Transcription factor GABP/NRF-2 controlling biogenesis of mitochondria regulates basal expression of peroxiredoxin V but the mitochondrial function of peroxiredoxin V is dispensable in the dog.

Peroxiredoxins (PRDXs) represent a conserved family of six antioxidant proteins which are widely expressed in different organisms. Human PRDX5 is detected in the cytosol and nucleus and can also target peroxisomes and mitochondria. However, it remains unknown if mitochondrial localization of PRDX5 is essential for its functions. Here we studied whether the known regulator of mitochondrial biogenesis, transcription factor GABP/NRF-2, is required for the basal expression of the human PRDX5 gene and what the significance is of the mitochondrial targeting of the PRDX5 protein. It was found that mutation-mediated inactivation of all potential binding sites for GAPB in the PRDX5 promoter lead to ∼80% inhibition of its basal activity in a reporter gene assay. Co-transfection of plasmids expressing GABP-alpha and GABP-beta stimulated activity of the non-mutated PRDX5 promoter but had no effect on the mutated promoter, suggesting that basal expression of the human PRDX5 gene is regulated by GABP. We found that the dog c-Myc-tagged PRDX5 did not target the mitochondria of human cells. Endogenously expressed PRDX5 also showed no association with mitochondria in the dog cells. It appears, therefore, that during evolution the dog PRDX5 gene lost its upstream ATG codon and mitochondrial targeting signal without major functional consequences.

The DL1 repeats in the genome of Diphyllobothrium latum.

Diphyllobothrium latum is a widespread intestinal parasite, which has a great clinical relevance, but there are no sequences of its nuclear genome. In this paper, a repetitive element in the D. latum genome is firstly described. The adult D. latum was obtained in the result of expulsion from intestinum of a patient suffering from diphyllobothriasis. Genomic DNA was isolated from several proglottids of this individual. PstI restriction products of D. latum genomic DNA were sequenced. Polymerase chain reaction (PCR) amplification of these products using genomic DNA and selected primers was carried out. Thereby a cluster of a repetitive element, called DL1, was discovered. For precise identification of a beginning and an end of the repeat, a product of PCR amplification of D. latum genomic DNA with one specific primer was sequenced. In discussion, several evidences that DL1 repeat is a member of the SINE family of retroposons were adduced.

Mitochondrial targeting of human peroxiredoxin V protein and regulation of PRDX5 gene expression by nuclear transcription factors controlling biogenesis of mitochondria.

Peroxiredoxin V (PRDX5) is a member of the family of mammalian proteins that neutralize reactive oxygen species. The PRDX5 gene is constitutively expressed at a high level in many human tissues, but functional elements of its promoter responsible for a high basal activity in the absence of oxidative stress have still not been identified. Among predicted binding sites for transcription factors in the human PRDX5 promoter are binding sites for nuclear respiratory factor 1 (NFR-1) and nuclear respiratory factor 2 (also called GABPA), which regulate the biogenesis of mitochondria. We constructed luciferase reporter gene plasmids containing stepwise deletions of the PRDX5 promoter and examined their activities in transient transfections. Our results suggest that basal PRDX5 promoter activity mostly depends on NFR-1 and GABPA sites. The latter, in the PRDX5 promoter, were conserved in the six mammalian genomes analyzed (human, chimpanzee, cow, mouse, rat and dog) and a fraction of human PRDX5 associates with the mitochondrial matrix. We also found that the N-terminal 50 amino acids of the full-length human PRDX5 (24 kDa) translated from its first AUG codon targets this protein exclusively to mitochondria. However, the short form of PRDX5 (17 kDa), translated from its second AUG codon, has cytoplasmic and nuclear localization, which is also typical for endogenously expressed protein. Together, our results indicate that high basal expression of the PRDX5 gene is coordinated with the expression of nuclear genes encoding mitochondrial proteins and that the PRDX5 protein might play a major role in permanent defense against reactive oxygen species produced by mitochondria.

Clusters of regulatory signals for RNA polymerase II transcription associated with Alu family repeats and CpG islands in human promoters.

Primate genomes contain a very large number of short interspersed GC-rich repeats of the Alu family, which are abundant in introns and intergenic spacers but also present in 5' flanking regions of genes enriched in binding motifs (BMs) for transcription factors and frequently containing CpG islands. Here we studied whether CpG islands located in promoters of human genes overlap with Alu repeats and with clusters of BMs for the zinc-finger transcription factors Sp1, estrogen receptor alpha, and YY1. The presence of estrogen-response elements in Alu was shown earlier and here we confirm the presence in the consensus Alu sequence of the binding sites for Sp1 and YY1. Analyzing >5000 promoters from the two databases we found that Alu sequences are underrepresented in promoters compared to introns and that approximately 4% of CpG islands located within the -1000 to +200 segments of human promoters overlap with Alu repeats. Although this fraction was found to be lower for proximal segments of promoters (-500 to +100), our results indicate that a significant number (>1000) of all human genes may be controlled by Alu-associated CpG islands. Analysis of clustering of potential BMs for the indicated transcription factors within some promoters also suggests that the Alu family contributed to the evolution of transcription cis-regulatory modules in the human genome. It is important that among Alu sequences overlapping with CpG islands in promoters a large fraction of members of the old Alu subfamilies is found, suggesting extensive retroposon-assisted regulatory genome evolution during the divergence of the primates.