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And Trans-Chromosomal Interactions Define Pericentric Boundaries In HIGHLIGHTED ARTICLE | INVESTIGATION Cis- and Trans-chromosomal Interactions Define Pericentric Boundaries in the Absence of Conventional Heterochromatin Lakshmi Sreekumar,* Priya Jaitly,* Yao Chen,† Bhagya C. Thimmappa,*,1 Amartya Sanyal,† and Kaustuv Sanyal*,2 *Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India and †School of Biological Sciences, Nanyang Technological University, Singapore, Singapore 637551 ORCID IDs: 0000-0002-1849-4374 (L.S.); 0000-0002-1660-6416 (P.J.); 0000-0003-2708-8674 (Y.C.); 0000-0002-2109-4478 (A.S.); 0000-0002-6611-4073 (K.S.) ABSTRACT The diploid budding yeast Candida albicans harbors unique CENPA-rich 3- to 5-kb regions that form the centromere (CEN) core on each of its eight chromosomes. The epigenetic nature of these CENs does not permit the stabilization of a functional kinetochore on an exogenously introduced CEN plasmid. The flexible nature of such centromeric chromatin is exemplified by the reversible silencing of a transgene upon its integration into the CENPA-bound region. The lack of a conventional heterochromatin machinery and the absence of defined boundaries of CENPA chromatin makes the process of CEN specification in this organism elusive. Additionally, upon native CEN deletion, C. albicans can efficiently activate neocentromeres proximal to the native CEN locus, hinting at the importance of CEN-proximal regions. In this study, we examine this CEN-proximity effect and identify factors for CEN specification in C. albicans. We exploit a counterselection assay to isolate cells that can silence a transgene when integrated into the CEN-flanking regions. We show that the frequency of reversible silencing of the transgene decreases from the central core of CEN7 to its peripheral regions. Using publicly available C. albicans high-throughput chromosome conformation capture data, we identify a 25-kb region centering on the CENPA-bound core that acts as CEN-flanking compact chromatin (CFCC). Cis- and trans-chromosomal interactions associated with the CFCC spatially segregates it from bulk chromatin. We further show that neocentromere activation on chromosome 7 occurs within this specified region. Hence, this study identifies a specialized CEN-proximal domain that specifies and restricts the centromeric activity to a unique region. KEYWORDS centromere; CENPA; Hi-C; Candida albicans; neocentromere N a majority of eukaryotes, centromere (CEN) specification gional CENs. The CENs in the Candida species are one of Iis fulfilled by the assembly of the histone H3 variant, CENPA, the most well-studied short regional CENs. Candida albicans and subsequent kinetochore stabilization. In most fungal has unique 3–5 kb CENPA-bound CEN DNA (Sanyal et al. species, CENs extend over to a region on every chromosome, 2004), as does C. dubliniensis (Padmanabhan et al. 2008) ranging in sizes from 3 to 300 kb (Friedman and Freitag and C. lusitaniae (Kapoor et al. 2015). On the other hand, 2017), which are categorized as short regional or long re- the 10- to 11-kb CENs in C. tropicalis consist of a 2- to 5-kb central core flanked by inverted repeats (Chatterjee Copyright © 2019 by the Genetics Society of America et al. 2016). The 40- to 110-kb-long regional CENs of doi: https://doi.org/10.1534/genetics.119.302179 Manuscript received April 4, 2019; accepted for publication May 22, 2019; published Schizosaccharomyces pombe contain a 10- to 14-kb-long CENPA- Early Online May 29, 2019. bound region flanked by outer pericentric repeats (Clarke et al. Supplemental material available at FigShare: https://doi.org/10.25386/genetics. 8197388. 1986). The filamentous yeast Neurospora crassa harbors 150– 1 Present address: Department of Biochemistry, Robert-Cedergren Centre for 300 kb of heterochromatic CENPA-rich DNA (Smith et al. Bioinformatics and Genomics, University of Montreal, Montreal, QC H3T1J4, Canada. 2011). Among the basidiomycetes, Cryptococcus neoformans con- 2 Corresponding author: Molecular Mycology Laboratory, Molecular Biology and tain 27–65 kb of transposon-rich CENPA-bound DNA sequences Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur Post, Bangalore 560064, India. E-mail: [email protected] (Yadav et al. 2018). Evidently, regional CENs in fungi do not Genetics, Vol. 212, 1121–1132 August 2019 1121 entirely depend on conserved DNA elements for kineto- CEN is inactivated. Therefore, neocentromeres are an aid to chorebinding,andthereforehavebeenproposedasexcel- study de novo CEN formation mechanisms. Neocentromeres lent models to study epigenetically regulated metazoan are formed at CEN-proximal loci in Drosophila (Maggert CENs. and Karpen 2001) and chicken cells (Shang et al. 2013). The centromeric chromatin is different