CH391L/S2013 Hala Ouzon March 20 2013

'Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair' [1]

Background

Approximately 2% to 8% of cytosines in mammals are methylated (2). Methylation occurs mostly in CpG sites; 60%–90% of the CpG sites in mammals are methylated .Most of the remaining unmethylated residues are clustered in CpG islands within functional gene promoters (3). DNA methylation in promoter elements represses gene transcription. Some bacterial species use DNA methylation as an immune response by selectively degrading unmethylated foreign DNA.(2) The DNA methylation process is understood but not much is known about the demethylation processes. The level and pattern of 5-meC are determined by both DNA methylation and demethylation (3) For some genes, targeted or specific methylation by methyltransferases may be sufficient to create their methylation patterns, without the need for demethylases; for others, promiscuous methylation would need to be pruned by demethylases to generate the desired methylation pattern (2). DNA demethylation can be passive and/or active. Passive DNA demethylation occurs when maintenance methyltransferases are inactive during the cell cycle following DNA replication, which results in a retention of the unmethylated state of the newly synthesized strand. Active DNA demethylation involves one or more enzymes and can occur independently of DNA replication.

Intrudoction

This paper, by Corterllino et.al. , studied Thymine DNA glycosylase, a repair enzyme involved in active DNA demethylation. Using biochemical and developmental data they showed the important role this enzyme plays in maintaining proper DNA methylation patterns and suggested a two step mechanism for active DNA demethylation in mammals. Examples of active demethylation in non-mammalians include: in Arabidopsis: the base excision repair (BER) protein Demeter and ROS1(direct demethylasetion through glycosylase activity); in Xenopus: demethylation initiated by GADD45a in a process dependent on BER protein XPG; in zebra fish embryos: DNA demethylation occurs in two coupled steps, enzymatic 5mC deamination to thymine by activation-induced deaminase (AID) or Apobec2b, 2a, followed by removal of the mismatched thymine by the zebra fish thymine glycosylase MBD4, with Gadd45 promoting the reaction. Many studies documented the possible involvement of Thymine DNA glycosylase(TDG) in transcription and active demethylation. Indeed, TDG interacts with several transcription factors, including retinoic acid receptor (RAR), retinoid X receptor (RXR), estrogen receptor α (ERα) , thyroid transcription factor 1 (TTF1) ,and histone acetyl-transferases p300 and CBP which encouraged this group do carry on this study.

Methods

In this paper the authors wanted to investigate the functional role of TDG in epigenetic regulation, DNA demethylation, and mammalian development. To do that this group generated a Tdg null mouse and found that the removal of mispaired thymine in G:T mismatch is completely abrogated in Tdg-/- homozygous mouse which suggests that TDG is the predominant G:T mismatch repair enzyme in mouse embryo fibroblasts (MEFs)

Results

Whereas heterozygous Tdg+/− mice are viable and fertile and show no obvious phenotype, homozygosity for the null Tdg allele leads to embryonic lethality. Many features of the phenotype of Tdg null embryos have been previously described for either Cbp−/− or p300−/− embryos (two proteins required for endogenous gene transcription (4)) or those of embryos deficient in various Rar and Rxr genes. These data made this group conclude that the lethality phenotype is likely related to the inactivation of a developmentally relevant, transcription-related function of TDG. To investigate this further they established mouse embryo fibroblast (MEF) lines from Tdg null embryos and used these for the rest of the study.

This study shows that the activities of RAR /RXR ,CBP/p300, and many other transcription factors, were reduced in Tdg null MEFs. And that P300 only forms a complex with RAR and RXR in the presence of TDG. In addition TDG binds directly to the promoter of two differentially expressed RAR-RXR target genes, Crabp2 and Rbp1, from which the authors infer TDG’s role in transcription regulation.

Genes that showed different expression in Tdg null MEFs were examined for DNA methylation patterns at their promoters using sodiumbisulfite/DNA sequencing. The down-regulated genes contain a CpG island within 2 kb of sequence upstream of the transcriptional start site and are hypermethylated. This strongly indicates the involvement of TDG in protecting from hypermethylation during early development and raises the question of whether TDG has a role in demethylation. To answer this question this group studied a gene that is known to be demethylated at its CpG sites in liver during liver development and found that in Tdg null liver these sites are actually methylated. They also studied another gene that is known to be demethylated by single-strand nicks 3′ to the 5mC to find that in Tdg null MEFs it was methylated, suggesting that demethylation leads to a base excision repair pathway.

To further assist TDG’s role in active DNA methylation, this group studied the transcriptional reactivation of an in vitro-methylated Oct4 gene in cancer cells and in the same cells plus either shRNA(C8) which targets TDG or shRNA(C7) as a negative control. When the Oct4 promoter is demethylated by the action of TDG, EGFP reporters get expressed. They found that the EGFP reporter was expressed in parental cancer but not with C8 cells and that expression was evident within 12 hours of transfection. Because of this short time frame of demethylation , and since the reporter plasmid actually lacks an origin of replication, they were able to rule out the possibility of passive demethylation and confirm TDG’s involvement in active DNA demethylation. The second experiment that supported this conclusion was the design of a knockin mouse strain with a TDG mutation in the glycosylase site to find that indeed this dramatically affected survival and it was actually more severe than a Tdg knockout. This inferred that catalytic activity of TDG is indeed essential for development and DNA demethylation.

Other DNA glycoselases in mammals were also shown to carry out demethylation in a two step reaction where one enzyme would deaminate the 5mC to T and another would remove the mismatched T. To check whether TDG mediates DNA demethylation in a similar fashion, this group performed a Co-IP experiment on TDG and found that it forms a complex with AID (the enzyme that deaminates 5mC) and GADD45a (the enzyme that promotes the removal of T). They also checked the interactions of catalytically mutant TDG with these proteins to find that these interactions are maintained regardless of the mutation which might explain why the catalytic mutant phenotype is more severe then the knockout since mutant tdg forming these complexes might be sequestering AID and GADD45a in nonfunctional, nonproductive complexes.

By examining TDG catalytic activity on demethylation intermediates this group found that TDG shows no activity against 5hmC but very high activity against double stranded 5hmU.

The authors predicted that if TDG removes mismatched T, then in the absence of TDG one should see an increase in the G: C to A: T transition mutation. However, sequencing promoters of genes that undergo TDG-dependent protection from hypermethylation or DNA demethylation showed no transition mutation. They suggested that these results could be because of a possible coordination between deamination and glycosylation such that deamination does not occur in the absence of TDG.

References

1.Cortellino, S., et al., Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair. Cell, 2011. 146(1): p. 67-79.

2.Kasper, L.H., et al., CBP/p300 double null cells reveal effect of coactivator level and diversity on CREB transactivation. EMBO J, 2010. 29(21): p. 3660-72.

3.Tucker, K.L., Methylated cytosine and the brain: a new base for neuroscience. Neuron, 2001. 30(3): p. 649-52.

4.Zhu, J.K., Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet, 2009. 43: p. 143-66.