Human Artificial Chromosomes for Cancer Research and Functional Genomics

点击翻译按钮获取中文摘要

HACs assembled from alpha-satellite 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, synthetic biology, 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. This HAC was successfully used to clarify a role of different types of chromatin in kinetochore function. During the past year, the same approach exploiting the tethering of the tetO-HAC kinetochore revealed that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory. We also demonstrated that CENP-B protein balances assembly of CENP-A centrochromatin or heterochromatin formation on satellite DNA. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of kinetochore. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the tetO-HAC. Previously, we have demonstrated functional expression of dozen genes loaded into the alphoidtetO-HAC. In recent study, we have developed a gene therapy model that employs the teO-HAC as a gene delivery vector in the treatment of the monogenic genetic disease hemophilia A in mice. It was demonstrated that the therapeutic HAC is maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. For gene expression/function analyses, a platform with multi-integrase recombination sites has been inserted into tetO-HAC. This platform allows assembly of unlimited number of genomic DNA segments in the HAC. Work is in progress to use this HAC for assembling of synthetic nucleolar organizer region (NOR) from human rDNA units and flanking regions recently isolated and characterized in our lab. It worth noting that despite the key role of rRNA genes, little is known about the extent of sequence variation in ribosomal DNA (rDNA). The rDNA clusters and flanking 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 human rDNA units within an individual NOR are only partially known. To address this lacuna, we first applied TAR cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Additional studies are required to clarify if some of the reported variations have functional consequences - for example, speculatively affecting the assembly of ribosomes; the selection or efficiency of translation of particular mRNAs; and/or the severity of "ribosomopathies" like Diamond-Blackfan anemia. It is worth noting that this is the first example of isolation of s YAC/BAC clones corresponding to rDNA array and long flanking sequences corresponding to PJ and DJ regions. tetO-HAC with multi-integrase recombination sites opens the opportunity to assemble the entire NOR of the chromosome 22. Such a HAC module that is transferable into any type of human cells will be used to investigate the role of NORs in nucleolar formation and function, and can initiate studies of the role of these regions in the well-known empirical association of nucleoli with pathology. We have also applied our tetO-HAC for measuring chromosome instability (CIN) in human cells. 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 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 300 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. In recent study the HAC-based assay was applied for analysis of newly developed microtubule-stabilizing compounds. The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In our recent work, we designed a new protocol to screen for, and rank the efficacy of, compounds specifically targeting telomeres and telomerase. The protocol is based on the use of two isogenic cell lines containing a circular HAC (lacking telomeres) and a linear HAC (containing telomeres): compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, we found several compounds (derivatives of Pt ttpy) that induce a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Cytological analysis showed that chromosome loss after drugs treatment correlated with the induction of telomere-associated DNA damage. Identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction 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 because not all genes controlling chromosome transmission have yet been identified. Indeed, 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. To address this problem, we have developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses tetO-HAC expressing the GFP transgene. When this assay was applied to screen a siRNA library of protein kinases we identified 6 new genes whose deletion promotes CIN. At present, the HAC-based assay is applied for screening the Ambion collection of different siRNAs libraries (cell cycle regulation, DNA damage response, epigenetics, transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells. .