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Heterochromatin normally has prescribed chromosomal positions and must not encroach on adjacent regions. We demonstrate that the fission yeast protein Epe1 stabilises silent chromatin, preventing the oscillation of heterochromatin domains. Epe1 loss leads to two contrasting phenotypes: alleviation of silencing within heterochromatin and expansion of silent chromatin into neighbouring euchromatin. Thus, we propose that Epe1 regulates heterochromatin assembly and disassembly, thereby affecting heterochromatin integrity, centromere function and chromosome segregation fidelity. Epe1 regulates the extent of heterochromatin domains at the level of chromatin, not via the RNAi pathway. Analysis of an ectopically silenced site suggests that heterochromatin oscillation occurs in the absence of heterochromatin boundaries. Epe1 requires predicted iron- and 2-oxyglutarate (2-OG)-binding residues for in vivo function, indicating that it is probably a 2-OG/Fe(II)-dependent dioxygenase. We suggest that, rather than being a histone demethylase, Epe1 may be a protein hydroxylase that affects the stability of a heterochromatin protein, or protein–protein interaction, to regulate the extent of heterochromatin domains. Thus, Epe1 ensures that heterochromatin is restricted to the domains to which it is targeted by RNAi.

Cellular senescence is a tumor-suppressing mechanism that is accompanied by characteristic chromatin condensation called senescence-associated heterochromatic foci (SAHFs). We found that individual SAHFs originate from individual chromosomes. SAHFs do not show alterations of posttranslational modifications of core histones that mark condensed chromatin in mitotic chromosomes, apoptotic chromatin, or transcriptionally inactive heterochromatin. Remarkably, SAHF-positive senescent cells lose linker histone H1 and exhibit increased levels of chromatin-bound high mobility group A2 (HMGA2). The expression of N-terminally enhanced green fluorescent protein (EGFP)–tagged histone H1 induces premature senescence phenotypes, including increased levels of phosphorylated p53, p21, and hypophosphorylated Rb, and a decrease in the chromatin-bound endogenous histone H1 level but not in p16 level accumulation or SAHF formation. However, the simultaneous ectopic expression of hemagglutinin-tagged HMGA2 and N-terminally EGFP-tagged histone H1 leads to significant SAHF formation (P < 0.001). It is known that histone H1 and HMG proteins compete for a common binding site, the linker DNA. These results suggest that SAHFs are a novel type of chromatin condensation involving alterations in linker DNA–binding proteins.

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-δ on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.

Heterochromatin protein 1 (HP1) was originally described as a non-histone chromosomal protein and is required for transcriptional gene silencing and the formation of heterochromatin. Although it is localized primarily at pericentric heterochromatin, a scattered distribution over a large number of euchromatic loci is also evident. Here, we provide evidence that Drosophila HP1 is essential for the maintenance of active transcription of euchromatic genes functionally involved in cell-cycle progression, including those required for DNA replication and mitosis. Depletion of HP1 in proliferating embryonic cells caused aberrant progression of the cell cycle at S phase and G2/M phase, linked to aberrant chromosome segregation, cytokinesis, and an increase in apoptosis. The chromosomal distribution of Aurora B, and the level of phosphorylation of histone H3 serine 10 were also altered in the absence of HP1. Using chromatin immunoprecipitation analysis, we further demonstrate that the promoters of a number of cell-cycle regulator genes are bound to HP1, supporting a direct role for HP1 in their active transcription. Overall, our data suggest that HP1 is essential for the maintenance of cell-cycle progression and the transcription of cell-cycle regulatory genes. The results also support the view that HP1 is a positive regulator of transcription in euchromatin.

Background

Cells that reach “Hayflick limit” of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining.

Methods

We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH).

Results

Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli.

Conclusions

Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.

Gerontology research carried out in different scientific centers of Georgia follows the basic directions of most work in this field: epidemiology, investigation of the mechanisms of aging, and finding ways to prevent senile pathologies and to prolong life. The genealogy and epidemiology of long-living peaple have been studied in areas with high occurrence of these people by considering the sex ratio and social status of the long-living, the influence of environmental factors, and the development of senile pathologies. According to the centrosome (centriole) model of aging, the centrosomes and the cytoskeleton, important structures in cellular differentiation and morphogenesis, may be involved in the initiation of the replication senescence mechanism. Our analysis of genetic studies shows that progressive chromosome heterochromatinization (condensation of eu- and heterochromatin regions) occurs in aging. Decreases in the repair processes and increases in the frequency of chromosome aberrations during aging are secondary to this progressive chromosome heterochromatinization. Chromosome heterochromatinization is a key factor in aging but may be reversible under the influence of bioregulators, some chemical substances, and heavy metal salts. The study of chromosome heterochromatinization may provide clues to the potential for prolonging the human lifespan.

Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci.

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 104-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions.

Summary

Chromatin is highly dynamic and subject to extensive remodeling under many physiological conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We find increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In skeletal muscle we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, post mitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to further investigate the progressive epigenetic changes that occur during aging.

The possible influence of the high polymorphic C heterochromatic regions of human chromosomes 1, 9, 16, and Y on meiotic chromosome segregation was investigated. Faulty chromosome segregation may be the result of either an abnormal quantity of C heterochromatin on the homologues, or disequilibrium between the homologues. The aim of our study was to determine whether either a variation in the amounts of total C heterochromatin or differences in the amounts of C heterochromatin between homologues could lead to faulty chromosome segregation. The study was performed on C banded metaphases obtained from peripheral lymphocyte cultures of 15 couples with recurrent early abortions and 15 control couples, all Caucasians. Analysis of variance was first performed on separate metaphases to measure intra-individual, inter-individual, and between population variation in a hierarchical model. Since the significant intra-individual differences covered the other parameters we performed, secondly, a one way analysis of variance on the mean values of metaphases per person in order to measure the inter-individual and between population variation. The results did not show a relationship between C heterochromatin lengths and occurrence of recurrent abortions.

The capacity of in vitro bioassays to detect the potential carcinogenicity of metal compounds is reviewed. The in vitro bioassays discussed include: bacterial reversion analysis to determine the capacity of metal salts to revert Salmonella typhimurium histidine auxotrophs or to revert Escherichia coli WP 2 tryp- to tryptophan prototrophy; examination of the ability of metal salts to preferentially inhibit cell growth in Bacillus subtilis cells deficient in DNA repair pathways; determination of the ability of metal salts to induce resistance to base analogs in mammalian cells; the capacity of metal salts to enhance viral transformation of mammalian cells or to transform cells in the absence of virus; and the ability of metal salts to induce chromosomal aberrations in mammalian cells. Using each of these in vitro bioassays, diverse metal compounds have been identified as potential carcinogens. Furthermore, the use of different compounds of a specific metal may allow a determination of the valence which may be required for carcinogenesis.

Instability of the heterochromatic centromeric regions of chromosomes 1, 9, and 16 associated with immunodeficiency was found in a four year old girl. Similar phenotypic and chromosomal abnormalities were described in a previous patient studied by us and in four other published cases. All these patients have facial anomalies in addition to combined immunodeficiency and chromosomal instability. Stretching of the heterochromatic centromeric regions of chromosomes 1, 16, and to a lesser extent, 9 and homologous and non-homologous associations of these regions were the most common cytogenetic findings in all the patients. Multi-branched configurations and whole arm deletions of chromosomes 1 or 16 or both were also found. Comparing clinical and chromosomal data we conclude that immunodeficiency, centromeric heterochromatin instability, and facial anomalies form a new syndrome, for which we propose the acronym ICF. A mutation interfering with the normal process of condensation of part of the centromeric heterochromatin is postulated as the basic chromosome defect in this syndrome.

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The immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Ighb scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy- 3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the VH gene repertoire of the GC B cells was strongly influenced by the source of the CD4+ T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies declined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germine-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.

Spontaneous and clastogen-induced chromosomal instability in a high-risk group (i.e, 33 patients with rectal carcinomas) was investigated using peripheral blood lymphocytes as target cells. In addition to the analysis of spontaneous and clastogen-induced chromosome aberrations, this study also included classical karyotype analysis and scoring of sister chromatid exchanges (SCE) in some of the patients. Diepoxybutane (DEB), 4-nitroquinoline-1-oxide (NQO), and bleomycin were used as standard clastogens. Lymphocytes of healthy control individuals were studied in parallel with each cancer patient. While only slight but significant differences could be detected of the average spontaneous, DEB- and bleomycin (G2)-induced chromosome breakage between patient and control lymphocytes, individual patients and two of the control individuals showed a more distinct increase in the frequency of the studied end points. These increases were documented by a variegated mosaicism of karyotypic changes and by an increased breakage rate induced by the clastogens. Neither the bleomycin-exposure in the G1 phase nor SCE was capable of detecting differences between the patients and controls. Of particular interest in the sense of high-risk individuals were seven patients and two control persons whose lymphocytes exhibited increased chromosomal sensitivity under more than one of the studied experimental conditions.

