Transcription Factors and DNA Repair Enzymes Compete for Damaged Promoter Sites

Abstract

Transcriptional regulation is a tightly regulated, vital process. The transcription factor cyclic AMP-response element-binding protein 1 (CREB1) controls ∼25% of the mammalian transcriptome by binding the CREB1 binding site consensus sequence (CRE) sequence (TGACGTCA). DNA lesions within CRE modulate CREB1 binding negatively and positively. Because appropriate DNA lesions also interact with base excision repair proteins, we investigated whether CREB1 and repair glycosylases compete with each other. We incubated 39-mer CRE-containing double-stranded oligonucleotides with recombinant CREB1 alone or with UNG2 or OGG1, followed by EMSA. The CpG islet within CRE was modified to contain a G/U or 8-oxoG (°G)/C mispair. OGG1 and CREB1 reversibly competed for CRE containing an °G/C pair. Also, OGG1 blocked CREB1 from dimerizing by 69%, even when total CREB1 binding was reduced only by 20-30%. In contrast, bound CREB1 completely prevented access to G/U-containing CRE by UNG2 and, therefore, to base excision repair, whereas UNG2 exposure prevented CREB1 binding. CREB1 dimerization was unaffected by UNG2 when CREB1 bound to CRE, but was greatly reduced by prior UNG2 exposure. To explore physiological relevance, we microinjected zebrafish embryos with the same oligonucleotides, as a sink for endogenous CREB1. As predicted, microinjection with unmodified or lesion-containing CRE, but not scrambled CRE or scrambled CRE with a G/U mispair, resulted in increased embryo death. However, only the G/U mispair in native CRE resulted in substantial developmental abnormalities, thus confirming the danger of unrepaired G/U mispairs in promoters. In summary, CREB1 and DNA glycosylases compete for damaged CRE in vitro and in vivo, thus blocking DNA repair and resulting in transcriptional misregulation leading to abnormal development.

Keywords: 8-Oxoguanine (8-oxoG); 8-Oxoguanine glycosylase (OGG1); CREB1 transcription factor; DNA base excision repair; DNA damage repair; DNA repair; G/U mispair; cAMP-response element-binding protein (CREB); development; embryo; glycosylase/transcription factor competition; transcription; uracil DNA glycosylase; zebrafish.