Differential CpG island methylation of murine adenine nucleotide translocase genes

Abstract

Adenine nucleotide translocase (Ant) mediates the exchange of ADP and ATP across the inner mitochondrial membrane in eukaryotes. Mice possess three distinct but highly homologous Ant isoforms, encoded by independent genes, whose transcription depends upon tissue type. Ant1 is expressed selectively in heart and skeletal muscles, Ant2 is ubiquitously expressed in most tissues but lower in skeletal muscle and testis, while Ant4 is exclusively expressed in the testis. Of interest, each of these Ant genes contains CpG islands in their proximal promoter regions. We investigated the methylation status of the three Ant genes in various tissues with active and inactive transcription. In contrast to the Ant4 gene in which CpG island methylation is essential for gene repression, the CpG islands of Ant1 and Ant2 are hypomethylated regardless of the gene expression status throughout the tissues of male mice. Despite the tissue specific expression profile of Ant1, CpG methylation is unlikely involved in the regulation of the gene. Consistent with these findings, addition of a CpG-demethylating agent, 5-aza-2'-deoxycitidine, to fibroblasts increased the expression of Ant4 but not Ant1 or Ant2 genes. This study provides insight regarding the differential regulation of Ant isoforms in mammals, whereby both the Ant1 and Ant2 genes are capable of expression, but the Ant4 gene is completely repressed throughout somatic tissues. To the best of our knowledge, this is a first example to clearly demonstrate a differential usage of CpG island methylation within a family of genes.

Figure 1. CpG islands of murine Ant1, Ant2 and Ant4 genes

The promoter regions along with exon 1 (-500 to +212) of Ant1, Ant2, and Ant4 were analyzed for the presence of CpG islands using the MethPrimer program. A CpG island is defined as a region of DNA greater than 200 bp, with a GC content above 0.5 and an observed/expected CpG ratio above 0.6.

Figure 2. Tissue specific expression of murine Ant1, Ant2 and Ant4 genes

RNA expression of Ant1, Ant2, and Ant4 genes in various tissues of adult C57BL/6 mice (~8 weeks) were analyzed using real-time RT-PCR. Relative expression levels of Ant genes to β-actin using the comparative Ct method are shown. Sk M: Skeletal Muscle, M: Male, F: Female

Figure 3. CpG methylation of murine Ant1, Ant2 and Ant4 genes (COBRA)

CpG methylation status of Ant1, Ant2, and Ant4 gene promoters in various tissues was analyzed using combined bisulfite restriction analysis (COBRA) as described in Materials and Methods. “U” represents unmethylated DNA fragments, while “M” represents methylated DNA fragments.

Figure 4. CpG methylation of murine Ant1, Ant2 and Ant4 genes (Bisulfite Sequencing)

CpG methylation status of (A) Ant1, (B) Ant2, and (C) Ant4 gene promoters in various tissues was analyzed using bisulfite sequencing analysis as described in Materials and Methods. % Methylation (% Met) for each CpG site investigated is demonstrated in the bar graphs. Actual CpG methylation status of the region in eight sequenced clones is also shown to the right of the graph. The open circles represents unmethylated CpG residues, while the closed circles represents methylated CpG residues.

Figure 4. CpG methylation of murine Ant1, Ant2 and Ant4 genes (Bisulfite Sequencing)

CpG methylation status of (A) Ant1, (B) Ant2, and (C) Ant4 gene promoters in various tissues was analyzed using bisulfite sequencing analysis as described in Materials and Methods. % Methylation (% Met) for each CpG site investigated is demonstrated in the bar graphs. Actual CpG methylation status of the region in eight sequenced clones is also shown to the right of the graph. The open circles represents unmethylated CpG residues, while the closed circles represents methylated CpG residues.

Figure 4. CpG methylation of murine Ant1, Ant2 and Ant4 genes (Bisulfite Sequencing)

CpG methylation status of (A) Ant1, (B) Ant2, and (C) Ant4 gene promoters in various tissues was analyzed using bisulfite sequencing analysis as described in Materials and Methods. % Methylation (% Met) for each CpG site investigated is demonstrated in the bar graphs. Actual CpG methylation status of the region in eight sequenced clones is also shown to the right of the graph. The open circles represents unmethylated CpG residues, while the closed circles represents methylated CpG residues.

Figure 5. The effects of 5-aza-2’-deoxycitidine and trichostatin A on Ant1, Ant2 and Ant4 gene expression

NIH3T3 mouse fibroblasts were treated with 5-aza-2’-deoxycitidine (5-aza-dC, 5 μM), trichostatin A (TSA, 200 nM) or both for 48 hrs. RNA expression of Ant1, Ant2, and Ant4 genes was analyzed using real-time RT-PCR as described above in Fig. 2. Relative expression levels of Ant genes compared to β-actin using the comparative Ct method are shown. The mean values for the Ant4 transcript are: No treatment (1.21 × 10^-3), 5-aza-dC (9.71), TSA (1.53 × 10^-3) and 5-aza-dC+TSA (1.89).