Human Artificial Chromosomes (HACs) assembled from alpha-satellte 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. We previously constructed a synthetic HAC (tetO-HAC) allowing tethering of its 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 or tTA, 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. We demonstrated that this HAC can be transferred and maintained in human iPSCs as autonomous chromosome without effecting pluripotent properties of the cells. In separate experiments, a platform with multi-integrase recombination sites has been inserted into tetO-HAC and has been successfully used for a gene assembly in the HAC. A new tetO-HAC system has several notable advantages that set it apart from other artificial chromosome-based systems, including assembly of unlimited number of genomic DNA segments and the opportunity to remove mis-incorporated DNA segments. Work is in progress to use multi-integrase system for assembling of synthetic nucleolar organizer region (NOR) in the HAC from human rDNA units isolated by transformation-associated recombination (TAR). Such a HAC module will be used to investigate the requirements for nucleolar location of rDNA repeats and effect of copy number of rDNA units on cell proliferation and stress response. Note that despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. In our recent work, we recovered ribosomal DNA segments from a single human acrocentric chromosome (chromosome 21) using TAR cloning in yeast. Accurate long-read sequencing of TAR-isolated units from the chromosome revealed substantial variation among tandem repeat rDNA copies and several palindromic structures. These clones revealed few hundred variant positions in the 45S transcription unit and in the intergenic spacer sequence. The large number of variants observed provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human diseases. Even after the completion of the human genome sequence few rDNA-containing BAC clones were sequenced and the genomic structure of entire rDNA clusters on human chromosomes 13, 14, 15, 21 and 22 are still unknown and these regions represent large gaps in the current genomic assembly. So far we do not know organization and divergence of rDNA units within individual NORs. To address this question, during the past year, we are focused on analysis of an individual NOR using TAR cloning strategy developed in our lab. This work resulted in assembly of the entire NOR sequence on human chromosome 22 along with long flanking proximal and distal sequences. Thus, we succeeded to close one of the gaps on acrocentric chromosomes. The loading of different variants of a human rDNA unit identified in our study into the tetO-HAC may help to clarify the peculiarity of PolI transcription machinery as well as the mechanism by which rDNA units are selected and targeted for chromatin changes leading to heterochromatinization in NORs. This work is now in progress. 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 themfrom 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 non-essential 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. The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In our recent work, we applied our HAC system to screen for, and rank the efficacy of, compounds specifically targeting telomeres and telomerase. The study revealed dozen compounds that selectively target telomerase or telomeres. Cytological analysis showed that chromosome instability after the drug treatment correlated with the induction of telomere-associated DNA damage. New compounds that greatly increase CIN may expedite the development of new therapeutic strategies for cancer treatment. In the vast majority of human tumors the molecular basis of CIN remains unknown, partially due to not all genes controlling proper chromosome transmission having been identified yet. We demonstrated the utility of the HAC-based assay for identification of new genes controlling chromosome transmission in human cells. In our recent work, we modified EGFP-HAC and converted the original assay into high-throughput CIN screen of chemical libraries and siRNA libraries of human genes. Analysis of siRNAs targeting each of 720 human protein kinase genes revealed new six CIN genes with no previous information on their role in chromosome transmission. This assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, transcription factors) to identify other genes involved in CIN. Each of these new CIN genes may be considered as a new target for cancer therapy. During the past year, we also worked on the development of a new strategy to built HACs with a functional kinetochore. All prior HACs required large arrays of alpha-satellite DNA repeats for their construction. Bypassing of this rule would have several clear benefits. First, HAC construction would be greatly facilitated. Traditional constructs for HAC formation contain 50-200 kb of highly repetitive DNA complicating handling at all steps, from their initial construction to their clonal stability during bacterial propagation. Second, mapping the chromatin features of HACs using sequencing-based approaches would become possible. In our recent work, we report the development of a type of HAC that functions independently of these constraints. Specifically, we demonstrated that human chromosomal regions corresponding to neocentromeres are competent in de novo kinetochore formation. Importantly, that these regions contain no any centromeric repeats. Thus our study reveals molecular requirements for centromere establishment and demonstrates that alpha-satellite DNA can be bypassed altogether, thereby greatly facilitating the construction of HACs and expending the toolbox for centromere biology studies, gene therapy applications and synthetic biology efforts.