Fig. 2: Epigenetic regulation of the Gilz gene (Tsc22d3) by DNA methylation in the second (F2) and third (F3) generations.

Genomic structure of the Tsc22d3 mouse gene (a). Each gray box represents exons of the Tsc22D3 gene. Black arrows represent the 2 promoters regulating the transcription of the Gilz isoforms, named P1 and P2. The region upstream of P2 contains 6 half-site glucocorticoid responsive elements (GREs) represented by black stars. The alternative splicing of exon 3 allows the transcription of Gilz isoform 2, translated as protein variant 2, which is responsible for water and sodium reabsorption in the kidney. We focused on the region upstream of exon 3, regulating the transcription of Gilz’s isoform 2 (b). Zero has been arbitrarily defined as the first base of exon 3. The 6 GRE half-sites are represented as described above, as well as the 12 pairs of primers used to amplify the P2 region (see below). Relative methylation profile of control (c) and preterm (d) male offspring at 6 months of age (M6). Methylation profiles were determined using amplification of methylated DNA with 12 pairs of primers in the region upstream of exon 3. Methylated DNA has been immunoprecipitated by a MeDIP technique; the results presented are the mean of the percent of methylated DNA compared to the input, normalized to the TSH2B-positive control gene for each primer pair, represented according to its position (in base pairs) in the P2 region. Gilz methylation index in control and preterm male offspring at M6 (e) in arbitrary units. The Gilz methylation index was determined by calculating the area under the curve (AUC) from the methylation profiles for each mouse. Each mouse in the control group is represented by a gray dot, and each mouse in the premature group is represented by a black square, with mean and SD for each group. Nonparametric Mann–Whitney U-tests. Correlations between the Gilz methylation index and Gilz renal mRNA expression in the F2 and the F3 generations (f) were obtained by Spearman regression analysis. MeDIP was performed by pooling samples from the F2 and F3 generations (after ensuring that the results were consistent in both generations), with n = 8 in the preterm group and n = 9 in the control group. *P < 0.05, **P < 0.01, ***P < 0.001.