Dissertation
A FUNCTIONAL TANDEM REPEAT VNTR2-1 IN HUMAN TELOMERASE GENE REGULATION
Washington State University
Doctor of Philosophy (PhD), Washington State University
01/2021
DOI:
https://doi.org/10.7273/000006444
Handle:
https://hdl.handle.net/2376/118962
Abstract
Repetitive DNA accounts for over half of the human genome, of which the function has not been fully understand. The introduction of long-read sequencing technology significantly improved the quality of genome sequencing. This allows us to take a closer look at those long DNA repeats which are also more prone to mutation and recombinational events. We have previously identified tandem repeat of 42-bp/unit, VNTR2-1, in intron 2 of the hTERT gene as a novel regulatory element important for hTERT transcription in cancer cells. hTERT gene, encoding the catalytic subunit of human telomerase, was found to be expressed in 90% of human tumors, which synthesizes telomere DNA and helps them escape from cellular senescence. In the current study, we found that repeats of 42-bp consensus sequence of VNTR2-1 activated the luciferase gene in a reporter plasmid. Mutation of predicted cis-regulatory elements within the 42-bp repeats, including an E-box motif, resulted in a partial or complete loss of its enhancer activity. MYC family proteins c-Myc, MAX regulated hTERT gene transcription through both VNTR2-1 and E-boxes at the proximal hTERT promoter, consistent with previous data from our laboratory. Chromatin segmentation analysis of published ChIP-sequencing data from K562 cells suggested VNTR2-1 was a bivalent enhancer. The hTERT promoter became repressed in a clone of telomerase-expressing human melanoma cell line MelJuSo with genomic deletion of VNTR2-1, as suggested by increases of H3K27me3 and H3K9me3 marks. Therefore, we provided additional evidence for VNTR2-1 as a functional regulatory element that regulated hTERT expression by MYC family transcription factors. Moreover, a CRISPR-mediated chromatin purification experiment was also performed in order to discover other proteins that interacted with VNTR2-1. We utilized its repetitive nature and delivered one single guide RNA that targets most 42-bp repeats, to capture all regulatory macromolecules bound to this region. The results we obtained here may help improve our knowledge on repetitive genomic DNA functions as well as the regulatory mechanism of human telomerase gene.
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Details
- Title
- A FUNCTIONAL TANDEM REPEAT VNTR2-1 IN HUMAN TELOMERASE GENE REGULATION
- Creators
- Xiaolu Zhu
- Contributors
- Jiyue Zhu (Advisor)Kathryn Meier (Committee Member)Lucia Peixoto (Committee Member)Shobhan Gaddameedhi (Committee Member)Weimin Li (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- College of Pharmacy and Pharmaceutical Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 98
- Identifiers
- 99900592056401842
- Language
- English
- Resource Type
- Dissertation