bioRxiv preprint doi: https://doi.org/10.1101/465880; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Orc4 spatiotemporally stabilizes centromeric chromatin 2 3 4 Lakshmi Sreekumar1, Kiran Kumari2,3,4, Asif Bakshi1, Neha Varshney1, Bhagya C. Thimmappa1, 5 Krishnendu Guin1, Leelavati Narlikar5, Ranjith Padinhateeri2, Rahul Siddharthan6, Kaustuv Sanyal1 6 7 1Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, 8 Bangalore, India; 2Department of Biosciences and Bioengineering, Indian Institute of Technology, 9 Bombay, Mumbai, India; 3IITB-Monash Research Academy, Mumbai, India; 4Department of 10 Chemical Engineering, Monash University, Melbourne, Australia 5Department of Chemical 11 Engineering, CSIR-National Chemical Laboratory, Pune, India; 6The Institute of Mathematical 12 Sciences/HBNI, Taramani, Chennai, India 13 14 15 *corresponding author 16 Kaustuv Sanyal 17 Molecular Biology & Genetics Unit 18 Jawaharlal Nehru Centre for Advanced Scientific Research 19 Jakkur, Bangalore - 560064 20 India 21 Email: [email protected] 22 Telephone : +91-80-2208 2878 23 Fax : +91-80-2208 2766 24 Homepage: http://www.jncasr.ac.in/sanyal 25 26 27 Present address: Asif Bakshi, Laboratory of Drosophila Neural Development, Centre for DNA 28 Fingerprinting and Diagnostics, Inner Ring Road, Uppal, Hyderabad 500039, India 29 Bhagya C. Thimmappa, Department of Biochemistry, Robert-Cedergren Centre for Bioinformatics 30 and Genomics, University of Montreal, 2900 Edouard-Montpetit, Montreal, H3T1J4, QC, Canada 31 32 33 34 1 bioRxiv preprint doi: https://doi.org/10.1101/465880; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Abstract (150 words) 2 Spatiotemporal regulation in DNA replication maintains kinetochore stability. The epigenetically 3 regulated centromeres (CENs) in the budding yeast Candida albicans have unique DNA sequences, 4 replicate early and are clustered throughout the cell cycle. In this study, the genome-wide occupancy 5 of replication initiation protein Orc4 reveals its abundance at all CENs in C. albicans. Orc4 associates 6 with four different DNA motifs, one of which coincides with tRNA genes. Hi-C combined with 7 genome-wide replication timing analyses identify enriched interactions among early or late replicating 8 Orc4-bound regions. A simulated polymer model of chromosomes reveals that early replicating and 9 strongly enriched Orc4-bound sites localize towards the kinetochores. Orc4 is constitutively localized 10 to CENs, and both Orc4 and Mcm2 stabilize CENPA. CENPA chaperone Scm3 localizes at the 11 kinetochore during anaphase, coinciding with the loading time of CENPA. We propose that this 12 spatiotemporal nuclear localization of Orc4, with Mcm2 and Scm3, recruits CENPA and stabilizes 13 centromeric chromatin. 14 15 16 17 18 19 20 21 22 23 24 25 26 2 bioRxiv preprint doi: https://doi.org/10.1101/465880; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Introduction 2 The timely duplication of the genetic material and faithful chromosome segregation maintains 3 genome stability. In eukaryotes, the origin recognition complex (ORC) comprising of Orc1-6 along 4 with Cdc6, Cdt1 and the minichromosome maintenance complex (Mcm2-7) initiate DNA replication 5 at multiple discrete sites on a chromosome that serve as DNA replication origins (Leonard and 6 Mechali 2013). ORC recognizes its cognate binding sites in a species-specific manner - an AT-rich 7 consensus sequence in the budding yeast Saccharomyces cerevisiae (Wyrick, Aparicio et al. 2001) to 8 AT-rich asymmetric sequences in the fission yeast Schizosaccharomyces pombe (Chuang and Kelly 9 1999) while its binding is non-specific to any DNA sequence in humans (Vashee, Cvetic et al. 2003). 10 The robust programing of the time of firing of replication origins during S phase is required to ensure 11 the availability of the limiting concentration of replication initiation factors (Aparicio 2013). Genomic 12 regions that replicate early during S phase have a higher propensity to attract ORC and are more 13 efficient in origin firing, whereas late replicating regions appear to be more stochastic contributing to 14 inefficient initiation events (Mesner, Valsakumar et al. 2011). The spatial organization of 15 chromosomes within the nucleus also favors the accessibility of initiation factors to complete DNA 16 replication in a timely manner (Aparicio 2013). Chromosome conformation capture (3C) analysis has 17 revealed higher interaction frequencies between early origins in S. cerevisiae (Duan, Andronescu et 18 al. 2010). Evidently, understanding the topology of the three-dimensional (3D) genome and the 19 factors that spatiotemporally regulate the chromosomal architecture is necessary to explain how the 20 genome is organized into various functional domains. 