TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation

Abstract

The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes.

Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation

“…Assay of the 11 mammalian glycosylases indicated that TDG and UNG2 displayed strong reactivation activity. NEIL1 seemed to have a weaker reactivation activity than TDG, as reported in a previous study (25). Protein expression of NEIL2 and MUTYH was hard to detect.…”

Prolonged re-expression of the hypermethylated geneEPB41L3using artificial transcription factors and epigenetic drugs

“…53 Double stranded DNA oligos (BIO BASIC, Markham, Canada) coding for the two 6-finger ZFPs predicted to bind the target sequences (21ab: GCAA-CAGGGGGCGGGGGG, 22ab: GGGGAGGAAGCCG-CAGCC) were subcloned into the pMX-IRES-GFP containing either the gene activator VP64, no effector domain (NoEf) or the catalytic domain (CD) of Tet2 24 or TDG. 54 Tet2 and TDG DNA fragments were created by PCR (Phusion Hot Start II High-Fidelity DNA polymerase, Thermo Scientific) using construct-specific PCR primers flanked with MluI and PacI restriction sites on pcDNA3-Flag-TET2CD or TDG 55 and ligated into the pMX-IRES-GFP by sticky-end ligation with T4 ligase (Thermo Scientific).…”

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

“…Moreover, increasing literature has further emphasized the importance of NEIL1's cellular repair activity, as NEIL1 deficiency has led to multiple abnormalities and is associated with severe human diseases, including cancer (14-19). Additionally, emerging evidence has also implicated a role of NEIL1 in active DNA demethylation (20)(21)(22).NEIL1 is capable of removing a wide array of oxidized pyrimidines and purines; representative substrates of extensive investigations include thymine glycol (Tg), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC), dihydrothymine (DHT), and dihydrouracil (DHU), as well as the formamidopyridines (FapyA and FapyG), spiroiminodihydantoin (Sp), and guanidinohydantoin (Gh) (23-27). Among these lesions, Tg is the most common pyrimidine base modification produced under oxidative stress and ionizing radiation (28)-arising from oxidation of thymine and 5-methylcytosine-and is also a preferred substrate of NEIL1 (3).…”

“…Moreover, increasing literature has further emphasized the importance of NEIL1's cellular repair activity, as NEIL1 deficiency has led to multiple abnormalities and is associated with severe human diseases, including cancer (14)(15)(16)(17)(18)(19). Additionally, emerging evidence has also implicated a role of NEIL1 in active DNA demethylation (20)(21)(22).…”