Background

Radiation-induced chromosome aberrations lead to a plethora of detrimental effects at cellular level. Chromosome aberrations provide broad spectrum of information ranging from probability of malignant transformation to assessment of absorbed dose. Studies mapping differences in radiation sensitivities between human chromosomes are seldom undertaken. Consequently, health risk assessment based on radio-sensitivities of individual chromosomes may be erroneous. Our efforts in this article, attempt to demonstrate differences in radio-sensitivities of human chromosome-1 and/or -2, both in interphase and metaphase spreads.

Methods

Upon blood collection, dosimetry and irradiation were performed. Lymphocytes were isolated after whole-blood irradiation with 60Co γ-rays in the dose range of 0–5 Gy for both interphase, and metaphase aberration studies. Induction of premature chromosome condensation in interphase cells was accomplished using a phosphatase inhibitor, calyculin-A. Metaphase spreads were harvested from short-term peripheral blood lymphocyte cultures following colcemid arrest and using an automated metaphase harvester and spreader. Aberration analysis in both interphase and metaphase spreads were done using FISH.

Results

In interphase, aberrant cell and aberration frequency involving chromosome 1 and/or 2 increased linearly with radiation dose. In metaphase, aberrations increased in a linear-quadratic manner with dose. Our studies ascertain that chromosome-2 is more radio-sensitive than chromosome-1 in both interphase and metaphase stages, albeit the DNA content of chromosome-2 is lesser than chromosome-1 by almost 10 million base pairs.

Conclusion

Differences in radio-sensitivities of chromosomes have implications in genetic damage, chromosome organization, and chromosome function. Designing research experiments based on our vital findings may bring benefit to radiation-induced risk assessment, therapeutics and development of chromosome specific biomarkers.

Telomeres, the physical ends of eukaryotic chromosomes, consist of tandem arrays of short DNA repeats and a large set of specialized proteins. A recent analysis has identified telomeric repeat-containing RNA (TERRA), a large non-coding RNA in animals and fungi, which forms an integral component of telomeric heterochromatin. TERRA transcription occurs at most or all chromosome ends and it is regulated by RNA surveillance factors and in response to changes in telomere length. TERRA functions that are emerging suggest important roles in the regulation of telomerase and in orchestrating chromatin remodelling throughout development and cellular differentiation. The accumulation of TERRA at telomeres can also interfere with telomere replication, leading to a sudden loss of telomere tracts. Such a phenotype can be observed upon impairment of the RNA surveillance machinery or in cells from ICF (Immunodeficiency, Centromeric region instability, Facial anomalies) patients, in which TERRA is upregulated because of DNA methylation defects in the subtelomeric region. Thus, TERRA may mediate several crucial functions at the telomeres, a region of the genome that had been considered to be transcriptionally silent.

Mice homozygous for a mutation (BrdtΔBD1/ΔBD1) lacking the first bromodomain of Brdt, a testis-specific member of the BET family of double-bromodomain containing proteins, are sterile and exhibit profound defects in chromatin remodeling during spermiogenesis. We have now observed that a prominent feature of the aberrant spermatid nuclei is a fragmented chromocenter, a structure comprised of peri-centromeric heterochromatin. There was a concomitant increase in the levels of heterochromatin protein 1 alpha (Hp1α), suggesting that the presence of multiple chromocenters was correlated with a spread of heterochromatin beyond the normal centromeric region. Brdt protein was normally present throughout the nucleus but was excluded from the chromocenter. A more densely staining region of Brdt protein appeared to separate sirtuin 1 (Sirt1) protein from contact with the chromocenter. Although still nuclear, this unique localization of Brdt protein was lost in BrdtΔBD1/ΔBD1 mutant spermatids and Brdt and Sirt1 overlapped around the chromocenters. There was also ectopic localization of the H1 histone family, member N, testis-specific (H1fnt) protein in BrdtΔBD1/ΔBD1 round spermatids, which may be linked to the previously reported loss of polarized localization of peri-nuclear heterochromatin foci. The extent of chromocenter fragmentation was more severe and penetrant in mutant testes on a pure 129Sv/Ev as compared to a pure C57Bl/6 background. Indeed, all aspects of the mutant phenotype were more severe on the 129Sv/Ev background. Contrary to previous studies in genetic models where fragmented chromocenters were observed in spermatids, the BrdtΔBD1/ΔBD1 mutant spermatids do not undergo apoptosis (on either background). These observations suggest that the first bromodomain of Brdt is critical in the formation and/or maintenance of an intact chromocenter and implicate this structure in proper remodeling of the chromatin architecture of the sperm head.

