Investigating the mechanisms of telomere maintenance in zebrafish tissues and human brain cancer cell lines
Author(s)
Advisor
Adams, Michelle MarieDate
2019-09Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
Telomeres are nucleoprotein complexes formed at each end of the chromosomes to
protect these ends from deterioration. In each round of cellular division, telomeric
sequences shorten due to the end replication problem of DNA polymerase. Progressive
telomere shortening results in replicative senescence in healthy somatic cells. To evade
replicative senescence, cells need to maintain their telomere length either by activating
the telomerase enzyme or through the alternative lengthening of telomeres (ALT).
Telomerase is a holoenzyme, which is composed of dyskerin, telomerase RNA subunit
(TR or TERC), and telomerase catalytic subunit (TERT). Dyskerin and TR are
constitutively expressed in all cells but TERT expression is silenced in adult somatic
cells. Thus, telomerase activity is dependent on the expression of TERT. Current
studies show that TERT re-activation is a common feature of cancer cells and 85-90%
of cancers utilize telomerase enzyme in maintaining telomeres to become immortal.
Remaining of cancer cells maintain their telomeres by the alternative lengthening of
telomeres (ALT), which is a DNA repair pathway dependent mechanism. Current
models suggest that ALT is achieved by homology-directed DNA repair, through the
interaction of multiple proteins. DNA methylation is regarded as a key player in epigenetic silencing of transcription.
DNA methyltransferase inhibitors are currently being used in cancer treatments.
Recent studies show that DNA methyltransferases and their expression levels impact
both telomerase- and ALT mediated lengthening of telomeres, and have different
outcomes in different tissue types.
In this study, we worked on the zebrafish brain and human brain cancer cell lines. In
zebrafish brain, we observed differences in methylated regions at Sp1 binding site
between young and old that can be associated with telomere shortening. By silencing
DNMT1 and DNMT3B in brain cancer cell lines, we investigated the changes in gene
expression levels of telomerase and ALT related genes, telomerase activity, population
doubling time and replicative senescence status. To further investigate TERT
regulation, we introduced mutations to the Sp1 binding sites in the promoter region
and measured the promoter activity with luciferase assay. Our results show that Sp1
methylation sites in the telomerase promoter region are critical in brain aging,
dependent on their position. We propose a therapeutical option for brain aging and
tumorigenesis.
Keywords
AgingZebrafish
Brain cancers
Telomerase
TERT
ALT
Sp1
Gene expression
qTRAP
Site-directed mutagenesis