Thesis title: Quantification of Telomerase Complex Proteins and Transcriptional Regulation of Tert
Human telomerase complex consist of the telomerase reverse transcriptase protein (hTERT) and telomerase RNA (hTR) and other associated molecules including Dyskerin 1, NOP10, GAR1, NHP2 and Tcab1. Amongst these, hTERT is the most important factor because during differentiation of a stem cell to the differentiated cell, hTERT is transcriptionally silenced, whereas levels of other telomerase components mostly remain unchanged. Moreover, hTERT has been shown to have non-telomeric roles like increasing cell proliferation, cell cycle regulation by binding to promoters and acting as a co-transcription factor. Therefore, determining hTERT amount in a cell is important to recapitulate telomerase activity as well as to regulate cell cycle and proliferation. Telomerase is re-activated in ~90% of the cancers and re-activation of the hTERT gene is the key mechanism. In the first part of my study, to understand the minimum requirement for the telomerase activity as well as non-telomeric functions, I have quantified hTERT molecule and other telomerase complex molecules in cancer cells. Our results indicated that there were at least ~650 hTERT molecules per cell to perform telomeric and non-telomeric functions in the cell. Moreover, other telomerase components were found in different ratios suggesting that each protein can work cooperatively for telomeric functions and also function individually for non-telomeric processes.
In the second part of my study I investigated the molecular mechanism of TERT reactivation in cancers where TERT expression is driven due to mutation in TERT promoter. Cancer-specific hTERT romoter mutations are observed in melanoma, glioblastoma, urothelial cancers and hepatacellular carcinoma with high frequencies. These mutations create ETS family binding motif and are, generally, associated with increase hTERT transcription. GABPA, one of the ETS family members, has been shown to bind to the mutant hTERT promoter. However, the mechanism of increased hTERT expression is yet to be understood. Recently, GABP has been shown to be associated with Topology Associated Domains in breast cancer, suggesting that GABP may take a role for the regulation of the genes in long distances. Therefore in the second part of the study, I investigated the epigenetic mechanisms including long-range interactions and histone marks enrichment of the mutant and wild-type hTERT promoter. In order to identify the effect of the mutation, I performed CRISPR/Cas9 genome editing in the mutant melanoma and glioblastoma cell lines and converted the mutant base to wild-type. Eventually, I generated isogenic cell lines which were different only at one nucleotide position. Our results revealed that mutant hTERT promoter formed a long-range interaction with a region located ~300kb upstream (T-INT1) (Chr5:1,556,087-1,558,758) of itself. Interestingly, this region harbors multiple GABPA motifs and I observed cooperative binding of GABPA proteins in mutant hTERT promoter and T-INT1 region. Our results revealed that long-range interaction between mutant hTERT promoter and T-INT1 region through GABPA, abolished auto-inhibition function of GABPA and increased the stability in hTERT promoter. Subsequently, active histone marks were enriched and BRD4 chromatin remodeling protein was recruited to the promoter. Finally, all these epigenetic changes recruited RNA polymerase 2 to the promoter for the transcription of the hTERT gene.
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