The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) is the only disease known to result from a mutated DNA methyltransferase gene, namely, DNMT3B. Characteristic of this recessive disease are decreases in serum immunoglobulins despite the presence of B cells and, in the juxtacentromeric heterochromatin of chromosomes 1 and 16, chromatin decondensation, distinctive rearrangements, and satellite DNA hypomethylation. Although DNMT3B is involved in specific associations with histone deacetylases, HP1, other DNMTs, chromatin remodelling proteins, condensin, and other nuclear proteins, it is probably the partial loss of catalytic activity that is responsible for the disease. In microarray experiments and real-time RT-PCR assays, we observed significant differences in RNA levels from ICF vs. control lymphoblasts for pro- and anti-apoptotic genes (BCL2L10, CASP1, and PTPN13); nitrous oxide, carbon monoxide, NF-κB, and TNFa signalling pathway genes (PRKCH, GUCY1A3, GUCY1B3, MAPK13; HMOX1, and MAP4K4); and transcription control genes (NR2F2 and SMARCA2). This gene dysregulation could contribute to the immunodeficiency and other symptoms of ICF and might result from the limited losses of DNA methylation although ICF-related promoter hypomethylation was not observed for six of the above examined genes. We propose that hypomethylation of satellite 2at1qh and 16qh might provoke this dysregulation gene expression by trans effects from altered sequestration of transcription factors, changes in nuclear architecture, or expression of noncoding RNAs.

Earlier reports indicated the presence of significant amounts of inorganic salts in the nucleus. In the present study the possibility that this might be related to the transcription process was tested on seminiferous epithelium of the adult mouse, using potassium pyroantimonate as a fixative. The results indicated that a correlation exists between the inorganic cations comprising the pyroantimonate-precipitable fraction and the RNA synthetic activity. During meiotic prophase an accumulation of cation-antimonate precipitates occurs dispersed through the middle pachytene nuclei, the stage in which RNA synthesis reaches a maximum. At other stages (zygotene to diplotene), where RNA synthesis falls to a low level, that pattern is not seen; cation-antimonate deposits are restricted to a few masses in areas apparently free of chromatin. The condensed sex chromosomes, the heterochromatin of the "basal knobs," the axial elements, and the synaptonemal complexes are devoid of antimonate deposits during the meiotic prophase. The Sertoli cells, active in RNA synthesis in both nucleoplasm and nucleolus, show cation-antimonate deposits at these sites. In the nucleoplasm some "patches" of precipitates appear coincident with clusters of interchromatin granules; in the nucleolus the inorganic cations are mainly located in the fibrillar and/or amorphous areas, whereas relatively few are shown by the granular component. The condensed chromatin bodies associated with the nucleolus were always free of antimonate precipitates. It is suggested that the observed sites of inorganic cation accumulation within the nucleus may at least partially indicate the presence of RNA polymerases, the activity of which is dependent on divalent cations.

Carcinogenic, water-insoluble Ni compounds are phagocytized by cells; and the particles undergo dissolution inside the cell, releasing Ni ions that interact with chromatin. Ni produces highly selective damage to heterochromatin. The longest contiguous region of heterochromatin in the Chinese hamster genome is found on the q arm of the X chromosome, and this region is selectively damaged by Ni. More than half of the male mice in which there were Ni-induced transformations of Chinese hamster cells exhibited complete deletion of the long arm of the X chromosome. The introduction of a normal X chromosome into these cells resulted in cellular senescence, suggesting that the Ni interacted with Chinese hamster genome to inactivate a senescence gene. Investigations were conducted into the mechanisms by which Ni produced damage to chromatin. Ni ions have a much higher affinity for proteins and amino acids than for DNA (by five to seven orders of magnitude). Therefore, Ni interacted with chromatin because of the protein present, not because of its reactivity for DNA. Studies have shown that Ni produced an increase in oxidative products in cells as indicated by oxidation of the fluorescent dye dichlorofluorescein; Ni has also been shown to produce oxidation of proteins in cells, as measured by carbonyl formation. Ni cross-linked certain amino acids and proteins to DNA. These covalent cross-links were not dissociated by EDTA and are inconsistent with direct Ni involvement, but they are consistent with Ni acting catalytically. Using subtractive hybridization, we have isolated a number of clones that are expressed in normal but not in Ni-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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Senescence is characterized by an irreversible cell proliferation arrest. Specialized domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), are thought to contribute to the irreversible cell cycle exit in many senescent cells by repressing the expression of proliferation-promoting genes such as cyclin A. SAHF contain known heterochromatin-forming proteins, such as heterochromatin protein 1 (HP1) and the histone H2A variant macroH2A, and other specialized chromatin proteins, such as HMGA proteins. Previously, we showed that a complex of histone chaperones, histone repressor A (HIRA) and antisilencing function 1a (ASF1a), plays a key role in the formation of SAHF. Here we have further dissected the series of events that contribute to SAHF formation. We show that each chromosome condenses into a single SAHF focus. Chromosome condensation depends on the ability of ASF1a to physically interact with its deposition substrate, histone H3, in addition to its cochaperone, HIRA. In cells entering senescence, HP1γ, but not the related proteins HP1α and HP1β, becomes phosphorylated on serine 93. This phosphorylation is required for efficient incorporation of HP1γ into SAHF. Remarkably, however, a dramatic reduction in the amount of chromatin-bound HP1 proteins does not detectably affect chromosome condensation into SAHF. Moreover, abundant HP1 proteins are not required for the accumulation in SAHF of histone H3 methylated on lysine 9, the recruitment of macroH2A proteins, nor other hallmarks of senescence, such as the expression of senescence-associated β-galactosidase activity and senescence-associated cell cycle exit. Based on our results, we propose a stepwise model for the formation of SAHF.

