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.

Chromosome engineering: prospects for gene therapy.

Recent advances in chromosome engineering and the potential for downstream applications in gene therapy were presented at the Artificial Chromosome Session of Genome Medicine: Gene Therapy for the Millennium in Rome, Italy in September 2001. This session concentrated primarily on the structure and function of human centromeres and the ongoing challenge of equipping human artificial chromosomes (HACs) with centromeres to ensure their mitotic stability. Advances in the 'bottom up' construction of HACs included the transfer into HT1080 cells of circular PACs containing alpha satellite DNA, and the correction of HPRT deficiency in cells using HACs. Advances in the 'top down' construction of HACs using telomere associated chromosome fragmentation in DT40 cells included the formation of HACs that are less than a megabase in size and transfer of HACs through the mouse germline. Significant progress has also been made in the use of human minichromosomes for stable trans-gene expression. While many obstacles remain towards the use of HACs for gene therapy, this session provided an optimistic outlook for future success.

Stable gene expression from a mammalian artificial chromosome.

We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing alpha-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both alpha-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.

A testis-specifically expressed gene is embedded within a cluster of maternally expressed genes at 89B in Drosophila melanogaster.

Differential screening was used to isolate a genomic clone, lambda89Ba located at 89B that hybridised preferentially with female cDNA. On further investigation, a 3.1-kb subfragment, 89Ba(3.1), was shown to contain a gene with male germline-specific expression (Mst89B) flanked by two genes (Mat89Ba and Mat89Bb) expressed predominantly in the ovaries and embryo of Drosophila melanogaster. Mat89Bb is separated from Mst89B by at most 100 bp; Mst89B and Mat89Ba are convergently transcribed and their 3' untranslated regions (UTRs) overlap by a minimum of 85 bp. Database searches with either the 89Ba(3.1) genomic DNA sequence or conceptual translations of Mst89B or Mat89Bb cDNAs failed to reveal any significant similarities with database entries. Using in situ hybridisation to ovaries, Mat89Ba and Mat89Bb were shown to be expressed in nurse cells at stages nine and ten of oogenesis and exported to the oocyte. In addition, Mat89Bb transcripts were detected in the follicle cells surrounding the oocyte. Mst89B transcripts were present throughout spermatogenesis in germline-derived cells, consistent with northern analysis which showed that they were absent in the offspring of tudor flies that lack a germline. The absence of Mst89B transcripts at the tip of the testis suggested that the somatic cells in this region do not express Mst89B. Two 12-bp sequences were identified in the 5' UTR of Mst89B with a strong similarity to translational control elements (TCEs) originally identified in the CGP gene family. This suggests that TCEs may be present in a wider class of testis transcripts.