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  • The present findings also show that paternal hyperglycemia i


    The present findings also show that paternal hyperglycemia induced hypermethylation of specific CpG dinucleotides in the Ppara promoter region of the offspring liver, rather than altering the methylation of all CpGs in the Ppara promoter, and the magnitude of variation in CpG methylation was sufficient to alter transcription. The CpG dinucleotides in the Ppara promoter coincided with the putative NCT-501 of a number of TFs that have an important regulatory role in a wide range of metabolic processes. Compared to imprinted genes, which usually have a high methylation level causing silencing of the genes inherited from one parent [48,49], epigenetic variations of Ppara expressed in somatic cells with relatively low methylation level allow fine control of transcription by changing the balance of TF regulation. This finding is consistent with the work of other researchers, where adverse environmental factors caused a small change in the relatively low methylation level of the promoter region of genes that have a crucial role in controlling mammalian metabolic function [29,46,47]. Imprinted genes are monoallelically expressed with one of the copies of the gene silenced in a parent of origin-dependent manner, and only one copy is functional; thus, any epigenetic alterations on one allele may lead to detrimental consequences, causing fetal growth disruption, lower birth weight, and cancer. However, DNA methylation alterations on biallelically expressed genes such as the one we discussed here seem to be less lethal and are more often linked to “soft” consequences such as metabolic disorders, e.g., gluconeogenesis [17] and lipogenesis [50] abnormalities, and impaired glucose tolerance and insulin secretion [42]. We confirmed that the methylation signature of the Ppara locus was also present in the liver samples of STZ-O fetuses well before they developed metabolic alterations that may secondarily lead to epigenetic de novo modifications. In accordance with this finding, we found lipid accumulation was present in the STZ-O group as early as the day after weaning, long before they gained more weight than the CB-O group [31]. Therefore, the presence of the same signature in the liver of STZ-O fetuses and STZ-O adults, and the lipid derangement in the liver of STZ-O fetuses as early as the day after weaning, strongly supports epigenetic marks caused by paternal lifestyle and the persistence of environmental factors from early life into adulthood. It would help to further elaborate epigenetic inheritance through the paternal lineage if the DNA methylation status in the sperm of STZ rats was studied. Genomes undergo a massive epigenetic reprograming during gametogenesis and postzygotic divisions, and consequently epigenetic modifications in the germ cells could be erased and then be re-established, except for in some regions, primarily IAPs, which remain substantially methylated in all stages of germ cell development [15,51]. Therefore investigating the methylation changes in the Ppara promoter region in sperm, which undergo the dynamic process of erasing and re-establishing their epigenetic profile, would be practically difficult, and proving the consistency of DNA methylation marks between sperm and mature somatic cells could even be obscure. DNA methylation is known to play a critical role in the expression of genes, and one of the major mechanisms is through methylation-dependent TF-DNA interaction. TFs usually bind to nonmethylated DNA motifs and promote or repress gene transcription. However, such interactions can be directly disrupted by methylation of the CpG sites in the motifs [52]. To further investigate the correlation of paternal hyperglycemia-induced changes in the Ppara promoter methylation status and lipid dysregulation in the offspring, we tested the effect of potential TFs on Ppara promoter activity. By using bioinformatics, SP1 has been identified as a potential TF binding to the hypermethylated CpG site 13 on the Ppara promoter. Following the deletion of this possible binding site of SP1, a luciferase reporter assay showed remarkably downregulated Ppara gene expression, which demonstrated that CpG site 13 is critically involved in Ppara gene activation and that an elevated methylation level at this site can negatively affect gene expression. Indeed, through in vitro studies, we demonstrated Ppara expression was effectively inhibited with methylation of CpG site 13. However, it should be noted that after M.HPII treatment, the methylation level of CpG site 13 changed from 0% to near 100%, but this dramatic change in vitro could be different from in vivo conditions. Our research showed that methylation of this specific CpG is potentially relevant to the phenotype we observed in the animal studies, but further investigations are needed to confirm its actual physiologic and pathogenic contribution.