Cancer-associated alteration of pericentromeric heterochromatin may contribute to chromosome instability.

Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.

Suitability of a CMV/EGFP cassette to monitor stable expression from human artificial chromosomes but not transient transfer in the cells forming viable clones.

Human artificial chromosomes (HACs) were generated by transfer of telomerized PAC constructs containing alpha satellite DNA of various human chromosomes. To monitor which cells took up constructs and subsequently formed stable clones under blasticidin S (BS) selection, a CMV/EGFP expression cassette was inserted into a HAC construct based on chromosome 5 alpha satellite DNA (142 kb). Lipofection into HT1080 cells resulted in a small proportion of cells exhibiting bright green fluorescence on day 1. Areas containing such early green cells were marked, and plates monitored over 2 weeks. In only one out of 41 marked areas, a viable clone developed. In the remaining 40 areas, the green cells ceased division at 1-8 cells. In contrast, outside the marked areas, 16 stable clones formed which did not exhibit green fluorescence during the first cell divisions, but all cells of each became green around day 4-6. Fluorescence in situ hybridization (FISH) analysis of isolated clonal lines demonstrated low copy HAC formation without integration. We conclude that transient expression of an EGFP marker on HAC DNA is not a suitable means for the identification of the proportion of transfected cells which are capable of forming viable clones. One explanation could be that the high copy number required to consistently detect transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and clone formation.

Analysis of parent of origin specific DNA methylation at SNRPN and PW71 in tissues: implication for prenatal diagnosis.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct developmental disorders caused by absence of paternal or maternal contributions of the chromosome region 15q11-q13, resulting from deletions, uniparental disomy (UPD), or rare imprinting mutations. Molecular cytogenetic diagnosis is currently performed using a combination of fluorescence in situ hybridisation (FISH), DNA polymorphism analysis, and DNA methylation analysis. Only methylation analysis will detect all three categories of PWS abnormalities, but its reliability in tissues other than peripheral blood has not been examined extensively. Therefore, we examined the methylation status at the CpG island of the small nuclear ribonucleoprotein associated polypeptide N (SNRPN) gene and at the PW71 locus using normal and abnormal lymphoblast (LB) cell lines (n = 48), amniotic fluid (AF) cell cultures (n = 25), cultured chorionic villus samples (CVS, n = 17), and fetal tissues (n = 18) by Southern blot analysis with methylation sensitive enzymes. Of these samples, 20 LB cell lines, three AF cultures, one CVS, and 15 fetal tissues had been previously diagnosed as having deletions or UPD by other molecular methods. Methylation status at SNRPN showed consistent results when compared with FISH or DNA polymorphism analysis using all cell types tested. However, the methylation pattern for PW71 was inconsistent when compared with other tests and should therefore not be used on tissues other than peripheral blood. We conclude that SNRPN, but not PW71, methylation analysis may be useful for diagnosis of PWS/AS on LB cell lines, cultured amniotic fluid, or chorionic villus samples and will allow, for the first time, prenatal diagnosis for families known to carry imprinting centre defects.