Download Epigenetics and DNA repair

Epigenetics and DNA repair
Is a chromatin remodeling process involved in the higher UV sensitivity of
nucleotide excision repair defective cells?
Marie Curie Project (FP7-IIF-221362/908362)
UVC irradiation induces two major DNA lesions: (I) cyclobutane pyrimidine dimers (CPDs) and (II)
pyrimidine-pyrimidine 6-4 photoproducts (6-4 PP). Nucleotide excision repair (NER) is one of the major
cellular pathways that remove the DNA lesions induced by UVC. Such lesions, if either unrepaired or
misrepaired, interfere with essential DNA metabolic activities resulting in mutation induction,
chromosomal instability and cell death (Friedberg et al., 1995).
The dramatic consequences of defective NER can be observed in the human hereditary diseases
Xeroderma Pigmentosum (XP) and Cockayne's Syndrome (CS). XP and CS patients are extremely
photosensitive and exhibit skeletal as well as neurological abnormalities. CS gene products are
components of a special sub-pathway of NER known as transcription coupled repair (TCR). CS cells are
then defective in the TCR pathway and are extremely sensitive to the cytotoxic effects of UVC irradiation
(Boer and Hoeijmakers, 2000). It has been demonstrated that CS cells are prone to form chromosomal
aberrations as well as to exhibit an enhanced apoptotic potential after UVC irradiation (De Santis et al.,
2001).
AA8 and UV61 are isogenic Chinese hamster cell lines which differ in TCR efficiency. AA8 is the
parental cell line, which is proficient in the genome overall repair of UVC radiation induced 6-4 PP and
CPDs from the transcribing strand of active genes. UV61 cells (the hamster homologue of human
Cockayne’s syndrome) originally isolated from AA8, exhibit proficient repair of 6-4 PP but are deficient
in CPDs removal by the TCR pathway.
TCR is responsible for the accelerated removal of DNA lesions from the transcribing strand of active
genes. Actively transcribing genes constitute 5-8% of the eukaryotic genome. The lesions repaired by
TCR in those regions might be very minimal as compared to the remainder of the genome. Therefore, it
can be expected only a slight increase in apoptosis and chromosomal aberrations in TCR defective UV61
cells as compared to AA8 cells. However, significant increase in the induction of both chromosomal
aberrations and apoptosis were observed in UV61 cells (De Santis et al., 2001).
Since in Chinese hamster ovary cells lines (CHO) the global genome repair (GGR) is not active, the
higher induction of chromosomal aberrations in CS cells seems to be associated with transcription
blockage (TCR failure) caused by UV-lesions located in the transcribed strands of the genome. However,
these lesions should not affect more than the 10% of the genome; therefore, it is proposed that the high
increase in the production of chromosomal aberrations in CS cells could be related to a more decondensed
chromatin in these cells after UVC exposure.
Preliminary experiments using the inhibitor of histone deacetylases trichostatin A (TSA), which produce
a chromatin decondensation, showed a significant increase of chromosomal aberrations in normal as well
as in CS cells treated with UVC in the presence of TSA. Since the formation of chromosomal aberrations
derives from the repair process of primary UVC induced lesions, it has been proposed to study the
influence of chromatin remodeling in the processing of UVC induced lesions in TCR proficient and
deficient cell lines. Several methodological approaches were applied to study cell survival and
proliferation with different doses of UVC and TSA to determine if any synergistic effect exists between
both treatments. Then, kinetics analysis of UVC-induced CPDs repair in the presence or absence of TSA
as well as the histone acetylation pattern along different recovery times after UVC irradiation were
carried out. Finally, chromatin status was studied employing different approaches based in nucleases
digestions on nuclei from treated and control cells to see whether there is any link between chromatin
remodeling and the higher UVC sensitivity observed in CS cells.