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Technology # Array version
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Tissue
Physiopathology
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User name	Sebastian Eilebrecht
Email	eilebrecht@ihes.fr
Phone / Fax number	+491755662397 /
Location	IHES (Systems Epigenomics Group) - 35, route de Chartres - 91440 Bures sur Yvette, France

Scientific description

The Thymine-DNA Glycosylase (TDG) is a multifunctional enzyme and is essential for embryonic development. It mediates the base excision repair (BER) of G:T and G:U DNA mismatches arising from the deamination of 5-methyl cytosine (5-MeC) and cytosine, respectively. Recent studies have pointed at a role of TDG during the active demethylation of 5-MeC within CpG islands. TDG interacts with the histone acetylase Creb Binding Protein (CBP) to activate CBP-dependent transcription and with the Retinoic Acid Receptor Î± (RARÎ±), resulting in the activation of RARÎ± target genes. Here we provide evidence for the existence of a functional tertiary complex containing TDG, CBP and activated RARÎ±. Using global transcriptome profiling, we uncover a coupling of de novo methylation-sensitive and retinoic acid-dependent transcription, which coinsides with a significant subset of CBP target genes. The introduction of a point mutation in TDG, which neither affects overall protein structure nor BER activity, leads to a significant loss in tertiary complex stability, resulting in the deregulation of retinoic acid targets involved in cellular networks associated to DNA replication, recombination and repair. We thus demonstrate for the first time a direct coupling of TDG's epigenomic and transcription regulatory function through tertiary complexes with CBP and RARÎ±.

Technical description

Microarray analyses, RNA amplification, labeling, hybridization and detection were performed following the protocols supplied by Applied Biosystems using the corresponding kits (Applied Biosystems). The microarray data were extracted using the bioconductor limma package [1] and median normalized. Data quality was determined using a QC procedure [2]. Data were normalized using NeONORM with k=0.02 [3,4,5]. Subtraction profiling was performed as in [6,7] using the CDS test [8]. 

1. Smyth, G.K., Michaud, J. and Scott, H.S. (2005) Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics}, 21, 2067-2075.

2. Brysbaert, G., Pellay, F.X., Noth, S. and Benecke, A. (2010) Quality assessment of transcriptome data using intrinsic statistical properties. Genomics Proteomics Bioinformatics, 8, 57-71.

3. Noth, S., Brysbaert, G. and Benecke, A. (2006) Normalization using weighted negative second order exponential error functions (NeONORM) provides robustness against asymmetries in comparative transcriptome profiles and avoids false calls. Genomics Proteomics Bioinformatics, 4, 90-109.

4. Noth, S., Brysbaert, G., Pellay, F.X. and Benecke, A. (2006) High-sensitivity transcriptome data structure and implications for analysis and biologic interpretation. Genomics Proteomics Bioinformatics, 4, 212-229.

5. Noth, S. and Benecke, A. (2005) Avoiding inconsistencies over time and tracking difficulties in Applied Biosystems AB1700/Panther probe-to-gene annotations. BMC Bioinformatics, 6, 307.

6. Eilebrecht, S., Benecke, B.J. and Benecke, A. (2011) 7SK snRNA-mediated, gene-specific cooperativity of HMGA1 and P-TEFb. RNA Biol, 8, 1084-1093.

7. Eilebrecht, S., Becavin, C., Leger, H., Benecke, B.J. and Benecke, A. (2011) HMGA1-dependent and independent 7SK RNA gene regulatory activity. RNA Biol, 8, 143-157.

8. Tchitchek, N., Dzib, J.F., Targat, B., Noth, S., Benecke, A. and Lesne, A. (2012) CDS: a fold-change based statistical test for concomitant identification of distinctness and similarity in gene expression analysis. Genomics Proteomics Bioinformatics, 10, 127-135.