Epigenetische Auswirkungen einzelner DNA-Basenmodifikationen
Zusammenfassung der Projektergebnisse
Results obtained during the second funding period contribute to understanding of regulation of gene transcription by DNA base modifications classified primarily as lesions (8-oxo-7,8-dihydroguanine, 5-hydroxymethyluracil) or regulatory marks (5-hydroxymethyl-, 5-formyl- and 5-carboxycytosine). Firstly, using synthetic DNA modifications specifically accommodated within the regulatory cis-elements (CRE and GC box), we characterised the impacts of single DNA modifications on the promoter activity. These effects were rather mild in CRE but very significant in GC box, where 5-carboxycytosine behaved as a strong repressive mark if present within the central CpG dinucleotide. Besides, we found that 8-oxo-7,8-dihydroguanine had positiondependent effects on GC box activity and, intriguingly, escaped base excision repair (BER) at the same positions, corroborating its putative function as a negative regulatory mark. Secondly, we investigated the role of BER by combining the reverse genetics (knockout of DNA glycosylases) and chemical biology (synthetic BER-resistant DNA lesions) tools. The results suggested that generation of apurinic/apyrimidinic (AP) lesions by DNA N-glycosylases provides a restriction point for subsequent decision whether the final repair outcome would be promoter activation or silencing. Thus, we found that the presence of an AP lesions does not compromise or even mildly enhances activities of the investigated cis-elements until the subsequent strand incision step takes place. Their conventional processing by a BER endonuclease (with all liklihood, APE1) would lead to promoter silencing. However, intrinsic variation or regulation of the APE1 efficiency (based on the DNA context, cell type or biochemical microenvironment) may contribute to highly variable transcriptional outcomes. Considering this, we questioned whether alternative pathways for processing of AP lesions exist in human cells and reported a significant contribution of nucleotide excision repair (NER) to the removal of AP lesions under physiological conditions. Importance of this finding is not limited to the maintenance of genetic stability. The results showed that, in contrast to APE1-mediated BER, NER does not lead to transcriptional silencing. Thereby, we propose that switching between the excision repair pathways acting on AP lesions may provide a decision mechanism for commitment of gene promoters to either activation or repression. Such a mechanism could be of extreme importance in the context of the epigenetic reprograming pathways, such as TET- and TDG-mediated active DNA demethylation.
Projektbezogene Publikationen (Auswahl)
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(2017). " Functional impacts of 5- hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter." Nucleic Acids Research 45(19): 11033-11042
Kitsera, N.; Allgayer, J.; Parsa, E.; Geier, N.; Rossa, M.; Carell, T.; Khobta, A.
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(2019). " Nucleotide excision repair of abasic DNA lesions." Nucleic Acids Research 47(16): 8537-8547
Kitsera, N.; Rodriguez-Alvarez, M.; Emmert, S.; Carell, T.; Khobta, A.
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(2021). "Regulation of GC box activity by 8-oxoguanine." Redox Biology 43: 101997
Muller, N. and Khobta, A.
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(2021). “Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis- Element in Response to 5-Formylcytosine and 5-Carboxycytosine.” International Journal of Molecular Sciences; 22(20):11025
Müller, N., Ponkkonen, E., Carell, T. and Khobta, A.
