Genome-wide analysis reveals a cell cycle–dependent mechanism controlling centromere propagation

Department of Genome Dynamics, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 01/2009; 183(5):805-18. DOI: 10.1083/jcb.200806038
Source: PubMed


Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.

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    • "Instead a CENP-A-specific chaperone, HJURP, binds soluble CENP-A-H4 complex before its chromatin incorporation during G1 phase (Dunleavy et al., 2009; Foltz et al., 2009) (Figure 6). The yeast and Drosophila homologs of HJURP are apparently Scm3 (Mizuguchi et al., 2007; Pidoux et al., 2009; Sanchez-Pulido et al., 2009; Williams et al., 2009) and Cal1 (Erhardt et al., 2008), respectively. Scm3 is found in hexameric Cse4 nucleosomes (Mizuguchi et al., 2007), which appear to be assembly intermediates (Camahort et al., 2007). "
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    ABSTRACT: Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function.
    Full-text · Article · Sep 2014 · Developmental Cell
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    • "Having established that Modulo is a CAL1 partner, we next investigated whether it plays a role in CAL1 function. To address this, RNAi knock-down of Modulo was performed in S2 cells expressing GFP-CAL1 and mCherry-tubulin [10]. Western blot analysis determined that Modulo protein levels decreased to undetectable levels 4 days after addition of double stranded RNA (dsRNA), whereas levels of CAL1 and CID were unaffected (Fig. 3A). "
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    ABSTRACT: The relationship between the nucleolus and the centromere, although documented, remains one of the most elusive aspects of centromere assembly and maintenance. Here we identify the nucleolar protein, Modulo, in complex with CAL1, a factor essential for the centromeric deposition of the centromere-specific histone H3 variant, CID, in Drosophila. Notably, CAL1 localizes to both centromeres and the nucleolus. Depletion of Modulo, by RNAi, results in defective recruitment of newly-synthesized CAL1 at the centromere. Furthermore, depletion of Modulo negatively affects levels of CID at the centromere and results in chromosome missegregation. Interestingly, examination of Modulo localization during mitosis reveals it localizes to the chromosome periphery but not the centromere. Combined, the data suggest that rather than a direct regulatory role at the centromere, it is the nucleolar function of modulo which is regulating the assembly of the centromere by directing the localization of CAL1. We propose that a functional link between the nucleolus and centromere assembly exists in Drosophila, which is regulated by Modulo.
    Full-text · Article · Sep 2012 · PLoS ONE
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    • "Interestingly, a BLASTp of the C terminus identified only the known Drosophila CAL1 orthologs, suggesting that the N terminus is more conserved than the C terminus across distant dipteran species. BLASTp of the middle region of CAL1 only identified CAL1 orthologs from D. yakuba and D. simulans, consistent with the previously reported divergence of this part of CAL1 (Erhardt et al. 2008; Schittenhelm et al. 2010). Additional BLASTp, BLASTn, and BLASTx (identification and comparison of protein coding sequences in genomic DNA) were carried out using the CAL1 sequence of D. melanogaster alongside the putative CAL1 ortholog from D. grimshawi, which, being a more distant relative of the D. melanogaster CAL1, could help to identify more distant homologs in the species tree. "
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    ABSTRACT: Centromeres are essential cis-elements on chromosomes that are crucial for the stable transmission of genetic information during mitotic and meiotic cell divisions. Different species employ a variety of centromere configurations, from small genetically defined centromeres in budding yeast to holocentric centromeres that occupy entire chromosomes in Caenorhabditis, yet the incorporation of nucleosomes containing the essential centromere-specific histone H3 variant CENP-A is a common feature of centromeres in all eukaryotes. In vertebrates and fungi, CENP-A is specifically deposited at centromeres by a conserved chaperone, called HJURP or Scm3, respectively. Surprisingly, homologs of these proteins have not been identified in Drosophila, Caenorhabditis, or plants. How CENP-A is targeted to centromeres in these organisms is not known. The Drosophila centromeric protein CAL1, found only in the Diptera genus, is essential for CENP-A localization, is recruited to centromeres at a similar time as CENP-A, and interacts with CENP-A in both chromatin and pre-nucleosomal complexes, making it a strong candidate for a CENP-A chaperone in this lineage. Here, we discuss the conservation and evolution of this essential centromere factor and report the identification of a "Scm3-domain"-like region with similarity to the corresponding region of fungal Scm3 as well as a shared predicted alpha-helical structure. Given the lack of common ancestry between Scm3 and CAL1, we propose that an optimal CENP-A binding region was independently acquired by CAL1, which caused the loss of an ancestral Scm3 protein from the Diptera lineage.
    Full-text · Article · Jul 2012 · Chromosome Research
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