Orc4 spatiotemporally stabilizes centromeric chromatin

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.

A phylogenetically-restricted essential cell cycle progression factor in the human pathogen Candida albicans

“…9b ). An increase in Cse4 levels could be an outcome of Cse4 loading at anaphase in C. albicans 63 , 64 . Finally, we analyzed the integrity of the mitotic spindle, as mentioned previously, in CSA6 PSD mutant.…”

“…In a recent study from our laboratory, Mcm7 has been identified as a subunit of the kinetochore interactome in a basidiomycete yeast Cryptococcus neoformans 95 . Another subunit of the Mcm2-7 complex, Mcm2, is involved in regulating the stability of centromeric chromatin in C. albicans 64 . Considering the growing evidence of the role of Mcm2-7 subunits beyond their canonical, well-established roles in DNA replication, the serendipitous identification of Mcm7 as a regulator of genome stability in our screen is striking.…”

“…Overexpression of genes from the tetracyline inducible promoter (P TET ) was achieved by the addition of anhydrotetracycline (Atc, 3 µg/ml) or doxycycline (Dox, 50 µg/ml) in YPDU medium at 30 °C 48 in the dark as Atc and Dox are light-sensitive. The CSA6 PSD strains were grown at 30 °C either in permissive (YPDU) or nonpermissive (YPDU + 5 mM methionine (M) + 5 mM cysteine (C)) conditions of the MET3 promoter 62 , 64 . E. coli strains were cultured at 30 °C or 37 °C in Luria-Bertani (LB) medium or 2YT supplemented with ampicillin (50 µg/ml or 100 µg/ml), chloramphenicol (34 µg/ml), kanamycin (50 µg/ml) and tetracycline (10 µg/ml).…”

A universal framework for detecting cis-regulatory diversity in DNA regions

“…We found no evidence of tRNA gene expression going down during these stages of development. However, tRNA genes are known to play a role in remodeling chromatin and act as insulators or harbor origins of replication in other organisms ( Su et al 2020 ; Sreekumar et al 2021 ). Modules 1 and 9 are the only other modules that are significantly closing.…”

Spindle pole body-associated Atg11, an autophagy-related protein, regulates microtubule dynamics essential for high-fidelity chromosome segregation

“…It is likely that depending on the types of mitoses, the players might differ in performing a non-canonical function. More examples of such non-canonical functions (Sreekumar et al, 2021; Varma et al, 2012), suggest how proteins organize between various processes to maintain cellular homeostasis. A series of evidence have established an interplay between errors in the spindle alignment with ageing and aneuploidy leading to the development of cancer and neurodegenerative diseases (Cheng et al, 2008; Knoblich, 2010; Lancaster & Knoblich, 2012; Manzano-Lopez et al, 2019; Yamashita et al, 2007).…”