Ni(II) affects ubiquitination of core histones H2B and H2A

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Ni(II) affects ubiquitination of core histones H2B and H2A Aldona A. Karaczyn 1 , Filip Golebiowski, Kazimierz S. Kasprzak⁎ Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Bldg. 538, Room 205E, Frederick, MD 21702-1201, USA

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

The molecular mechanisms of nickel-induced malignant cell transformation include effects

Received 15 November 2005

altering the structure and covalent modifications of core histones. Previously, we found that

Revised version received

exposure of cells to Ni(II) resulted in truncation of histones H2A and H2B and thus

19 June 2006

elimination of some modification sites. Here, we investigated the effect of Ni(II) on one such

Accepted 20 June 2006

modification, ubiquitination, of histones H2B and H2A in nuclei of cultured 1HAEo− and

Available online 22 June 2006

HPL1D human lung cells. After 1–5 days of exposure, Ni(II) up to 0.25 mM stimulated monoubiquitination of both histones, while at higher concentrations a suppression was found. Di-

Keywords:

ubiquitination of H2A was not affected except for a drop after 5 days at 0.5 mM Ni(II). The

Nickel(II)

decrease in mono-ubiquitination coincided with the appearance of truncated H2B that lacks

Histones

the K120 ubiquitination site. However, prevention of truncation did not avert the decrease of

Ubiquitination

H2B ubiquitination, indicating mechanistic independence of these effects. The changes in

Histone H2B

H2B ubiquitination did not fully coincide with concurrent changes in the nuclear levels of

Histone H2A

the ubiquitin-conjugating enzymes Rad6 and UbcH6. Overall, our results suggest that

Lung cells

dysregulation of H2B ubiquitination is a part of Ni(II) adverse effects on gene expression and

Rad6

DNA repair which may assist in cell transformation.

UbcH6

Introduction Histone proteins associated with nuclear DNA to form chromatin appear to be a major target for carcinogenic nickel [1–5]. In our previous studies, we have demonstrated that Ni(II) exposure resulted in structural modifications of histones H2A and H2B in cultured cells, consisting of truncation of H2A molecule at its C-terminal end, and H2B molecule at both Nand C-terminal ends. In some follow-up experiments, we also noticed that the appearance of the truncated versions of both histones coincided with a marked decrease in the level of their mono-ubiquitination that implied a possible mechanistic association between those effects. Unlike the poly-ubiquitination, whose function is tagging proteins for proteosomal degradation, mono-ubiquitination is believed to be a part of

Published by Elsevier Inc.

multifunctional covalent modifications of histones which also include acetylation, methylation, phosphorylation, and others [6–9]. They are all involved in organizing chromatin into transcriptionally active and inactive regions. In mammalian cells, ubiquitin can be bound to the K119 residue of H2A and K120 residue of H2B [10–12]. These sites are close to the sites of Ni(II)-mediated histone truncation: E121 in histone H2A [4,13,14] and K116 in histone H2B [2], thus suggesting a possible accomplice role for ubiquitin in the truncation effect. It has been suggested that along with other modifications, histone ubiquitination may affect chromatin structure [6,15,16], and this could assist in the truncation, e.g., by presenting the target peptide bonds for hydrolytic/enzymatic cleavage. Nickel compounds have been demonstrated in several in vitro experiments to affect histone acetylation and

⁎ Corresponding author. Fax: +1 301 846 5946. E-mail address: [email protected] (K.S. Kasprzak). 1 Current address: Wise Laboratory of Environmental and Genetic Toxicology, Department of Applied Medical Sciences, University of Southern Maine, 96 Falmouth Street, 178 Science Building, Portland, ME 04103, USA. 0014-4827/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.yexcr.2006.06.025

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methylation [17–19], whereas influence of this metal on histone ubiquitination has been thus far reported only briefly [20]. Therefore, the aim of the present study was to test nickel effect on the ubiquitination of histones H2B and H2A in more detail, including the dependence on concentration and exposure time, and especially the temporal coincidence and possible mechanistic connection of ubiquitination with the observed truncation of histone H2B.

