Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine.

Nadine Müller,Eveliina Ponkkonen,Andriy Khobta,Thomas Carell

doi:10.3390/ijms222011025

Abstract

Stepwise oxidation of the epigenetic mark 5-methylcytosine and base excision repair (BER) of the resulting 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) may provide a mechanism for reactivation of epigenetically silenced genes; however, the functions of 5-fC and 5-caC at defined gene elements are scarcely explored. We analyzed the expression of reporter constructs containing either 2′-deoxy-(5-fC/5-caC) or their BER-resistant 2′-fluorinated analogs, asymmetrically incorporated into CG-dinucleotide of the GC box cis-element (5′-TGGGCGGAGC) upstream from the RNA polymerase II core promoter. In the absence of BER, 5-caC caused a strong inhibition of the promoter activity, whereas 5-fC had almost no effect, similar to 5-methylcytosine or 5-hydroxymethylcytosine. BER of 5-caC caused a transient but significant promoter reactivation, succeeded by silencing during the following hours. Both responses strictly required thymine DNA glycosylase (TDG); however, the silencing phase additionally demanded a 5′-endonuclease (likely APE1) activity and was also induced by 5-fC or an apurinic/apyrimidinic site. We propose that 5-caC may act as a repressory mark to prevent premature activation of promoters undergoing the final stages of DNA demethylation, when the symmetric CpG methylation has already been lost. Remarkably, the downstream promoter activation or repression responses are regulated by two separate BER steps, where TDG and APE1 act as potential switches.

Highlights

Epigenetic regulation of the genome function is crucial for concerted realization of gene expression programs during development and for maintenance of the lineage-specific gene expression patterns in adulthood
Synthetic oligonucleotides containing all types of cytosine modifications were incorporated into the pGCbox-W vector, with efficiencies closely approaching 100% (Figure 1b,c)
Expression values showed an inverse relationship between the activities of the GC box box constructs containing base excision repair (BER)-resistant versus BER-sensitive modifications, wherein the constructs containing BER-resistant versus BER-sensitive modifications, wherein the expression was significantly enhanced by 20 -deoxy 5-caC compared to its 20 -fluorinated expression was significantly enhanced by 2′-deoxy 5-caC compared to its 2′-fluorinated counterpart in both the purine-rich (p = 1.2 × 10−3−3) and the pyrimidine-rich DNA strands counterpart in both the purine-rich (p = 1.2 × 10 ) and the pyrimidine-rich DNA strands (p = 7.2 × 10−4 ).−4 These results indicate that removal of 5-caC reactivated the GC box (p = 7.2 × 10 )

Summary

Introduction

Epigenetic regulation of the genome function is crucial for concerted realization of gene expression programs during development and for maintenance of the lineage-specific gene expression patterns in adulthood. During these processes, functions of the whole genome and of the individual genes are dynamically regulated by deposition and removal or maintenance of epigenetic marks, which include specific chromatin components as well as chemically modified DNA bases [1,2]. Functions of the whole genome and of the individual genes are dynamically regulated by deposition and removal or maintenance of epigenetic marks, which include specific chromatin components as well as chemically modified DNA bases [1,2]. The methyl mark sometimes acts by directly preventing transcription factor binding to their target sites [10,11,12]

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Discussion

Conclusion