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.

Mutant huntingtin impairs Ku70-mediated DNA repair.

DNA repair defends against naturally occurring or disease-associated DNA damage during the long lifespan of neurons and is implicated in polyglutamine disease pathology. In this study, we report that mutant huntingtin (Htt) expression in neurons causes double-strand breaks (DSBs) of genomic DNA, and Htt further promotes DSBs by impairing DNA repair. We identify Ku70, a component of the DNA damage repair complex, as a mediator of the DNA repair dysfunction in mutant Htt-expressing neurons. Mutant Htt interacts with Ku70, impairs DNA-dependent protein kinase function in nonhomologous end joining, and consequently increases DSB accumulation. Expression of exogenous Ku70 rescues abnormal behavior and pathological phenotypes in the R6/2 mouse model of Huntington's disease (HD). These results collectively suggest that Ku70 is a critical regulator of DNA damage in HD pathology.

Chapter 6. Application of new methods for detection of DNA damage and repair.

New methods for detecting DNA damage and repair are reviewed and their potential significance is discussed. These include methods based on analysis of DNA damage-induced chromatin modifications, cytological detection of DNA repair synthesis, damage-induced immobilization of repair proteins and living cell imaging. Special attention is paid to current methods of detection of modifications of histones and other proteins associated with DNA double-strand breaks which represent most dangerous genome damage. New methods of analysis of DNA damage and repair may be useful in biodosimetry, early cancer diagnostics and in the analysis of efficiency of cancer radiation therapy and chemotherapy.

Regulation of mammalian gene expression by retroelements and non-coding tandem repeats.

Genomes of higher eukaryotes contain abundant non-coding repeated sequences whose overall biological impact is unclear. They comprise two categories. The first consists of retrotransposon-derived elements. These are three major families of retroelements (LINEs, SINEs and LTRs). SINEs are clustered in gene-rich regions and are found in promoters of genes while LINEs are concentrated in gene-poor regions and are depleted from promoters. The second class consists of non-coding tandem repeats (satellite DNAs and TTAGGG arrays), which are associated with mammalian centromeres, heterochromatin and telomeres. Terminal TTAGGG arrays are involved in telomere capping and satellite DNAs are located in heterochromatin, which is implicated in transcription silencing by gene repositioning (relocalization). It is unknown whether interstitial TTAGGG sequences, which are present in many vertebrates, have a function. Here, evidence will be presented that retroelements and TTAGGG arrays are involved in regulation of gene expression. Retroelements can provide binding sites for transcription factors and protect promoter CpG islands from repressive chromatin modifications, and may be also involved in nuclear compartmentalization of transcriptionally active and inactive domains. Interstitial telomere-like sequences can form dynamically maintained three-dimensional nuclear networks of transcriptionally inactive domains, which may be involved in transcription silencing like classic heterochromatin.

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.

Inhibition of transcription at radiation-induced nuclear foci of phosphorylated histone H2AX in mammalian cells.

Double-strand DNA breaks (DSBs) induced by ionizing radiation can be visualized in human cells using antibodies against Ser-139 phosphorylated histone H2AX (gamma-H2AX). Large gamma-H2AX foci are seen in the nucleus fixed 1 hour after irradiation and their number corresponds to the number of DSBs, allowing analysis of these genome lesions after low doses. We estimated whether transcription is affected in chromatin domains containing gamma-H2AX by following in vivo incorporation of 5-bromouridine triphosphate (BrUTP) loaded by cell scratching (run-on assay). We found that BrUTP incorporation is strongly suppressed at gamma-H2AX foci, suggesting that H2AX phosphorylation inhibits transcription. This is not caused by preferential association of gamma-H2AX foci with constitutive or facultative heterochromatin, which was visualized in irradiated cells using antibodies against histone H3 trimethylated at lysine-9 (H3-K9m3) or histone H3 trimethylated at lysine-27 (H3-K27m3). Apparently, formation of gamma-H2AX induces changes of chromatin that inhibit assembly of transcription complexes without heterochromatin formation. Inhibition of transcription by phosphorylation of histone H2AX can decrease chromatin movement at DSBs and frequency of misjoining of DNA ends.

Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange.

Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.

Slow elimination of phosphorylated histone gamma-H2AX from DNA of terminally differentiated mouse heart cells in situ.

