Human Artificial Chromosomes for Cancer Research and Functional Genomics

Division Of Basic Sciences - Nci

Investigators

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Abstract

Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for the study assembly and maintenance of human kinetochore as well as for gene therapy, screening of anticancer drugs and biotechnology. As known, HACs are generated after transfection of alphoid DNA arrays into human cells where the HAC seeding DNA undergoes spontaneous lesions and multimerization. How and when they occur remains unknown. In our recent work, we have performed the first systematic study of rearrangements that occur during HAC formation and determined how they alter the epigenetic landscape in the HAC centromere and how they impact HAC segregation in mitosis. We demonstrated that dramatic DNA rearrangements involving a process resembling chromothripsis can occur early during HAC formation and that once formed, they are stably maintained across many cell generations. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes. We previously constructed a synthetic HAC (tetO HAC) with a unique gene loading site allowing tethering of the HAC kinetochore by different chromatin modifies fused with the tet repressor protein. The tetO HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, such as the tTS, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the tetO HAC. In separate experiments, a platform with multi integrase recombination sites has been inserted into tetO HAC for assembly large genetic loci in the HAC. Work is in progress to assemble entire nucleolar organizer region (NOR) in the HAC using rDNA units isolated from acrocentric chromosomes. Note that the rDNA clusters and flanking proximal (PJ) and distal (DJ) sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of the 43 kb human rDNA units within an individual nucleolar organizer region (NOR) are only partially known. To address this lacuna, we first applied transformation associated recombination (TAR) cloning to isolate individual rDNA segments from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA (both in the transcribed and spacer regions), raising the possibility of corresponding variations in ribosome dynamics. The average rate of variations in rDNA (roughly 7.5 variants per kb) is comparable to typical estimates of variations across the human genome. The large number of rDNA units on chromosome 21 (56 copies) made it very difficult to determine a sequence of the entire NOR along with flanking PJ and DJ regions. In recent work, we have focused on the NOR for chromosome 22, which typically carries a low number of rDNA repeats, and isolated the entire block of rDNA units (two tandem 43 kb repeats) with their flanking PJ and DJ regions using a TAR cloning technique. Multiple variations among the rDNA units were again seen both in the transcribed and spacer regions. Characteristic variants included SNPs, short INDELs, and variable lengths of short repeat motifs. Most of the variants found correspond to the SNPs previously identified in rDNA units on chromosome 21 and transcriptomes sampled from multiple individuals. Comparable intra- and inter-individual divergence of rDNA units on the same and different chromosomes support the concerted evolution of rDNA units. TAR clones carrying an entire rDNA array and flanking PJ and DJ regions from chromosome 22 provides a platform for assembly of a synthetic NOR region in the tetO HAC vector containing multiple integrase sites. This can enable detailed study of the sequence requirements and mechanism of nucleolar formation in human cells, including the role of PJ and DJ in this process. We have also applied our tetO HAC for measuring chromosome instability (CIN) in human cells. Whole chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a characteristic feature of most types of cancer, thus distinguishing them from their normal counterparts. Although CIN is generally considered a driver of tumor growth, a threshold level exists whereby further increase in CIN frequency becomes a barrier against tumor growth and therefore can be exploited therapeutically. However, drugs known to increase CIN beyond this therapeutic threshold are currently few in number. In our previous work, we have developed a new quantitative assay for measuring CIN based on the use of a nonessential HAC carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows measurement of HAC loss rate in response to drug treatment by routine flow cytometry. We used this assay to rank more than 200 anticancer drugs on their effect on HAC loss. The strongest effect was observed for microtubule stabilizing agents and inhibitors of topoisomerase TOP1, developed in our branch. We also demonstrated the utility of the assay for screening of new CIN genes. Presently, approximately 400 human genes that control proper chromosome transmission have been annotated with gene ontology (GO) terms, while systematic CIN gene screens in the yeast S. cerevisiae have revealed more than 700 genes. Therefore, it may be supposed that many human CIN genes remain unidentified. In our recent work, we modified EGFP HAC and converted the original assay into high throughput CIN screen of siRNA libraries of human genes. Analysis of siRNAs targeting each of 720 human protein kinase genes revealed dozen CIN genes with no information on their role in chromosome transmission. At present, in collaboration with the Functional Genomics Laboratory at NCATS, NIH, we are analyzing the Ambion collection of siRNAs covering whole human genome. This analysis should identify all unknown genes involved in proper chromosome transmission. Each of these new CIN genes may be considered as a new target for cancer therapy. Telomerase/telomere targeting therapy is a potentially promising approach for cancer treatment because even transient telomere dysfunction can induce chromosomal instability (CIN) and may be a barrier to tumor growth. However, till now only a limited number of chemical compounds that target telomerase or telomeres have been identified and only a few are in clinical trials. Two years ago, we developed a dual HAC assay that enables identification and ranking of compounds that induce CIN as a result of telomere dysfunction. This assay is based on the use of two isogenic cell lines, one carrying a linear HAC (containing telomeres) and the other carrying a circular HAC (lacking telomeres). Disruption of telomeres in response to drug treatment results in specific destabilization of the linear HAC. Recently, we used the dual HAC assay for the analysis of the terpyridine platinum derived G4 ligands developed at the Institute Curie, France. Our analysis revealed four compounds, Pt tpy, Pt ttpy, Pt vpym and Pt cpym, that induce a specific loss of the linear HACs. Increased CIN after treatment by these compounds correlates with the induction of double stranded breaks (DSBs) predominantly localized at telomeres and reflecting telomere associated DNA damage. This family of G4 ligands are promising drug candidates for treatment of cancer alone or in combination with ionizing radiation.