Genetic linkage studies implicate a gene or genes at Xq27 in hereditary prostate cancer susceptibility (HPCX). The corresponding region spans 750 kb and includes five SPANX genes, which encode proteins that are expressed in sperm nuclei and a variety of cancer cells. Each SPANX gene is embedded in a recently formed segmental duplication (SD) up to 100 kb in size, resulting in extensive enrichment in long stretches of repeated DNA in this region. Our previous analysis revealed frequent gene deletion, duplication and homology-based sequence transfers involving SPANX genes at Xq27, suggesting that SD-mediated homologous recombination in this region might be a source for predisposition to hereditary prostate cancer. In support of this hypothesis, a large inversion was detected in this region by three color FISH. During the past year, we have concentrated on mapping the breakpoint(s) of an inversion in prostate cancer patients. Several candidate breakpoint regions have been identified within a 750 kb sequence corresponding to the SPANX gene cluster. This analysis was performed using a combination of Southern blot-hybridization, long PCR, TAR cloning and DNA sequencing. Verification of these candidate regions is in progress. We hypothesize that such type of rearrangements leads to activation of expression of SPANX proteins that may play a role in cancer progression in multiple human cell types. The development and detailed studies of Human Artificial Chromosomes (HACs) offer new approaches for: 1) elucidating the mechanisms for de novo centromere/kinetochore formation and its structural/functional organization, and 2) development of gene delivery vectors with potential therapeutic applications. The role of chromatin structure in kinetochore function has been studied intensively but still remains poorly understood. Three years ago we have generated a HAC in human HT1080 cells with a conditional centromere, which we expect to be instrumental in resolving many questions. The HAC includes approximately 6,000 copies of the tetracycline operator (tetO) sequence. Such configuration allows a specific manipulation of the protein complement of a single kinetochore in vivo by targetting with tetR fusion proteins. This approach has been previously used to target chromatin modifying proteins into the HAC and to demonstrate that a balance between open and condensed chromatin is critical for kinetochore function. The strongest effect on the synthetic kinetochore was observed after tethering transcriptional repressors inducing HP1alpha repressive chromatin. Our collaborative studies with William Earnshaws laboratory showed that the disruption of kinetochore structure by a transcriptional repressor reflects a hierarchical disassembly of kinetochore components reflecting a pattern of protein interactions within kinetochore. During the past year, we demonstrated that tethering a minimal NFKB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of H3K9ac and a 10 fold elevation in transcript levels, but had no significant effect on kinetochore assembly or function. In contrast, tethering the herpes virus VP16 activation domain produced similar chromatin changes but resulted in a 150 fold elevation in transcripts approaching the level of transcription at an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing loss of assembled CENPA and blocking further CENPA assembly. Our data uncover a remarkable plasticity of functional centromeres in vivo: kinetochores tolerate profound changes in their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription. In addition, we discovered that de novo CENPA assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl switch. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENPA assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar effects were observed after tethering of CENPA deposition factors hMis18a or HJURP, to the alphoid array. In contrast, tethering of H3K9 tri-methylase (Suv39h1) to the array causes methylation of H3K9, preventing de novo CENPA assembly and kinetochore formation. HAT tethering bypasses the need for hMis18a, but not HJURP for de novo CENPA, CENPT, CENPI, and CENPE assembly at the ectopic site. CENPA arrays assembled de novo by this mechanism can form kinetochores of human artificial chromosomes (HACs) that are propagated indefinitely in human cells. HAC based vectors offer a promising system for delivery and expression of full length human genes of any size. 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 introduced a unique gene acceptor site into the synthetic tetO HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. In our recent work, we demonstrate 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 also show 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. It is a well established fact that the Pol III transcribed tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III transcribed genes exhibit barrier activity in mammals. Our recent results present evidence for a similar phenomenon in the mouse genome, which contains approximately 1000-times more putative RNA Pol III transcribed genes than the yeast genome. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells. Synthetic tRNA genes derived barrier elements as well as other known barrier elements are now being incorporated into a (HAC) based gene delivery system with a conditional centromere. Ultimately, we expect to develop a HAC based system that achieves consistent regulated expression of full size human genes, with minimal risk of uncontrolled epigenetic silencing of Pol II dependent transcription units.
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