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called gamma-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of gamma-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% gamma-H2AX-positive cells and in tissues fixed 1h after X-irradiation gamma-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of gamma-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the gamma-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of gamma-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.

Constitutive expression of the human peroxiredoxin V gene contributes to protection of the genome from oxidative DNA lesions and to suppression of transcription of noncoding DNA.

Peroxiredoxins belong to a family of antioxidant proteins that neutralize reactive oxygen species. One member of this family, peroxiredoxin I (PRDX1), suppresses DNA oxidation. Peroxiredoxin V (PRDX5) has been cloned as a transcriptional corepressor, as a peroxisomal/mitochondrial antioxidant protein, and as an inhibitor of p53-dependent apoptosis. Promoters of mammalian PRDX5 genes contain clusters of antioxidant response elements, which can bind the transcription factor NRF2. However, we found that expression of the human PRDX5 gene in situ was not stimulated by the oxidative agent menadione. Silencing of the NRF2 gene in the absence of oxidative stress by specific siRNA did not decrease PRDX5 protein concentration. We also constructed clones of human lung epithelial cells A549 with siRNA-mediated knockdown of the PRDX5 gene. This led to a significant increase in 8-oxoguanine formation in cell DNA. In the PRDX5 knockdown clone, an increase in transcripts containing sequences of alpha-satellite and satellite III DNAs was also detected, suggesting that this protein may be required for silencing of heterochromatin. Together, these results suggest that constitutively expressed PRDX5 gene plays an important role in protecting the genome against oxidation and may also be involved in the control of transcription of noncoding DNA.

Peroxiredoxin V is essential for protection against apoptosis in human lung carcinoma cells.

Sensitivity of tumor cells to treatment with anticancer drugs depends on expression and function of antiapoptotic and antioxidant proteins. The goal of our study was to determine the functional role of the novel antioxidant protein Peroxiredoxin V (PrxV), in protection of human lung carcinoma cell lines against apoptosis. Analysis of expression of PrxV in multiple lung carcinoma cell lines revealed a positive correlation between the expression of PrxV and radioresistance in vitro. Clones of the lung carcinoma cells U1810 with down-regulated expression of PrxV, or with its impaired enzymatic function (expression of redox-negative PrxV), demonstrated increased sensitivity to treatment with anticancer drugs etoposide and adriamycin. Pre-treatment of these clones with antioxidant N-acetyl-cysteine did not change their sensitivity to adriamycin, suggesting the involvement of a non-redox activity of PrxV. Expression of the redox-negative PrxV mainly affected the mitochondrial pathway of apoptosis, as assessed by cytochrome c release assay. Impairment of the PrxV enzymatic function also affected transmembrane potential and calcium loading capacity of mitochondria, as well as mitochondrial morphology. Altogether, these findings suggest that PrxV is a multifunctional protein, which is essential for protection against apoptosis induced by anticancer drugs.

Chromosome architecture in the decondensing human sperm nucleus.

Whereas recent studies demonstrated a well-defined nuclear architecture in human sperm nuclei, little is known about the mode of DNA compaction above the elementary structural unit of nucleoprotamine toroids. Here, using fluorescence in-situ hybridization (FISH) with arm-specific DNA probes of chromosomes 1, 2 and 5, we visualized arm domains and established hierarchical levels of sperm chromatin structures. The compact chromosome territories, which in sperm have a preferred intranuclear localization, have an extended conformation represented by a 2000 nm chromatin fiber. This fiber is composed of a 1000 nm chromatin thread bent at 180 degrees near centromere. Two threads of 1000 nm, representing p-arm and q-arm chromatin, run in antiparallel fashion and join at the telomeres. Each 1000 nm thread, in turn, resolves into two rows of chromatin globules 500 nm in diameter interconnected with thinner chromatin strands. We propose a unified comprehensive model of chromosomal and nuclear architecture in human sperm that, as we suggest, is important for successful fertilization and early development.

High mobility of flap endonuclease 1 and DNA polymerase eta associated with replication foci in mammalian S-phase nucleus.

Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase eta (Pol eta). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Pol eta and GFP-Fen1 are mobile with residence times at RFi (t(m)) approximately 2 and approximately 0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and t(m) increased, but it then recovered. The mobilities of focal GFP-Pol eta and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks.

