JCB_201608084 2645..2656

During early development, mitotic chromosome size scales to cell size; repeated cell division in the absence of embryo growth reduces cell size, and concomitantly, chromosome size decreases (Kieserman and Heald, 2011; Hara et al., 2013; Ladouceur et al., 2015). Chromosome size scaling is critical for life, as a single mitotic chromosome in an early embryo can be as large as the entire metaphase plate of smaller cells late in development (unpublished data). Previously, nuclear transport via the Ras-related nuclear protein (RAN) pathway was shown to play an important role in regulating mitotic chromosome size, implicating nuclear proteins in chromosome-size regulation (Hara et al., 2013; Ladouceur et al., 2015). To find proteins whose nuclear localization is critical for mitotic chromosome scaling, we devised and executed an RNAi screen of the Caenorhabditis elegans nucleome in a sensitized setting. We performed our screen in a C. elegans strain that harbors an exceptionally long chromosome as the result of a telomere fusion event between the two longest chromosomes (5 and X; Lowden et al., 2008, 2011). This strain is viable in laboratory conditions and contains a genome size not significantly different from the wild-type parent strain. We predicted that depletions with a robust effect on chromosome length would lead to embryonic lethality in our long-chromosome strain but not in control animals. This strategy identified the centromere-specific histone H3 variant CENP-A (HCP-3/CPAR-1 in C. elegans, hereafter CENP-A) and topoisomerase-II (TOP-2 in C. elegans, hereafter topo-II), among 13 other proteins, as being required for viability in the presence of an exceptionally long chromosome (Fig. 1, a and b). CENP-A–containing nucleosomes define centromeres and make up an estimated 2–5% of the metazoan genome. In C. elegans, as well as many other organisms, centromeres are not restricted to a single region of each chromosome, resulting in CENP-A dispersed along each chromosome (holocentric; Maddox et al., 2006; De Rop et al., 2012). Current hypotheses in both monocentric (regionalized) and holocentric organisms predict that patches of CENP-A chromatin assemble to form a unified centromeric chromatin region in mitosis (Blower et al., 2002). The mechanism of mitotic centromere assembly is unknown; however, CENP-A nucleosomes have increased internal rigidity that could potentially drive self-assembly on the outer face of mitotic chromosomes forming a linear array. In agreement with this hypothesis, in vitro reconstitution of synthetic arrays showed that CENP-A–containing nucleosomes are more condensed than The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans, CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening. CENP-A and topoisomerase-II antagonistically affect chromosome length