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Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability

Abstract

Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA1. However, TDG was also found to interact with transcription factors2,3, histone acetyltransferases4 and de novo DNA methyltransferases5,6, and it has been associated with DNA demethylation in gene promoters following activation of transcription7,8,9, altogether implicating an engagement in gene regulation rather than DNA repair. Here we use a mouse genetic approach to determine the biological function of this multifaceted DNA repair enzyme. We find that, unlike other DNA glycosylases, TDG is essential for embryonic development, and that this phenotype is associated with epigenetic aberrations affecting the expression of developmental genes. Fibroblasts derived from Tdg null embryos (mouse embryonic fibroblasts, MEFs) show impaired gene regulation, coincident with imbalanced histone modification and CpG methylation at promoters of affected genes. TDG associates with the promoters of such genes both in fibroblasts and in embryonic stem cells (ESCs), but epigenetic aberrations only appear upon cell lineage commitment. We show that TDG contributes to the maintenance of active and bivalent chromatin throughout cell differentiation, facilitating a proper assembly of chromatin-modifying complexes and initiating base excision repair to counter aberrant de novo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells.

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Figure 1: Embryonic essential function of Tdg in epigenetic gene regulation.
Figure 2: Complementation of the loss of gene expression depends on the chromatin state of the promoter.
Figure 3: TDG-dependent differences in gene expression and chromatin status arise during cell differentiation.
Figure 4: Structural and catalytic functions of TDG in epigenetic maintenance.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Gene expression array data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE20693.

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Acknowledgements

We thank D. Klewe-Nebenius for preparations of mouse primary fibroblast, and F. Mohn and D. Schübeler for discussions and assistance in the setup and evaluation of the ChIP experiments. The work was supported by project grants from the Swiss National Science Foundation (3100A0-108436; 3100A0-122574/) and the Association For International Cancer Research (01-330).

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D.C. established and performed the ChIP and MeDIP analyses and the in vitro differentiation experiments, and contributed to writing the paper; C.K. established and characterized MEF lines, designed and performed gene expression and DNA methylation analyses, and contributed to writing the paper; J.S. did blastocyst injections, established the first heterozygous Tdg knockout mice, characterized the lethal phenotype of the Tdg null embryos and provided SV40-immortalized MEFs; T.L. and Y.S. generated Tdg-targeting constructs and established heterozygous and homozygous Tdg knockout ES cell lines; Y.S. established in vitro differentiation protocols; E.MacD. performed the Big Blue mutation assays; A.W. performed ChIP experiments; D.S. isolated primary MEFs and performed RT–PCR validations of gene expression differences and the PARP inhibitor experiments; A.L.J. established and performed immunofluorescence experiments including XRCC1 foci analyses; F.S. performed bioinformatic analyses of gene expression array data; R.S. affinity-purified anti-TDG antibodies for ChIP; J.J. contributed genomic Tdg clones and supervised initial work of T.L.; A.B. was involved in study design (mutation analyses) and supervised the work of J.S. and E.MacD.; P.S. designed, coordinated and supervised the study, analysed the data and wrote the paper. All authors discussed the results and commented on the paper.

Corresponding author

Correspondence to Primo Schär.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-11 with legends and Supplementary Tables 1-4. This file was replaced on 07 October, 2011. (PDF 6270 kb)

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Cortázar, D., Kunz, C., Selfridge, J. et al. Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature 470, 419–423 (2011). https://doi.org/10.1038/nature09672

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