[Show abstract][Hide abstract] ABSTRACT: Poly (ADP-ribose) polymerase-1 (Parp1) has been implicated in DNA base excision repair, single- and double-strand break repair pathways, as well as in cell death by apoptosis or necrosis. We used Parp1(-/-) lacZ plasmid-based transgenic mice to investigate whether Parp1 deficiency influences the in vivo mutagenic and clastogenic response to the alkylating agent N-ethyl-N-Nitrosourea (ENU) in somatic and germ-cell tissues. The comparison of the lacZ mutant frequencies (MFs) between Parp1(+/+) and Parp1(-/-) mice showed that the ablation of Parp1 does not affect the spontaneous or ENU-induced MFs in liver and testis. In addition, the spectrum of the ENU-induced mutations was not dependent on the Parp1 status, given that similar spectra, consisting mostly of point mutations and a small fraction of deletions/insertions, wereobserved in organs of both Parp1(-/-) and Parp1(+/+) mice. Sequencing of point mutations revealed a consistent significant increase in A:T --> T:A base substitutions, typically induced by ENU. Overall, we observed that neither the frequency nor the spectrum of ENU-induced mutations demonstrated a specificity that could be attributed to the Parp1 impairment in mice organs. The analysis of micronucleus frequency in peripheral blood reticulocytes showed that ENU was clastogenic in both Parp1(-/-) and Parp1(+/+) mice and had a strong cytotoxic effect in Parp1(-/-) mice only. The present data suggest that, at a whole-organism level, Parp1-independent repair mechanisms may be operative in the removal of ENU-induced DNA lesions or that highly damaged cells may be preferentially committed to death when Parp1 is inactivated.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an important epigenetic modification that has profound roles in gene expression and, in particular, is thought to be crucial for regulation of tissue-specific genes in animal cells. The pivotal E(1)alpha subunit of human pyruvate dehydrogenase complex, an essential and rate-limiting enzyme system in energy metabolism, is encoded by two distinct genes: PDHA1 gene, located on chromosome X is expressed in somatic tissues, whereas PDHA2 gene, located on chromosome 4, is exclusively expressed in spermatogenic cells. The objective of this study is to elucidate the role of DNA methylation as an epigenetic mechanism controlling the regulation of PDHA2 gene expression in human tissues, namely its repression in somatic tissues and its activation in testicular germ cells. Genomic DNA was isolated from human somatic tissues (circulating lymphocytes and gastric cells) and from testis, including isolated fractions of haploid and diploid germ cells. After primer design with appropriate software, it was performed the sodium bisulfite PCR sequencing of the PDHA2 promoter and coding regions. Total RNA of the same tissues was isolated, reverse transcribed and PDHA1and PDHA2 transcripts were amplified with specific primers and analysed by agarose gel electrophoresis. The analysis of the genomic sequence of the PDHA2 gene revealed the presence of 61 CpG sites whose distribution matches the criteria for the presence of two CpG islands. Sequence analysis of both CpG islands upon bisulfite treatment displayed several differences, either between islands or among tissues. In particular, the methylation pattern of one of the CpG islands revealed a perfect correlation with transcriptional activity of the PDHA2 gene either in testis or in somatic tissues. Surprisingly, it is the full demethylation of the CpG island located in the coding region that seems to play a crucial role upon PDHA2 gene transcription in testis.