Orc4 spatiotemporally stabilizes centromeric chromatin
- Lakshmi Sreekumar1,8,
- Kiran Kumari2,3,4,
- Krishnendu Guin1,
- Asif Bakshi1,9,
- Neha Varshney1,10,
- Bhagya C. Thimmappa1,11,
- Leelavati Narlikar5,
- Ranjith Padinhateeri2,
- Rahul Siddharthan6 and
- Kaustuv Sanyal1,7
- 1Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India;
- 2Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India;
- 3IITB-Monash Research Academy, Mumbai 400076, India;
- 4Department of Chemical Engineering, Monash University, Melbourne 3800, Australia;
- 5Department of Chemical Engineering, CSIR-National Chemical Laboratory, Pune 411008, India;
- 6The Institute of Mathematical Sciences/HBNI, Taramani, Chennai 600113, India;
- 7Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
Abstract
The establishment of centromeric chromatin and its propagation by the centromere-specific histone CENPA is mediated by epigenetic mechanisms in most eukaryotes. DNA replication origins, origin binding proteins, and replication timing of centromere DNA are important determinants of centromere function. The epigenetically regulated regional centromeres in the budding yeast Candida albicans have unique DNA sequences that replicate earliest in every chromosome and are clustered throughout the cell cycle. In this study, the genome-wide occupancy of the replication initiation protein Orc4 reveals its abundance at all centromeres in C. albicans. Orc4 is associated with four different DNA sequence motifs, one of which coincides with tRNA genes (tDNA) that replicate early and cluster together in space. Hi-C combined with genome-wide replication timing analyses identify that early replicating Orc4-bound regions interact with themselves stronger than with late replicating Orc4-bound regions. We simulate a polymer model of chromosomes of C. albicans and propose that the early replicating and highly enriched Orc4-bound sites preferentially localize around the clustered kinetochores. We also observe that Orc4 is constitutively localized to centromeres, and both Orc4 and the helicase Mcm2 are essential for cell viability and CENPA stability in C. albicans. Finally, we show that new molecules of CENPA are recruited to centromeres during late anaphase/telophase, which coincides with the stage at which the CENPA-specific chaperone Scm3 localizes to the kinetochore. We propose that the spatiotemporal localization of Orc4 within the nucleus, in collaboration with Mcm2 and Scm3, maintains centromeric chromatin stability and CENPA recruitment in C. albicans.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.265900.120.
- Received May 12, 2020.
- Accepted January 27, 2021.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see https://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
