCR amplification. The gel electrophoresis was performed after PCR amplification. Result showed that all the samples yielded a clear smear between 500 bp and 2 kb. The PCR products were then used to prepare sequencing library for Illumina sequencing. iii. Analysis pipeline for DamID-Seq and analysis of Dam::CENP-A Data In total, 11 DamID-seq samples were generated for sequencing, two 10-worms Dam::CENP-A samples, six single-worm Dam::CENP-A samples, one GFP::Dam 10-worms sample, one GFP::Dam single-worm sample, and one 10-worms Dam::lamin sample. First, sequencing raw data in fastq format from each sample went through a series of quality control to find and keep the “DamID reads”. Each read (250 bp) is scanned for the presence of the different parts specialized for DamID-seq: I. the first 4 random bases (“NNNN”); followed by II. 15 bp of adapter sequence (“GTGGTCGCGGCCGAG”); followed by the III. DpnI motif (“GATC”). “DamID reads” are cut after the adapter sequence “GTGGTCGCGGCCGAG” to remove the DamID adapters. Then the remaining sequences are mapped to the C. elegans reference genome (wbcel235) using BWA. iv. CENP-A proteins are highly enriched at discrete, dispersed loci on C. elegans genome Finding out the chromatin domains to which CENP-A associates is a central part of DamID-seq data analysis, although by mapping to the reference genome I have obtained the information of CENP-A binding sites. For this, I needed to assign regions with significant numbers of mapped reads (Peak Calling). As peak calling software packages have typically been developed for ChIP-seq, we must choose a peak calling tool considering the balance between specificity and sensitivity, and adapt it to the CENP-A DamID-seq. To obtain CENP-A enriched domains, we do the peak calling by using MACS2 (threshold q value < 0.05) under narrow peak setting. The number of peaks with different peak width in each sample is then calculated. From the summary of peak calling we can find that 10-worms samples have around 900 CENP-A peaks, which were significantly more compared to the single-worm samples, which have around 600 peaks. We checked the distribution of peak width on both 10-worms and single-worm samples. We find that under narrow peak setting, they have similar pattern, in which most of the CENP-A peaks are around 400 to 1500 bp in length of DNA. We then located each of the CENP-A peaks onto the C. elegans genome to obtain the genome-wide distribution of peaks for each sample. The number of the peaks mapping to the chromosome and its distribution can reflect the distribution of the CENP-A binding sites. CENP-A peaks are found distributed throughout the C. elegans genome as discrete, dispersed loci. CENP-A peaks are enriched towards the arm regions relative to the centers of the autosomes, however, the distribution of CENP-A peaks on the X chromosome is relatively even. This peaks distribution pattern is very similar to the previously published C. elegans CENP-A N-ChIP-seq data (Steiner, 2019, eLife), which identified a total of 707 well-defined CENP-A high occupancy loci discretely located on the whole genome, despite using a different methodology. Furthermore, we compared genome-wide distribution of CENP-A peaks among the 10-worms samples and single-worm samples. In general, the pattern of CENP-A peaks on each chromosome is similar between two 10-worms samples, with some variations on specific positions. On each chromosome, single-worm samples have less CENP-A peaks than 10-worms samples. In addition, each single-worm samples has different CENP-A peak distribution pattern on each of the chromosomes, although most of the CENP-A peaks on the different single-worm samples can be found on the 10-worms samples. This finding suggests that the CENP-A distribution may have worm-to-worm variation.
