Nikolai Tomilin

The angiotensin converting enzyme I/D polymorphism in Russian athletes

The deletion (D) allele of the human ACE gene is associated with higher ACE activity than the insertion (I) allele. There is controversy as to whether the ACE genotype may be associated with elite athletic status; recent studies have identified no significant associations amongst those drawn from mixed sporting disciplines. However, such lack of association may reflect the mixed nature of such cohorts, given that an excess frequency of the I allele has been reported amongst elite endurance athletes, and an excess of the D allele amongst those engaged in more power-orientated sports. We examined this hypothesis by determining ACE I/D allele frequency amongst 217 Russian athletes (swimmers, skiers, triathletes and track-and-field participants) prospectively stratified by performance (‘outstanding’ or ‘average’), and the duration of their event (SDA (<1 min), MDA (1 to 20 min), and LDA (>20 min): short, middle and long distance athletes respectively). ACE genotype and allele frequencies were compared to 449 controls. ACE genotype frequency amongst the whole cohort, or the outstanding athletes alone, was no different to that amongst sedentary controls. However, there was an excess of the D allele (frequency 0.72, P=0.001) amongst the outstanding SDA group, and an excess of the I allele (frequency 0.63, P=0.032) amongst the outstanding MDA group. These findings were replicated in the outstanding swimmers, with track and field SDA similarly demonstrating an excess of the D allele (P=0.01). There was no association found between the outstanding LDA and ACE genotype (P=0.27). These data not only confirm an excess of the D allele in elite SDA, and I allele in elite MDA, but also offer an explanation as to why any such association may be hard to detect amongst a heterogeneous cohort of mixed athletic ability and discipline.

Dephosphorylation of Histone γ-H2AX during Repair of DNA Double-Strand Breaks in Mammalian Cells and its Inhibition by Calyculin A

Abstract Nazarov, I. B., Smirnova, A. N., Krutilina, R. I., Svetlova, M. P., Solovjeva, L. V., Nikiforov, A. A., Oei, S-L., Zalenskaya, I. A., Yau, P. M., Bradbury, E. M. and Tomilin, N. V. Dephosphorylation of Histone γ-H2AX during Repair of DNA Double-Strand Breaks in Mammalian Cells and its Inhibition by Calyculin A. Radiat. Res. 160, 309–317 (2003). The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called γ-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that γ-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of γ-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of γ-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the γ-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, γ-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of γ-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from γ-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and γ-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

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° 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.

Control of genes by mammalian retroposons.

Available data on possible genetic impacts of mammalian retroposons are reviewed. Most important is the growing number of established examples showing the involvement of retroposons in modulation of expression of protein-coding genes transcribed by RNA polymerase II (Pol II). Retroposons contain conserved blocks of nucleotide sequence for binding of some important Pol II transcription factors as well as sequences involved in regulation of stability of mRNA. Moreover, these mobile genes provide short regions of sequence homology for illegitimate recombinations, leading to diverse genome rearrangements during evolution. Therefore, mammalian retroposons representing a significant fraction of noncoding DNA cannot be considered at present as junk DNA but as important genetic symbionts driving the evolution of regulatory networks controlling gene expression.

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. BioEssays 30:338–348, 2008.

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.

Association of some potential hormone response elements in human genes with the Alu family repeats

Short interspersed repeats of the Alu family located in promoters of some human genes contain high-affinity binding sites for thyroid hormone receptor, retinoic acid receptor and estrogen receptor. The standard binding sites for the receptors represent variants of duplicated AGGTCA motif with different spacing and orientation (direct, DR, or inverted, IR), and Alu sequences were found to have functional DR-4, DR-2 or variant IR-3/IR-17 elements. In this study we analyzed distribution and abundance of the elements in a set of human genomic sequences from GenBank and their association with Alu repeats. Our results indicate that a major fraction of potentially active DR-4, DR-2 and variant IR-3/IR-17 elements in the genes is located within Alu repeats. Alu-associated DR-2 elements are conserved in primate evolution. However, very few Alu have potential DR-3 glucocorticoid-response elements. Gel-shift experiments with the probe (AUB) corresponding to the consensus Alu sequence just upstream of the RNA polymerase III promoter B-box and containing duplicated AGGTCA motif indicate that the probe interacts in a sequence-specific manner with human nuclear proteins which bind to standard IR-0, DR-1, DR-4 or DR-5 elements. The AUB sequence was also able to promote thyroid hormone-dependent trans-activation of a reporter gene. The results support the view that Alu retroposons played an important role in evolution of regulation of the primate gene expression by nuclear hormone receptors.

Transcription and replication silencer element is present within conserved region of human Alu repeats interacting with nuclear protein

Human cells contain a nuclear protein interacting with Alu repeats, and this protein seems to recognize a conserved sequence motif, GGAGGC, present within the RNA polymerase III promoter and within the SV40 T‐antigen‐dependent ARS‐like element. To study the potential functional role of this element, we have inserted the sequence into a chloramphenicolacetyltransferase (CAT) expression vector with a SV40 promoter and enhancer element from the up‐stream region of the human c‐myc gene, and transfected HeLa cells with the resulting plasmid. Analysis of expression by the CAT assay indicates that the Alu‐derived sequence supresses transcription of the CAT gene driven by the c‐myc enhancer/SV40 promoter. The Alu‐derived sequence also inhibits ARS activity of the c‐myc enhancer. The data allow the explaination of the transcriptional inactivity of Alu repeats in HeLa cells, and suggest the existence of a negative control of Alu transcription.

Visualization of Focal Nuclear Sites of DNA Repair Synthesis Induced by Bleomycin in Human Cells

Abstract Tomilin, N. V., Solovjeva, L. V., Svetlova, M. P., Pleskach, N. M., Zalenskaya, I. A., Yau, P. M. and Bradbury, E. M. Visualization of Focal Nuclear Sites of DNA Repair Synthesis Induced by Bleomycin in Human Cells. Radiat. Res. 156, 347–354 (2001). In this study, we examined DNA repair synthesis in human cells treated with the radiomimetic drug bleomycin, which efficiently induces double-strand breaks (DSBs). Using tyramide-biotin to amplify fluorescent signals, discrete nuclear foci from the incorporation of 5-iododeoxyuridine (IdU) were detected in proliferating human cells treated with bleomycin. We believe this comes from the repair of DSBs. An increase in the number of foci (>5 per nucleus) was detected in a major fraction (75%) of non-S-phase cells labeled for 30 min with IdU 1 h after the end of bleomycin treatment. The fraction of cells with multiple IdU-containing foci was found to decrease 18 h after treatment. The average number of foci per nucleus detected 1 h after bleomycin treatment was found to decrease twofold between 1 and 3.5 h, indicating that the foci may be associated with the slow component of DSB repair. The presence of DSBs in bleomycin-treated cells was confirmed using antibodies against phosphorylated histone H2AX (γ-H2AX), which is strictly associated with this type of DNA damage. After treatment with bleomycin, non-S-phase cells also displayed heterogeneous nuclear foci containing tightly bound proliferating cell nuclear antigen (PCNA), suggesting an ongoing process of unscheduled DNA synthesis. PCNA is known to be involved in base excision repair, but a fraction of the PCNA foci may also be associated with DNA synthesis occurring during the repair of DSBs.

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 (γ-H2AX). Large γ-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 γ-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 γ-H2AX foci, suggesting that H2AX phosphorylation inhibits transcription. This is not caused by preferential association of γ-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 γ-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.