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mace:id
Technology # Array version
Species
Organ
Tissue
Physiopathology
Type
Name

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User name	helene leger
Email	leger@ihes.fr
Phone / Fax number	+33(0)160926683 /
Location	IHES (systems epigenomics group) - 35 route de chartres - 91440 bures-sur-yvette, France

Scientific description

Because it excises thymine from Gâ€¢T mismatches, TDG was proposed to counter mutagenesis by 5-methylcytosine deamination. Yet, TDG was also observed to attack 5-methycytosine itself, making it a candidate DNA demethylase, and interactions with transcription factors implicated additional functions in gene regulation. Unlike other
DNA glycosylases, TDG is essential for embryonic development.
Fibroblasts from Tdg null embryos show massively impaired gene regulation, and this correlates with imbalanced histone modification and CpG methylation. TDG associates with the promoters of affected genes in MEFs and in embryonic stem cells, but epigenetic aberrations appear only in differentiated cells. TDG also contributes to the
maintenance of active and bivalent chromatin during cell differentiation, using its DNA glycosylase activity to counter aberrant de novo methylation. Thus, TDG dependent DNA repair stabilizes epigenetic states during cell differentiation.

Cortazar et al., Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature 470, 419-423 (2011)

Technical description

For gene expression analysis of ESCs and in vitro differentiated neuronal progenitor cells, total RNA was extracted from independent triplicates using the Trizol reagent (Invitrogen). RNA was quantified using the Quant-iT RiboGreen RNA Assay (Invitrogen) and 500 ng of total RNA subjected to cDNA synthesis and subsequent in vitro transcription to biotiylated cRNA using the Illumina. 

Total Prep RNA Amplification Kit (Ambion, USA). cRNA (1.5 mg) was hybridized to MouseWG-6v2 slides (Illumina) according to the manufacturerâ€™s protocol.
Bead arrays were washed and stained using FluoroLink Cy3 Streptavidin (GE Healthcare). Fluorescent signals were imaged using the iScan system (Illumina).
Scanner images files were processed to probe intensity files by the manufacturerâ€™s software and further processed with the genome studio software (Illumina) without normalization and background correction.

Affymetrix data and Illumina probe intensity data were either processed by robust multi-array average or variance stabilization transformation, respectively, using R/Bioconductor software and â€˜affyâ€™ or â€˜lumiâ€™ libraries, followed by quantile normalization. Significance of effects for probes (Illumina) or probe-sets (Affymetrix) was tested in R/Bioconductor (â€˜limmaâ€™ library) using a moderated t-test and the false discovery rate (55%) method of Benjamini andHochberg formultiple testing correction. No unspecific filter was applied and multiple probe-sets per gene or probe-sets with ambiguous genomic targets were retained.

Comparison of Tdg-/- and Tdg+/- ES cells and respective in vitro differentiated neuronal progenitors. 
Each condition is represented by 3 replicas.

References

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/21278727

Cortazar et al., Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature 470, 419-423 (2011)