Materials and methods Cell culture Human pulmonary HPL1D cells, a generous gift of Dr. T. Takahashi, Nagoya, Japan, and human airway epithelial lung 1HAEo− cells, obtained from Dr. D.C. Gruenert [21], were cultured for 1 to 5 days with 0.05–0.5 mM Ni(II) acetate (SigmaAldrich, St. Louis, MO) added to the culture media at approximately 70% of cell confluence. The cells were grown at 37°C under 5% CO2-containing air in the EMEM, or HAM'S F12 media (Biofluids Inc., Rockville, MD) supplemented with 10% of fetal bovine serum (Gemini Bio-Source, Woodland, CA) for 1HAEo− and HPL1D cells, respectively. In one experiment, cells were also grown with and without Ni(II) in the presence of the calpain inhibitor PD150606 (EMD Biosciences, San Diego, CA), as described elsewhere [2]. At termination of exposure, the media were discarded, the cells were rinsed twice with icecold PBS, pH 7.4, and only viable cells attached to the bottom of the flasks were collected for histone analysis. The untreated control cells were harvested when the cultures approached 95% confluence. As found by the Trypan blue exclusion method and proliferation after Ni(II) exposure, the viability of the adherent cells after 5 days ranged from approximately 90% at the lowest to 55–65% at the highest Ni(II) concentration tested, with the HPL1D cells being slightly more sensitive to nickel toxicity than 1HAEo− cells [2].

Protein extraction and gel electrophoresis Histones and other proteins were isolated from cell nuclei by extraction with 0.5 M HCl or 0.42 M NaCl (UbcH6 only), as described previously [22,23], and their total concentration in the extracts was measured using the Bradford method with albumin calibration (Pierce, Rockford, IL). The proteins were then separated by gel electrophoresis using a 10% or 4–12% gradient NuPage SDS Bis–Tris gels (Invitrogen, Carlsbad, CA) under reducing conditions provided by 10% v/v β-mercaptoethanol. Equal amounts of total protein (5 to 20 μg/well) were loaded on the gels and separated in 2-(-N-morpholino) ethano-sulfonic acid (MES) running buffer at 70–150 V for 1.5 h.

Western blotting Nuclear proteins separated by electrophoresis were transferred at 4°C onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ) in 25 mM [3-[(1,1dimethyl-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid] (AMPSO) buffer (Sigma-Aldrich), pH 9.5, at 35 V for 1.5 h;

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or in NuPage transfer buffer (Invitrogen, Carlsbad, CA) with 20% methanol, at 55 V for 2 h (UbcH6 blots only). The histone bands were visualized with the use of primary rabbit anti-H2B and anti-H3 antibodies (Upstate Biotechnology, Lake Placid, NY) or mouse anti-ubiquitinated-H2A antibodies (Upstate Biotechnology). Anti-ubiquitin monoclonal mouse antibodies (Covance Research Products, Inc., Berkeley, CA) were used to detect ubiquitinated histone fractions. To detect methylated histone H3, monoclonal anti-dimethyl-K4/H3 histone antibodies from Upstate Biotechnology were employed. The antiRad6 rabbit antibodies (against the C-terminal region of human, mouse, and rat Rad6 proteins) were from Zymed Laboratories (Invitrogen). The anti-UbcH6 rabbit antibodies (against full-length human UbcH6 protein) were purchased from Boston Biochem (Cambridge, MA). The mouse anti-β actin antibodies originated from Abcam (Cambridge, MA). Anti-rabbit (Cell Signaling Technology, Beverly, MA) or antimouse antibodies (Amersham Pharmacia Biotech), labeled with horseradish peroxidase, served as the secondary antibodies together with Super Signal (Pierce, Rockford, IL) reagents to detect the luminescent signal.

Results Ni(II) affects histone ubiquitination in a hormetic manner Western blots of nuclear histones separated from 1HAEo− and HPL1D cells, shown in Figs. 1 and 2, revealed that the ubiquitinated histone H2B was present in only one, the monoubiquitinated form. Ni(II) effect on the H2B ubiquitination level could be either stimulatory or inhibitory, depending on the dose. In addition, the magnitude of the effect depended on cell line and time of exposure. Thus, in 1HAEo− cells, maximum stimulation of the ubiquitination was reached in 24 h at 0.25 mM Ni(II), and this pattern persisted for 5 days of exposure. In HPL1D cells, the maximum stimulation was likewise reached at 0.25 mM Ni(II) in 24 h with, however, a conspicuous shift to 0.1 mM Ni(II) at later times. Ni(II) at concentrations above 0.25 mM tended to suppress the ubiquitination below the control level, especially in HPL1D cells and after longer exposures. The suppression concurred with the appearance of truncated histone H2B (2), q-H2B (Fig. 2). The identity of the ubiquitinated histone H2B band was established with the use of a combination of specific antihistone H2B antibodies and anti-ubiquitin antibodies. The latter revealed the presence of a second ubiquitinated protein band near the band reactive with anti-H2B antibodies, and also a third band between the 30 and 40 kDa markers, i.e., in a region where no bands reactive with anti-H2B antibodies were observed. Specific anti-ubiquitinated histone H2A antibodies helped us to find out that these additional bands represented ubiquitinated histone H2A (Figs. 1 and 3). Importantly, unlike histone H2B, histone H2A presented two ubiquitinated forms, one modified by mono- and the other by di-ubiquitination. For shorter exposures, the changes in mono-ubiquitination of histone H2A induced by Ni(II) were smaller and did not follow the corresponding Ni(II) dose-dependent changes in histone H2B. However, in the 3- and 5-day experiments, the changes in mono-ubiquitination levels became quite similar for both