Summary of objectives addressed:
Objectives Addressed Percentage achieved
1.Determine how histone chaperone LIN-53 assembles HCP-3 to holocentromeric chromatin in vivoYes90%
2.2. Elucidate the physical interaction between LIN-53 and HCP-3 in vitroYes85%
3.Decipher the effect of LIN-53 and transcription on the genome-wide localization of holocentromeres by DNA Adenine Methyltransferase Identification (DamID)Yes98%
Research Outcome
Major findings and research outcome: This project aims to understand how histone chaperone RbAp46/48/LIN-53 assemble CENP-A/HCP-3 to the permitted holocentric locations. Through in vivo reciprocal co-immunoprecipitation assays, we found that RbAp46/48/LIN-53 interacts with CENP-A/HCP-3 and the licensing factor KNL-2. By in vitro pull down assays, we found that RbAp46/48/LIN-53 can bind to CENP-A/HCP-3, H3, H4, (H3-H4)2 tetramers. Interestingly, the interaction with CENP-A/HCP-3 can be out competed by titrating in H3. By using artificial chromosome (AC) system in C. elegans, we found that RbAp46/48/LIN-53, together with HAT-1, a histone acetyltransferease, facilitates the acetylation of H3 and H4 acetylation on the AC, initial deposition of CENP-A/HCP-3 to the newly formed artificial chromosomes. Interestingly, while RbAp46/48/LIN-53 does not affect KNL-2 centromeric localization on endogenous chromosomes, it does affect the initial deposition of KNL-2 onto the newly formed artificial chromosomes. This suggests that while Mis18BP1KNL-2 could be a self-directing factor for centromere maintenance in the existing centromeres, it is downstream of RbAp46/48LIN-53 in de novo centromere establishment. We have published this work in Nuclei Acids Research 49:9154–9173. On the genome map, we have applied a low-input transgenic method called DNA Adenine Methyltransferase Identification (DamID) to analyze protein-DNA interaction. We have constructed Dam::CENP-A/HCP-3, in which Dam will methylate the G in GTAC in the vicinity of CENP-A/HCP-3. We have analyzed the CENP-A/HCP-3-enriched regions and compared among 10-worm samples and 1-worm data. Interestingly, there are worm-to-worm variations in the CENP-A/HCP-3-enriched regions. We also show exclusion of germline gene body in most CENP-A/HCP-3-enriched regions. We are preparing the manuscript for publication.
Potential for further development of the research
and the proposed course of action:
For the biochemical analyses, we will further identify the binding domain between HCP-3 and LIN-53, by expressing protein truncates of HCP-3 and LIN-53 from bacteria, for testing the required protein domain(s) involved in their interactions in pull-down assays. For the DamID analyses, we will further analyze and compare the DamID of LIN-53 and KNL-2 with that of HCP-3. This will allow us to understand the binding landscape of the upstream factors that affect HCP-3.
Layman's Summary of
Completion Report:
This project set out to understand how histone chaperone RbAp46/48/LIN-53 assembles CENP-A/HCP-3 onto specific permitted holocentromere locations on the genome. Through in vivo and in vitro biochemical analyses, we gain insight into the interactions of RbAp46/48/LIN-53 with CENP-A/HCP-3 and the centromere licensing factor KNL-2. On the other hand, RbAp46/48/LIN-53 also acts as H3-H4 chaperone. The binding of RbAp46/48/LIN-53 with KNL-2 on centromeric chromatin, and the interaction of CENP-A with KNL-2 may have brought CENP-A/HCP-3 specifically to existing holocentromere locations. Consistently, for new centromeres on artificial chromosomes, KNL-2 also depends on RbAp46/48/LIN-53 and the initial deposition of CENP-A/HCP-3. For the where question, we have applied a low-input transgenic method called DNA Adenine Methyltransferase Identification (DamID) to analyze protein-DNA interaction, specifically the localization of CENP-A/HCP-3 on the genome. Using as little as a single worm in a sample, we were able to identify CENP-A/HCP-3-enriched regions and show worm-to-worm variations in the pattern. The findings of this project has shed light on the assembly dependency of CENP-A in endogenous chromosomes and in new artificial chromosomes. In addition, the low input localization analysis shows where CENP-A/HCP-3 localizes in a single worm, and how the localization can overlap but also vary from worm-to-worm.
Research Output
Peer-reviewed journal publication(s)
arising directly from this research project :
(* denotes the corresponding author)
Year of
Publication
Author(s) Title and Journal/Book Accessible from Institution Repository
2021 Zhongyang Lin and Karen Wing Yee Yuen*  RbAp46/48(LIN-53) and HAT-1 are required for initial CENP-A(HCP-3) deposition and de novo holocentromere formation on artificial chromosomes in Caenorhabditis elegans embryos  No 
Jing Zhu and Karen Wing Yee Yuen*  Centromeric protein A (CENP-A) localization in holocentric Caenorhabditis elegans exhibits worm-to-worm variation using low-input analysis  No 
Recognized international conference(s)
in which paper(s) related to this research
project was/were delivered :
Month/Year/City Title Conference Name
Suzhou Epigenetic regulation of centromere establishment in Caenorhabditis elegans and holocentric but not everywhere—Determine the localization of centromeric protein A on the genome by single cell DamID  Cold Spring Harbor Asia Conference: Systems Biology of Gene Regulation & Genome Editing 
Singapore Elucidating the localization of centromeric protein A (CENP-A) in single cells in holocentric C. elegans  Cell Symposia | Single Cells: Technology to Biology 
Other impact
(e.g. award of patents or prizes,
collaboration with other research institutions,
technology transfer, etc.):

  SCREEN ID: SCRRM00542