Mechanistic study of the effects of 8-oxo-7,8-dihydroguanine (8-oxoG) on reporter gene expression in mammalian cells

DNA damage causes replication errors, leading to genetic instability or cell death. Besides that, many types of DNA base modifications have been shown to interfere with transcriptional elongation if they are located in the transcribed DNA strand of active genes, acting as roadblocks for RNA polymerases. It is widely assumed that transcription blockage by endogenous DNA damage is responsible for the early cell senescence in organs and accelerated ageing observed in individuals with compromised nucleotide excision repair.rnThe aims of this work were to design new experimental systems for testing transcription blocking potentials of DNA base modifications in an individual gene and to apply these test systems to the investigation of the effects of a frequent endogenously generated base modification, namely 8-oxo-7,8-hydroxyguanine (8-oxoG), on the gene transcription in cells. Several experimental strategies were employed for this purpose. First, I constructed an episomal vector encoding for a short-lived EGFP-ODC fusion protein and measured expression of the reporter gene in permanently transfected clonal cell lines exposed to DNA damaging agents. Second, the expression of plasmid-borne EGFP gene damaged with photosensitisers to obtain one or several oxidative purine modifications per plasmid molecule was determined in transiently transfected human and mouse host cells in an approach known as “host cell reactivation”. As a prerequisite for these experiments, a robust method of precise quantitative measurement of the EGFP gene expression in transiently transfected cells by flow cytometry was developed and validated. Third, I elaborated a very efficient procedure for insertion of synthetic oligonucleotides carrying 8-oxoG into plasmid DNA, avoiding any unwanted base damage and strand breaks. The consequences of 8-oxoG placed in defined positions in opposing DNA strands of the EGFP gene for transcription were measured by host cell reactivation in cells with functional 8-oxoguanine DNA glycosylase (OGG1) gene and in OGG1 null cells.rnThe results obtained in Ogg1-/- cells demonstrated that unrepaired 8-oxoG, even if situated in the transcribed DNA strand, does not have any negative effect on the reporter gene transcription. On the other hand, as few as one 8-oxoG was sufficient to cause a significant decrease of the gene expression in OGG1-proficient cell lines, i.e. in the presence of base excision repair. For two analysed positions of 8-oxoG in the plasmid DNA, the inhibition of gene transcription by the base modification correlated with the efficiency of its excision by purified OGG1 protein under cell-free conditions. Based on these findings, it has to be concluded that the observed decrease of transcription is mediated by excision of the base modification by OGG1 and probably caused by the repair-induced single-strand breaks. The mechanism of transcription inhibition by 8-oxoG is therefore clearly distinct from stalling of elongating RNA polymerase II complexes at the modified base.

Identification of target genes of the T-box proteins in Drosophila melanogaster

In a prior bioinformatic analysis by Hüyseyin Binbas, potential Tbx targets sequences in wing-related genes have been identified. Guided by this information, enhancer trap/reporter lacZ insertions were characterized by X-gal staining first in wildtype and then in l(1)omb imaginal discs.rnIn several lines I observed an increase in reporter expression in a l(1)omb mutant background. Since Omb is assumed to function predominantly as a transcriptional repressor, this may indicate direct regulation. Repression by Omb was observed e.g. for brk and tkv. These genes are negatively regulated by Dpp, while omb is induced by Dpp. Omb which mediates the effects of Dpp on proliferation could, thus, also mediate the Dpp effect on patterning of the wing disc. However, brk and tkv were not completely derepressed in l(1)omb indicating that Dpp represses these genes also by an Omb-independent mechanism.rnMore frequently I observed loss of reporter expression in an l(1)omb mutant background. In these cases, regulation by Omb presumably is indirect. For example, STAT92E-lacZ expression in the wildtype eye was symmetrically expressed at the dorsal and ventral margins. In l(1)omb, ventral expression was selectively lost. Loss of omb is known to cause ventral overproliferation of the eye by activation of the Jak/STAT pathway. STAT92E expression is negatively regulated by Jak/STAT signaling suggesting that loss of omb activates Jak/STAT further upstream in the pathway.rnRegional overproliferation of eye and wing in the l(1)omb mutant background proved a complicating issue in the search for Omb targets. This effect made it difficult to decide whether an expanded reporter expression pattern was due to tissue expansion or reporter gene derepression. For instance hth-lacZ appeared to expand along the ventral eye disc margin in l(1)omb. Without addtional experiments it cannot be concluded whether this is due to de-repression or to activation in association with the proliferative state. Parallel to my experiments, evidence accumulated in our laboratory that loss of omb may attenuate Wg and Hegehog signaling. Since these diffusible proteins are the main patterning molecules in the wing imaginal disc, with dpp being downstream of Hh, many of the observed effects could be secondary to reduced Wg and Hh activity. Examples are ab-lacZ, Dll-lacZ and vgBE-lacZ (reduced expression on the dorso-ventral boundary) and inv-lacZ (late larval expression in the anterior wing disc compartment is lost) or sal-lacZ. Epistasis experiment will be required to clarifiy these issues.rnFurthermore, loss of omb appeared to induce cell fate changes. It was reported previously that in an omb null mutant, the dorsal determinant apterous (ap) is ectopically expressed in the ventral compartment (an effect I did not observe with the strongly hypomorphic l(1)omb15, indicating strong dose dependence). Ventral repression of ap is maintained by epigenetic mechanisms. The patchy and variable nature of ectopic expression of ap or grn-1.1-lacZ points to an effect of omb on epigenetic stability.rnIn the second part of my thesis, an analysis of Omb expression in the Drosophila embryonic ventral nervous system was performed. Omb was found co-expressed with Eve in the medial aCC and RP2 motorneurons as well as the fpCC interneuron and the mediolateral CQ neurons. Additionally, Omb was detected in the Eg positive NB7-3 GW serotonergic motoneuron and the N2-4 neurons. Omb was not found in Repo positive glial cells. During embryonic stage 14, Omb showed some coepression with Dpn or Pros. At the embryonic stage 16, Omb was expressed in minor subset of Mid and Wg positive cells.