Human Artificial Chromosomes for Cancer Research and Functional Genomics

Division Of Basic Sciences - Nci

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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 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. Efficiency of HAC formation is quite low that requires analysis of a large number of the transfectants. In our recent collaborative work, we have performed the first systematic study of structural requirements of the alphoid DNA arrays for efficient HAC formation. We demonstrated that combination of CENP-B box positive and negative synthetic alpha satellite repeats greatly improves de novo HAC formation. Previously we constructed a synthetic HAC (tetO-HAC) allowing tethering of the HAC kinetochore by different chromatin modifies fused with the Tet repressor protein. The tetO-HAC, whose centromere contains tetO repeats, has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin. In our recent study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO and lacO, i.e., a lacO/tetO-HAC. This new HAC can be used for detailed epigenetic engineering studies and to study a mechanism of centromere repositioning and inactivation in dicentric chromosomes. 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. In separate experiments, a platform with multi-integrase recombination sites has been inserted into the tetO-HAC for assembly of large genetic loci in the HAC. Our work is in progress to assemble a synthetic nucleolar organizer region (NOR) in the HAC to clarify a molecular mechanism of NORs association with nucleoli. For this purpose, 45 kb rDNA units and flanking proximal junction (PJ) and distal junction (DJ) sequences were isolated from human chromosomes 21 and 22 by a transformation-associated recombination (TAR) cloning method developed in our lab. It is worth noting that analysis of the rDNA units revealed an unexpectedly high number of sequence variants in the 45S RNA region. Some variants in 18S and 28S rRNA regions may affect either ribosome RNA folding or interaction with ribosomal proteins. An entire rDNA array along with the flanking PJ and DJ regions isolated from chromosome 22 provides a platform for assembly of a synthetic NOR region. This can enable a detailed study of the sequence requirements and the mechanism of nucleolar formation in human cells, including the role of PJ and DJ in this process. To characterize the sequence requirements for nucleolar formation in human cells, we inserted different fragments of a NOR from chromosome 22 corresponding to a complete 45 kb rDNA unit, DJ and PJ regions into a gene loading site of the tetO-HAC propagated in human HT1080. To determine the nucleoli association status of the HACs carrying different TAR constructs in interphase cells, we applied 3D immune-FISH. The HACs were visualized with the PNA probe for the tetO-alphoid array. Nucleoli were visualized using an antibody against Nop52. The nucleoli association was observed for the HAC carrying the 45 kb entire rDNA repeat and the 58 kb DJ region. In contrary, the percentage of nucleoli association of the HAC carrying the 53 kb PJ fragment did not differ from the control HAC carrying the GFP transgene. Collectively, our analysis of the HACs carrying different regions of a NOR from chromosome 22 revealed that a single rDNA repeat, and a 58 kb of the DJ DNA fragment are sufficient to drive nucleolar association of these HACs. Work is in progress to clarify a molecular mechanism of NORs association with nucleoli using a HAC vector carrying different fragments of the rDNA unit and DJ region. An abnormal chromosome number is a feature of most solid tumors and is often accompanied by an elevated rate of chromosome instability (CIN). Gain or loss of entire chromosomes leads to large-scale changes in gene copy number and expression levels. Mutations in CIN genes are thought to be an early event in tumor development. At present, approximately 400 human genes that control proper chromosome transmission have been annotated with gene ontology terms, while systematic CIN gene screens in yeast have revealed more than 900 genes. Therefore, it may be supposed that many human CIN genes remain unidentified. In our previous work, we developed a high-throughput assay for identification of new human CIN genes using the tetO-HAC expressing a degron-destabilized EGFP. In the current study, we are using an available at NCATS Ambion collection of 19,000 siRNAs covering the whole human genome for screening new CIN genes. As a result, 250 new CIN candidate genes were identified. The experiments on reconfirmation of 250 CIN candidate genes are in progress. The first step is the genes knock-down using newly developed siRNAs. Identification of new CIN genes should create opportunities for the development of new therapeutic strategies to target the CIN phenotype of cancer cells. 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. Three 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. In recent work, we used a dual HAC assay for analysis of five Hsp90 molecular chaperone inhibitors, 17-AAG, TAS-116, XL888, SNX-2112 and STA-9090, some of which are in clinical trials. It was previously demonstrated that human Hsp90 is associated with a functional telomerase complex. Unexpectedly, all five chaperone inhibitors induce a very high loss of a linear HAC with no effect on stability of a circular HAC. Additional experiments demonstrated a shortage of telomeric repeats in the treated cells. It means that the analyzed compounds are very specific inhibitors of telomerase and are promising drug candidates for treatment of cancer. One of them, TAS-116, has been recently approved for treatment of gastrointestinal stromal tumors (GIST) in Japan.