Centromeres, the sites of spindle attachment during mitosis and meiosis, are located in specific positions in the human genome, normally coincident with diverse subsets of alpha satellite DNA. While there is strong evidence supporting the association of some subfamilies of alpha satellite with centromere function, the basis for establishing whether a given alpha satellite sequence is or is not designated a functional centromere is unknown, and attempts to understand the role of particular sequence features in establishing centromere identity have been limited by the near identity and repetitive nature of satellite sequences. Utilizing a broadly applicable experimental approach to test sequence competency for centromere specification, we have carried out a genomic and epigenetic functional analysis of endogenous human centromere sequences available in the current human genome assembly. The data support a model in which functionally competent sequences confer an opportunity for centromere specification, integrating genomic and epigenetic signals and promoting the concept of context-dependent centromere inheritance.

Human centromeres are defined by megabases of homogenous alpha-satellite DNA arrays that are packaged into specialized chromatin marked by the centromeric histone variant, centromeric protein A (CENP-A). Although most human chromosomes have a single higher-order repeat (HOR) array of alpha satellites, several chromosomes have more than one HOR array. Homo sapiens chromosome 17 (HSA17) has two juxtaposed HOR arrays, D17Z1 and D17Z1-B. Only D17Z1 has been linked to CENP-A chromatin assembly. Here, we use human artificial chromosome assembly assays to show that both D17Z1 and D17Z1-B can support de novo centromere assembly independently. We extend these in vitro studies and demonstrate, using immunostaining and chromatin analyses, that in human cells the centromere can be assembled at D17Z1 or D17Z1-B. Intriguingly, some humans are functional heterozygotes, meaning that CENP-A is located at a different HOR array on the two HSA17 homologs. The site of CENP-A assembly on HSA17 is stable and is transmitted through meiosis, as evidenced by inheritance of CENP-A location through multigenerational families. Differences in histone modifications are not linked clearly with active and inactive D17Z1 and D17Z1-B arrays; however, we detect a correlation between the presence of variant repeat units of D17Z1 and CENP-A assembly at the opposite array, D17Z1-B. Our studies reveal the presence of centromeric epialleles on an endogenous human chromosome and suggest genomic complexities underlying the mechanisms that determine centromere identity in humans.kinetochore | dicentric | epigenetics | polymorphism | heterochromatin

Current techniques for identifying mutations that convey a small increased cancer risk or those that modify cancer risk in carriers of highly penetrant mutations are limited by the statistical power of epidemiologic studies, which require screening of large populations and candidate genes. To identify dosage-sensitive genes that mediate genomic stability, we performed a genomewide screen in Saccharomyces cerevisiae for heterozygous mutations that increase chromosome instability in a checkpoint-deficient diploid strain. We used two genome stability assays sensitive enough to detect the impact of heterozygous mutations and identified 172 heterozygous gene disruptions that affected chromosome fragment (CF) loss, 45% of which also conferred modest but statistically significant instability of endogenous chromosomes. Analysis of heterozygous deletion of 65 of these genes demonstrated that the majority increased genomic instability in both checkpoint-deficient and wild-type backgrounds. Strains heterozygous for COMA kinetochore complex genes were particularly unstable. Over 50% of the genes identified in this screen have putative human homologs, including CHEK2, ERCC4, and TOPBP1, which are already associated with inherited cancer susceptibility. These findings encourage the incorporation of this orthologous gene list into cancer epidemiology studies and suggest further analysis of heterozygous phenotypes in yeast as models of human disease resulting from haplo-insufficiency. C ANCERS are commonly characterized as having an abnormal number of chromosomes, termed aneuploidy, which arises due to genomic instability. Aneuploidy has been proposed as a mutagenic mechanism and a driving force of tumor progression and not just a phenotype of the disease (Weaver and Cleveland 2006). Many of the genes whose disruption results in aneuploidy were first identified in budding yeast on the basis of analysis of haploid strains containing null mutations. Aneuploidy can be caused by inherited or somatic mutations in genes that function in the maintenance of genomic stability, such as those involved in centrosome formation,chromosomemetabolism,andcellcyclecheckpoints. Among these, cell cycle checkpoint pathways play an especially important role in maintaining genomic integrity when cells are challenged with genotoxic agents or other stressors (Zhou and Elledge 2000).Mutations in checkpoint genes including, BRCA1, PTEN, ATM, and CHEK2, lead to increases in breast cancer susceptibility (Li et al. 1997;Nathanson and Weber 2001;Meijers-Heijboer et al. 2002;King et al. 2003). Women who inherit BRCA1 mutations have an increased lifetime breast cancer risk of 50-80% (12% risk in the general population) and an ovarian cancer risk of 15-65% (1.5% risk in the general population). However, the age of cancer onset and type and number of cancers can vary, even among women carrying the same mutation. One mechanism for this variation is the presence of unlinked genetic loci, which modify this risk (Rebbeck 2002). For instance, ovarian cancer ...

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