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controls. Thus, this pattern did only partially resemble the corresponding changes in histone ubiquitination.

Decrease in ubiquitination coincides with, but is not caused by Ni(II)-induced truncation of histone H2B Ni(II) exposures that decrease histone H2B ubiquitination also cause truncation of this histone (Fig. 2). Since the Ni(II)mediated H2B truncation is executed by calcium-activated proteases, calpains [2], it seemed possible that the truncated H2B (q-H2B) originated from the pool of mono-ubiquitinated H2B; the ubiquitin tag could make H2B sensitive to calpain attack. To test this possibility, we looked at the ubiquitination status of H2B in extracts derived from cells exposed to 0.25 and 0.5 mM Ni(II) for 5 days in the presence of a specific calpain inhibitor PD150606 (3-(4-Iodophenyl)-2-mercapto-(Z)-2 propenoic acid; [25]). The results are shown in Fig. 4. As expected, 0.5 mM Ni(II) alone eliminated the ubiquitination and induced the truncation. However, calpain inhibition prevented only

Fig. 1 – Histone H2B ubiquitination following 24-h and 48-h exposure of 1HAEo− (A, B) and HPL1D (C, D) cells to 0 (Ctrl) or 0.05–0.5 mM Ni(II). Histones were extracted with acid from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting with anti-histone H2B antibodies (A, C) and visualization with secondary antibodies labeled to produce a chemiluminescent signal, as described in Materials and methods. The identity of the mono-ubiquitinated histone H2B (Ub-H2B) band was confirmed with the use of specific anti-ubiquitin antibodies (B, D) that also revealed the presence of a second band below the Ub-H2B band. The latter was identified as mono-ubiquitinated histone H2A with the use of specific anti-ubiquitinated-H2A (Ub-H2A) antibodies (compare Fig. 3).

histones, including a clear increase followed by an abrupt decrease with the rising Ni(II) doses. In contrast, the diubiquitination of histone H2A was less responsive to Ni(II), showing practically no change from the control level for up to 3 days of exposure. A conspicuous loss of H2A di-ubiquitination was observed only after 5 days in cells treated with 0.5 mM Ni(II) (Fig. 3).

Ni(II) effects on histone ubiquitination are associated with changes in K4 methylation of histone H3 Histone H2B ubiquitination controls the outcome of methylation at a specific lysine residue, K4, of histone H3 in yeast [24]. Therefore, to check if the changes in histone ubiquitination induced by Ni(II) would likewise affect histone H3 methylation in mammalian cells, we used the same nuclear extracts as those tested for ubiquitination, and analyzed them by Western blotting with anti-K4/H3 antibodies. What we found, as presented in Fig. 2C, was an increase in K4 methylation of H3 with Ni(II) concentration up to 0.1 mM, followed by a decrease below the control level at and above 0.25 mM Ni(II); histone H3 and β-actin served as loading

Fig. 2 – Histone H2B ubiquitination (Ub-H2B) following 3- or 5-day exposure of 1HAEo- (A) and HPL1D (B) cells to 0 (Ctrl) or 0.05 B 0.5 mM Ni(II). The marked decrease in the ubiquitination level at 0.5 mM Ni(II) is accompanied by histone H2B truncation (q-H2B) and a decrease in di-methylation of lysine 4 in histone H3 (Me(K4)H3) (C; 1HAEo- cells). Histones were extracted from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting with antibodies against histones H2B, H3, H3 di-methylated at lysine 4, or b-actin, and visualization with secondary antibodies labeled to produce a chemiluminescent signal, as described in Materials and methods. Blots in panel C were from the same gel; histone H3 and b-actin served as loading controls.

