Characterisation of Transcriptional Regulation of the Human Telomerase RNA Gene

Zhao, Jiangqin (2000) Characterisation of Transcriptional Regulation of the Human Telomerase RNA Gene. PhD thesis, University of Glasgow.

Abstract

The human telomerase core enzyme consists of an essential structural RNA (hTERC) with a template domain for telomeric DNA synthesis and of a catalytic protein (hTERT) with reverse transcriptase activity. Expression of the hTERC and hTERT are essential for telomerase activity. Variation in telomerase activity is correlated with cellular senescence and tumour progression. Recent studies indicate that the regulation of telomerase activity is multifactorial in mammalian cells. The primary mode of control of hTERT appears to be transcriptional regulation but very little is known about the molecular mechanisms involved in the regulation of hTERC transcription. In this study, I have cloned and characterised the genomic sequences and promoter of the hTERC gene to provide evidence that transcriptional mechanisms are involved in hTERC gene regulation. Transient transfection with a series of 5'-deletion mutants demonstrated that between -5.0 kb and -51 by of the hTERC gene is responsible for high promoter activity, the minimal promoter region was defined as 176 by (-107 to +69 bp). With the aid of in vitro DNase I footprinting, electrophoretic mobility shift assays (EMSAs) and mutagenesis analysis, four Sp1 binding sites and one CCAAT-box bound by the transcription factor NF-Y were identified to be involved in regulation of hTERC transcription. Co-transfection experiments showed that Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. Mutation of the CCAAT-box or the coexpression of a dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by pRb and Sp1, suggesting that NF-Y binding is a prerequisite for pRb and Sp1 to activate the hTERC promoter. The different transcriptional regulators appear to act in a species-specific manner. Whilst Sp1 and Sp3 act on the human, bovine and mouse TERC promoters, pRb activates only the human and bovine promoter and NF-Y is important for the human TERC gene. The hTERC gene is expressed during embryogenesis and then down-regulated during normal development but is re-expressed in tumour cells, the hTERC promoter activity was therefore further investigated and a higher promoter activity in immortal cells than in two mortal cell strains (WI38 and IMR90) was shown. In conclusion, hTERC promoter contains sequence elements that allow interactions with several different transcription factors. The interplay between NF-Y, pRb, Sp1 and Sp3 within the architecture of the hTERC promoter may combine to enable a wide variety of cell types from mortal to immortal to regulate hTERC expression through transcriptional control.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Nicol W Keith
• Keywords: Biochemistry
• Date of Award: 2000
• Depositing User: Enlighten Team
• Unique ID: glathesis:2000-76131
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 16:36
• Last Modified: 19 Nov 2019 16:36
• URI: https://theses.gla.ac.uk/id/eprint/76131