21 The faithful segregation of the duplicated genome is facilitated by centromeres (CENs) which act as 22 the binding platform for kinetochore proteins. In most eukaryotes, CENs are specified by a CEN- 23 specific histone H3 variant, CENPA through epigenetic mechanisms, where the underlying DNA 24 sequence appears dispensable for CEN establishment (Malik and Henikoff 2009). The epigenetic 25 specification of CENs in most eukaryotes is evident as CEN DNA are the most rapidly evolving loci 26 of the genome (Malik and Henikoff 2009). The targeted loading of CENPA is temporally separated 27 from bulk H3 chromatin assembly in the cell cycle and is mediated by the specific chaperone Holliday 28 junction recognition protein (HJURP) (Kato, Sato et al. 2007) and its analogs in various species. The 29 yeast homolog Scm3 loads CENPA during S phase in S. cerevisiae (Williams, Hayashi et al. 2009), 30 whereas in S. pombe CENPA loading occurs during G2 (Shukla, Tong et al. 2018). The functional 31 homolog of HJURP in Drosophila, CAL1 loads CENPA during late telophase (Dunleavy, Beier et al. 32 2012). In humans, the G1 loading of CENPA by HJURP (Foltz, Jansen et al. 2009) is regulated by the 33 Mis18 complex (Barnhart-Dailey, Trivedi et al. 2017). In S. pombe, RNAi and heterochromatin are 34 required for CEN establishment (Folco, Pidoux et al. 2008). Unlike metazoa, most fungi have early 35 replicating CENs (Raghuraman, Winzeler et al. 2001, Kim, Dubey et al. 2003, Koren, Tsai et al. 3 bioRxiv preprint doi: https://doi.org/10.1101/465880; this version posted September 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2010). Vertebrate CENs replicate between mid-late S phase (Ten Hagen, Gilbert et al. 1990). CENPA 2 nucleosome disruption following DNA replication transiently creates gaps or nucleosome-free regions 3 which have to be reassembled for CEN propagation. The placeholder model proposes that the gaps 4 generated upon parental CENPA eviction are occupied by placeholder molecules like H3 in S. pombe 5 (Shukla, Tong et al. 2018) and H3.3 in Drosophila (Dunleavy, Almouzni et al. 2011). Hence, CENPA 6 replenishment by its replication-independent loading is necessary for its maintenance. 7 Once established, CENPA chromatin can epigenetically self-propagate through multiple cell 8 divisions, best studied in the case of ectopic or neocentromere activation at non-centromeric loci upon 9 native CEN inactivation (Warburton 2004). However, mechanisms contributing to maintenance of 10 centromeric chromatin is relatively unclear. Growing evidence suggests a role of replication initiation 11 proteins in CEN function (Natsume, Muller et al. 2013). Fungal CENs replicate early in S phase 12 (Pohl, Brewer et al. 2012) to maintain kinetochore integrity and pericentromeric cohesion (Kitamura, 13 Tanaka et al. 2007, Natsume, Muller et al. 2013). In humans, Orc2 localizes to CENs (Prasanth, 14 Prasanth et al. 2004), and HJURP along with Mcm2 is required for CENPA inheritance during S 15 phase (Zasadzinska, Huang et al. 2018). In vitro experiments suggest that Mcm2 and Asf1 16 cochaperone dimers of H4 with both the canonical and variant forms of H3 through its histone- 17 binding mode (Huang, Stromme et al. 2015, Richet, Liu et al. 2015). Recently, DNA replication has 18 been implicated to remove ectopically loaded non-centromeric CENPA for the precise reloading of 19 centromeric CENPA during G1 in humans (Nechemia-Arbely, Miga et al. 2019). Also, replication 20 fork termination seen at CEN promotes CEN DNA loop formation which is ultimately required for 21 kinetochore assembly (Cook, Bennett et al. 2018). Hence, there is an implicit crosstalk of replication 22 initiation proteins for CEN function. 23 In yeast, CENs of all chromosomes cluster close to the spindle pole body (SPB) embedded into the 24 nuclear membrane and establish inter-chromosomal