DNA modifications repaired by base excision repair are epigenetic

Abstract

CREB controls ∼25% of the mammalian transcriptome. Small changes in binding to its consensus (CRE) sequence are likely to be amplified many fold in initiating transcription. Here we show that DNA lesions repaired by the base excision repair (BER) pathway modulate CREB binding to CRE. We generated Kd values by electrophoretic mobility shift assays using purified human CREB and a 39-mer double-stranded oligonucleotide containing modified or wild-type CRE. CRE contains two guanine residues per strand, one in a CpG islet. Alterations in CRE resulted in positive or negative changes in Kd over two orders of magnitude depending on location and modification. Cytosine methylation or oxidation of both guanines greatly diminished binding; a G/U mispair in the CpG context enhanced binding. Intermediates in the BER pathway at one G residue or the other resulted in reduced binding, depending on the specific location, while there was no change in binding when the single G residue outside of the CpG islet was oxidized. CREB recruits other partners after dimers form on DNA. Only UpG increased DNA.CREB dimer formation. Since oxidation is ongoing and conversion of cytosine to uracil occurs spontaneously or at specific times during differentiation and development, we propose that BER substrates are epigenetic and modulate transcription factor recognition/binding.

Keywords: (hm)C; (hm)U; (m)C; 5-hydroxylmethyluracil; 5-methylcytosine; 8-oxo-7,8-dihydroguanine; AID; AP; APEX1; ATF-1, ATF-2; BER; CRE; CREB; CREM; Chromatin; DNA base excision repair; DNA–protein interaction; Epigenetics; OGG1; RNS; ROS; THF; Transcription factors; abasic; activating transcription factor-1,-2; activation induced deaminase; base excision repair; cAMP response element; cAMP response element modulator; cyclic AMP response element binding protein 1; human AP endonuclease 1; hydroxymethylcytosine; oxoguanine glycosylase; reactive nitrogen species; reactive oxygen species; tetrahydrofuran; °G.