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2+. On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.

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Background

All prostheses with metallic components release metal debris that can potentially activate the immune system. However, implant-related metal hyper-reactivity has not been well characterized. In this study, we hypothesized that adaptive immunity reaction(s), particularly T-helper type 1 (Th1) responses, will be dominant in any metal-reactivity responses of patients with total joint replacements (TJAs). We tested this hypothesis by evaluating lymphocyte reactivity to metal "ions" in subjects with and without total hip replacements, using proliferation assays and cytokine analysis.

Methods

Lymphocytes from young healthy individuals without an implant or a history of metal allergy (Group 1: n = 8) were used to assess lymphocyte responses to metal challenge agents. In addition, individuals (Group 2: n = 15) with well functioning total hip arthroplasties (average Harris Hip Score = 91, average time in-situ 158 months) were studied. Age matched controls with no implants were also used for comparison (Group 3, n = 8, 4 male, 4 female average age 70, range 49–80). Group 1 subjects' lymphocyte proliferation response to Aluminum+3, Cobalt+2, Chromium+3, Copper+2, Iron+3, Molybdenum+5, Manganeese+2, Nickel+2, Vanadium+3 and Sodium+2 chloride solutions at a variety of concentrations (0.0, 0.05, 0.1, 0.5, 1.0 and 10.0 mM) was studied to establish toxicity thresholds. Mononuclear cells from Group 2 and 3 subjects were challenged with 0.1 mM CrCl3, 0.1 mM NiCl2, 0.1 mM CoCl2 and approx. 0.001 mM titanium and the reactions measured with proliferation assays and cytokine analysis to determine T-cell subtype prominence.

Results

Primary lymphocytes from patients with well functioning total hip replacements demonstrated a higher incidence and greater magnitude of reactivity to chromium than young healthy controls (p < 0.03). Of the 15 metal ion-challenged subjects with well functioning total hip arthroplasties, 7 demonstrated a proliferative response to Chromium, Nickel, Cobalt and/or Titanium (as defined by a statistically significant >2 fold stimulation index response, p < 0.05) and were designated as metal-reactive. Metals such as Cobalt, Copper, Manganese, and Vanadium were toxic at concentrations as low as 0.5 mM while other metals, such as Aluminum, Chromium, Iron, Molybdenum, and Nickel, became toxic at much higher concentrations (>10 mM). The differential secretion of signature T-cell subsets' cytokines (Th1 and Th2 lymphocytes releasing IFN-gamma and IL-4, respectively) between those total hip arthroplasty subjects which demonstrated metal-reactivity and those that did not, indicated a Th1 type (IFN-gamma) pro-inflammatory response.

Conclusion

Elevated proliferation and production of IFN-gamma to metals in hip arthroplasty subjects' lymphocytes indicates that a Th1 (vs. Th2) type response is likely associated with any metal induced reactivity. The involvement of an elevated and specific lymphocyte response suggests an adaptive (macrophage recruiting) immunity response to metallic implant debris rather than an innate (nonspecific) immune response.

Summary

Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytological appearance of large-scale chromatin structures.

Graphical Abstract

Highlights

► High-resolution chromatin analysis of active and inactive X chromosomes ► Gene promoters have a transcription-dependent open chromatin structure ► Active and inactive X chromosomes have similar bulk 30 nm chromatin fiber structures ► Transcription promotes the decompaction of large-scale chromatin structures