Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. However, the susceptibility gene in this region has not yet been identified. The strongest linkage peak of prostate cancer overlies a variable region of 750-kb at Xq27 that is enriched by large SDs carrying a cluster of SPANX genes. No mutations were detected in SPANX genes. It suggests that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. Since the last Annual Report, we have concentrated on the analysis of the 750-kb region in several X-linked families with the strongest linkage to HPCX1. Direct isolation of a set of overlapping genomic segments carrying SDs that completely covers the 750-kb region by in vivo recombination in yeast (a TAR cloning technique) were used to perform a mutational analysis of this region. The subsequent analysis of isolated fragments excluded the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. When our work was completed and submitted for publication (Genes, Chromosomes &Cancer J 2012, 51:933-948), the International Consortium for Prostate Cancer Genetics re-evaluated previous linkage data and concluded that there is no strong evidence for a major prostate cancer susceptibility gene located at Xq27-28 (Bailey-Wilson JE et al;International Consortium for Prostate Cancer Genetics. Analysis of Xq27-28 linkage in the international consortium for prostate cancer genetics (ICPCG) families BMC Med Genet 2012, 13: 46.) After exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy, the studies related to this sub-project have been completed in our lab and we have focused on other sub-projects linked with Human Artificial Chromosomes (HACs). (HACs) assembled from alphoid DNA arrays represent a novel episomal gene delivery vector for functional genomics and gene therapy. HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. We previously constructed a synthetic HAC (tetO-HAC) that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This HAC is the most advanced vector for expression of full-length genes and entire loci and for correction of genetic deficiencies in human cells. The tetO-HAC was also used as a unique system to study a role of epigenetic modifications in the human kinetochore function. The broad use of the tetO-HAC requires the knowledge of its structural organization. During the past year, we completed physical characterization of a megabase- size synthetic alphoid DNA array in the HAC that has been formed from a synthetic tetO-array. Our results provide a tool to control structural integrity of tetO-HAC during gene loading and HAC transfer into different host cells. They also shed light on a mechanism for de novo HAC formation in human cells. We previously demonstrated the utility of the synthetic HAC for delivery of full size genes and correction of genetic deficiencies in human cells. Specifically genomic copies of two cancer-associated genes, VHL mutated in von Hippel Lindau syndrome (VHL) and NBS1 mutated in Nijmegen breakage syndrome (NBS) were successfully transferred into gene deficient cells. We have also shown that phenotypes arising from stable gene expression from the HAC can be reversed when cells are cured of the HAC by inactivating its kinetochore in proliferating cell populations. During the past year, several other human genes were loaded into the HAC for gene transfer/gene expression studies, including BRCA1 and mtTOP1 gene previously discovered in LMP. In the tetO-HAC with a conditional centromere, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While this domain is permissivefor transcription, there are no studies on a long-term transgene expression within centrochromatin. In our recent study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA and tDNA, on the expression of an EGFP transgene loaded into the tetO-HAC vector. Unexpectedly, insulator function was essential for stable expression of the transgene in centrochromatin that represents open chromatin structure. A tDNA insulator (discovered in our lab) consisting of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements, such as gamma-satellite DNA and tDNA, that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. 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 alphoidtetO-HAC. However, inactivation of the HAC kinetochore requires transfection of cells by a retrovirus vector to achieve a high level of tTS expression, a step that potentially may lead to insertional mutagenesis. In our recent work, we described an approach to re-engineering the alphoidtetO-HAC vector that allows verification of phenotypic changes attributed to expression of genes from the HAC without transfecting exogenous chromatin modulators. In the new HAC vector, a tTS cassette is inserted into a gene-loading site along with a gene of interest. In the absence of doxycycline, expression of the tTS generates a self-regulating fluctuating heterochromatin on the alphoidtetO-HAC that induces a fast and strong silencing of the genes on the HAC without significant effect on HAC segregation. This silencing is reversible, and therefore expression of the HAC-encoded genes can be readily recovered by adding doxycycline. The newly modified alphoidtetO-HAC-based system has the potential for multiple applications in gene function studies. We have also applied our HAC system for screening of drugs affecting chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth. Our goal was to develop a new quantitative assay for identification of drugs that elevate CIN in cancer cells. For this purpose, the EGFP transgene was loaded into the tetO-HAC using Cre-loxP recombination. The presence of EGFP allows measuring of the HAC loss by flow cytometry. We have successfully used this assay to measure increased mis-segregation of chromosomes in response to different anticancer drugs. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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