Differential incorporation of halogenated deoxyuridines during UV-induced DNA repair synthesis in human cells.

Double labeling of interphase and metaphase chromosomes by 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) has been used in studies of the dynamics of DNA replication. Here, we have used this approach and confocal microscopy to analyze sites of DNA repair synthesis during nucleotide excision repair (NER) in quiescent human fibroblasts. Surprisingly, we have found that when both precursors are added at the same time to UV-irradiated cells they label different sites in the nucleus. In contrast, even very short periods of simultaneous IdU+CldU labeling of S-phase cells produced mostly overlapped IdU and CldU replication foci. The differential labeling of repair sites might be due to compartmentalization of I-dUTP and Cl-dUTP pools, or to differential utilization of these thymidine analogs by DNA polymerases delta and epsilon (Poldelta and Polepsilon). To explore the latter possibility we used purified mammalian polymerases to find that I-dUTP is efficiently utilized by both Poldelta and Polepsilon. However, we found that the UV-induced incorporation of IdU was more strongly stimulated by treatment of cells with hydroxyurea than was incorporation of CldU. This indicates that there may be distinct IdU and CldU-derived nucleotide pools differentially affected by inhibition of the ribonucleotide reductase pathway of dNTP synthesis and that is consistent with the view that differential incorporation of IdU and CldU during NER may be caused by compartmentalization of IdU- and CldU-derived nucleotide pools.

DNA damage-induced accumulation of Rad18 protein at stalled replication forks in mammalian cells involves upstream protein phosphorylation.

Rad18 protein is required for mono-ubiquitination of PCNA and trans-lesion synthesis during DNA lesion bypass in eukaryotic cells but it remains unknown how it is activated after DNA damage. We expressed GFP-tagged human (h)Rad18 in Chinese hamster cells and found that it can be completely extracted from undamaged nuclei by Triton X-100 and methanol. However, several hours after treatment with methyl methanesulfonate (MMS) Triton-insoluble form of GFP-hRad18 accumulates in S-phase nuclei where it colocalizes with PCNA. This accumulation is suppressed by inhibitors of protein kinases staurosporine and wortmannin but is not effected by roscovitine. We also found that methyl methanesulfonate induces phosphorylation of Ser-317 in protein kinase Chk1 and Ser-139 in histone H2AX and stimulates formation of single-stranded DNA at replication foci. Together, our results suggest that MMS-induced accumulation of hRad18 protein at stalled forks involves protein phosphorylation which may be performed by S-phase checkpoint kinases.

Downregulation of peroxiredoxin V stimulates formation of etoposide-induced double-strand DNA breaks.

Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.

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.

Protection of internal (TTAGGG)n repeats in Chinese hamster cells by telomeric protein TRF1.

Chinese hamster cells have large interstitial (TTAGGG) bands (ITs) which are unstable and should be protected by an unknown mechanism. Here, we expressed in Chinese hamster V79 cells green fluorescent protein (GFP)-tagged human TRF1, and found that a major fraction of GFP-TRF1 bound to ITs is diffusionally mobile. This fraction strongly decreases after treatment of cells with wortmannin, a protein kinase inhibitor, and this drug also increases the frequency of chromosome aberrations. Ionizing radiation does not induce detectable translocation of GFP-TRF1 to the sites of random double-strand breaks visualized using antibodies against histone gamma-H2AX. TRF1 is known to be eliminated from telomeres by overexpression of tankyrase 1 which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding tankyrase 1 and found that the frequency of chromosome rearrangements is increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 is involved in sequence-specific protection of internal nontelomeric (TTAGGG)n repeats.

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus.

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization.

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.

End-joining of reconstituted histone H2AX-containing chromatin in vitro by soluble nuclear proteins from human cells.

Non-homologous end-joining is an important pathway for the repair of DNA double-strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser-139 in the histone variant H2AX to form gamma-H2AX. Here we report efficient in vitro end-joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end-joining is not suppressed by the PI-3 kinase inhibitor wortmannin. During the end-joining reaction H2AX is phosphorylated at Ser-139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end-joining reaction appears to be independent of H2AX phosphorylation.