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Fig. 3 – Histone H2A ubiquitination following 3-day (A) or 5-day (B) exposure of 1HAEo− cells to 0 (Ctrl) or 0.05–0.5 mM Ni (II). The blot shows the positions of mono-ubiquitinated (Ub-H2A) and di-ubiquitinated (diUb-H2A) histone bands. Histones were extracted with acid from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting with anti-ubiquitinated histone H2A antibodies and visualization with secondary antibodies labeled to produce a chemiluminescent signal, as described in Materials and methods.

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Fig. 5 – The levels of Rad6 protein following 48-h and 5-day exposures of 1HAEo− cells to 0 (Ctrl) or 0.05–0.5 mM Ni(II). Proteins were extracted from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting. Anti-Rad6 and anti-histone H2B antibodies (loading control) and visualization with secondary antibodies labeled to produce a chemiluminescent signal were used as described in Materials and methods.

zymes in the histone-ubiquitinating systems [16,26,27]. To check this presumption, we stained Western blots of the nuclear proteins separated from 1HAEo− cells with anti-Rad6 and anti-UbcH6 antibodies. As we found, the level of Rad6 decreased steadily with increasing Ni(II) concentration, down to below the control level at 0.35 and 0.5 mM Ni(II) for both 2- and 5-day exposures (Fig. 5). In contrast, under the same conditions, the level of UbcH6 showed a gradual increase (Fig. 6).

the truncation but had no influence, whatsoever, on Ni(II) effects on ubiquitination including both, the increase by 0.25 mM and decrease by 0.5 mM Ni(II). Therefore, the decline in H2B ubiquitination could not be ascribed solely to the loss of K120, the only ubiquitination site in this protein, due to truncation.

Discussion

Decrease in ubiquitination concurs with decreasing nuclear Rad6 but not UbcH6 levels

We have demonstrated previously that Ni(II) mediates truncation of both N- and C-termini of histone H2B at two

The mechanisms of the observed hormetic effect of Ni(II) on histone H2B ubiquitination status might involve enhancement or inhibition of Rad6 and/or UbcH6, considered as the E2 en-

Fig. 4 – Inhibition of the Ni(II)-mediated truncation of histone H2B does not prevent the concurrent deubiquitination of this histone. 1HAEo− cells grown for 5 days with Ni(II) in the absence and presence 0.01 mM concentration of the calpain inhibitor PD150606 (Inh.) that prevented the truncation. Histones were extracted with acid from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting with anti-histone H2B antibodies and visualization with secondary antibodies labeled to produce a chemiluminescent signal, as described in Materials and methods.

Fig. 6 – The levels of UbcH6 protein following 48-h (A) and 5-day (B) exposures of 1HAEo− to 0 (Ctrl) or 0.05–0.5 mM Ni(II). Proteins were extracted from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting. Anti-UbcH6 and anti-β-actin antibodies (loading control) and visualization with secondary antibodies labeled to produce a chemiluminescent signal were used as described in Materials and methods.