from bulk chro- The assembly of ectopic CENPA as a “CENPA-rich zone” or matin and is epigenetically specified in most regional CENs “CENPA cloud” surrounding the endogenous CEN and prox- (Sullivan and Karpen 2004). While the core CEN and ad- imity of neocentromere hotspots to the native CEN in these jacent pericentric regions are poorly transcribed, pervasive organisms indicates that CENPA is peppered on CEN-adjacent levels of transcription at fungal CENs are known to influ- loci and can get rapidly incorporated into the CEN upon evic- ence CEN activity (Choi et al. 2011; Ohkuni and Kitagawa tion (Fukagawa and Earnshaw 2014). Also, the site of neo- 2011; Ling and Yuen 2019). Even though CENPA limits its centromere activation is found to be incompatible with localization on a chromosome, the functional region re- transcription (Scott and Sullivan 2014). These epigenetic quired for chromosome segregation is much larger and mechanisms ensure stable propagation of active CENs across involves pericentric regions. This is largely exemplified generations. in S. pombe minichromosomes, which are stabilized in CENs cluster next to the spindle pole bodies throughout the the presence of an outer pericentric repeat in addition to cell cycle in budding yeast species including Saccharomyces the central core (Baum et al. 1994). Centric and pericentric cerevisiae (Jin et al. 2000; Haase et al. 2013) and sev- chromatin differ notably in fungal systems. In S. pombe,H3 eral species of Candida (Sanyal and Carbon 2002; nucleosomes are nearly absent from the CENPA-rich cen- Padmanabhan et al. 2008; Burrack et al. 2016; Chatterjee tral core (Thakur et al. 2015). RNA interference (RNAi)- et al. 2016). Clustered centromeric regions were shown to directed heterochromatin assembly at the outer repeat be in physical proximity by a genome-wide chromosomal mediates targeting of CENPA by Clr4-mediated di- interaction study in S. cerevisiae, giving rise to physical inter- methylation at H3K9 (Volpe et al. 2002; Folco et al. actions between different CENs (Duan et al. 2010). High- 2008; Allshire and Ekwall 2015). These features are throughput chromosome conformation capture (Hi-C) and shared in C. neoformans,wheretheextensivelymethyl- related studies in S. cerevisiae have revealed chromosome ated CEN DNA is enriched with H3K9me2 and maintained substructures in which domains with similar contact proba- with the help of the RNAi machinery (Yadav et al. 2018). bilities have higher interactions than the ones that interact In contrast, the CENPA-bound regions in N. crassa have due to random diffusion (Tjong et al. 2012; Eser et al. 2017). heterochromatic properties containing H3K9me3 nucleo- Recently, chromosome conformation capture-on-chip (4C) somes and 5-methylcytosine (5mC) (Smith et al. 2011). analysis in vertebrates revealed that clustered CENs are pre- Their pericentric regions are 5- to 20-kb long and enriched sent in a compact chromatin environment (Nishimura et al. in H3K4me3 and 5mC (Smith et al. 2011; Friedman and 2018). The neocentromeres in these cells were commonly Freitag 2017). All known variants of fungal CEN chroma- associated with specific heterochromatin-rich regions in the tin in regional CENs are more similar to heterochromatin three-dimensional (3D) nuclear space. Hence, the 3D archi- than euchromatin, largely owing to the presence of silenc- tecture of the chromosome, its scaffolds, and its associated ing histone marks and components of RNAi machinery chromatin within the nucleus provide the spatial cues re- (Friedman and Freitag 2017). Intriguingly, the pericentric quired to specify CEN location. boundaries are often not well defined in organisms having Nonrepetitive CENs serve as excellent models to study short regional CENs, as features like pericentric repeats, characterization of centromeric chromatin. In C. albicans, ev- associated histone marks, or RNAi machinery are either ery CEN harbors a unique CEN DNA sequence (Sanyal et al. lacking or cryptic. 2004), each of which cannot stabilize a CEN plasmid (Sanyal One of the hallmarks of the epigenetic control at CENs is et al. 2004; Baum et al. 2006). The activation of neocentro- the reversible silencing of a transgene positioned within meres at hotspots proximal to the native CEN location the CENPA-binding region. Transgenes inserted at the central (Thakur and Sanyal 2013) and presence of CEN-proximal core and outer repeats of the S. pombe CENs undergo tran- replication origins (Koren et al. 2010; Mitra et al. 2014) in- scriptional silencing that are clonally inherited. Compared to dicate the prominent role of CEN-proximal or pericentric re- the outer repeats that are highly heterochromatinized, trans- gions for CEN function. Moreover, there is no functional gene silencing
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