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identical -KAVTK- motifs [2]. The outgoing C-terminal peptide, 117AVTKYTSSK, is taking away K120 that is the target of histone H2B ubiquitination in mammalian cells [11,28]. Indeed, our preliminary experiments revealed that under conditions leading to H2B truncation, loss of ubiquitination of this histone was also observed. To test if the latter was solely due to the loss of K120 or, perhaps, developed independently, in the present study, we first cultured human lung cells with Ni(II) for only 24 and 48 h, when the truncation does not occur. What we found was a hormetic effect of Ni(II) exposure on H2B ubiquitination, peaking around 0.2 mM Ni(II), and declining above this concentration in spite of the lack of truncation. The same appeared to be true for the longer exposures that induced the H2B truncation. Thus, apparently, these effects develop independently. For up to 5 days, the pattern of changes in histone H2B ubiquitination depended on Ni(II) concentration and to a limited extent also on cell type (we also observed similar changes in rat kidney epithelial NRK-52E cells (ATCC-CRL 1571); not shown), but not on the duration of exposure. In contrast, the occurrence of the truncation required longer exposures. The present results also revealed that Ni(II) up to a certain dose caused a gradual increase in H2B ubiquitination. This could be due to up-regulation of the ubiquitin gene and/ or activation of specific enzymes, i.e., mammalian homologues of yeast E1-, E2-, and/or E3-type enzymes, including the Rad6 and UbcH6 proteins. Rad6 and its mammalian homologues have been shown in several in vitro experiments to conjugate ubiquitin with histones H2B and H2A [16,26,29,30], while UbcH6 has been identified most recently as the E2 enzyme specific for histone H2B ubiquitination in mammalian cells [27]. Inactivation of deubiquitinating enzymes by Ni(II) should also be considered. Our experiments failed to reveal Rad6 activation by Ni(II) doses increasing H2B ubiquitination. Instead, a steady decrease in Rad6 level with increasing Ni(II) concentration was observed. In contrast, under the same conditions, UbcH6 levels tended to increase. This could possibly indicate a direct cause/effect relationship between the UbcH6 expression and H2B ubiquitination. However, such a simple conclusion cannot be drawn because of what happened at the higher, 0.5 mM, Ni (II) concentration: a significant increase in UbcH6 level was not accompanied by any conspicuous increase in H2B ubiquitination versus control. Thus, the exact mechanisms of the observed Ni(II) effects remain elusive. Since Rad6 and UbcH6 are only two of many elements of the ubiquitinating systems [31], full spectrum of Ni(II) effects on the latter remain to be investigated. To this juncture, special attention should be paid to mammalian homologue(s) of yeast Bre1 protein, the ubiquitin isopeptide ligase (E3) responsible for substrate H2B recognition [32–34]. Proteins of this class contain Zn(II)-binding RING fingers and may thus be functionally sensitive to interference from Ni(II) and other divalent metals [14,35]. Unfortunately, the mammalian homologues of Bre1 have been identified only very recently [27,36] and no specific antibodies were available to us for testing. Interestingly, in one of our previous studies on HPL1D cells [37], we found up-regulation of the ubiquitin gene by

low Ni(II) doses (0.1 and 0.2 mM), but down-regulation by higher doses (>0.4 mM), a pattern very closely resembling the H2B ubiquitination pattern of the present experiment. The concurrent slight increase in UbcH6 level by low Ni(II) doses might further enhance H2B ubiquitination facilitated by the increased expression of ubiquitin. The down-regulation of the ubiquitin gene by higher Ni(II) concentrations could, in turn, trigger a compensatory up-regulation of the UbcH6 expression, as observed. This likely scenario does not exclude other probable effects of Ni(II), such as (a) activation by Ni(II) of the variety of deubiquitinating enzymes, including some components of the SAGA histone acetylation complex [16,38,39], or (b) the loss of the K120 ubiquitination site due to H2B truncation. However, the first possibility looks unlikely if we consider the fact that Ni(II) at concentrations 0.4 mM and higher was found to lower histone acetylation through inhibition of relevant conjugating enzymes (histone acetyl transferases) but not through enhancement of histone deacetylases [17,19,40]. Therefore, it is possible that Ni(II) at higher concentrations can inhibit UbcH6 enzymatic activity, too (and the observed overexpression of UbcH6 was to compensate for the lost activity). Mechanism (b), above, can be excluded as the sole cause of the observed decrease in ubiquitination by the results of our short-term experiments, in which the truncation does not occur, and more definitively by the experiment with calpain inhibitor PD150606. In concordance with our previous findings [2], PD 150606 abolished the truncation but did not detectably alter the suppression of H2B ubiquitination by high-dose Ni(II). Therefore, Ni(II) effects on histone H2B ubiquitination and truncation are not interdependent and ubiquitination cannot be considered a signal for calpain attack on histone H2B. Besides the mono-ubiquitinated histone H2B, Western blotting analysis revealed the presence of mono- and diubiquitinated versions of histone H2A in our histone extracts. Both forms of ubiquitinated H2A have already been reported in the literature [12]. Interestingly, the dose- and timedependent changes in mono-ubiquitination of histones H2A generally followed those of histone H2B despite the fact that ubiquitination of these two histones may have opposite effect on gene transcription [41]. However, the di-ubiquitination of H2A was hardly affected by Ni(II), except for its marked decline after 5 days at the highest Ni(II) dose, concurrently with the drastic loss of Rad6. In cells, the Ni (II)-induced truncation of the C-terminal of histone H2A [4] does not eliminate the ubiquitination site. Truncation can be, therefore, ruled out as the cause of H2A ubiquitination decrease. This, again, points at Ni(II) effects on the availability of ubiquitin and on the balance between activities of specific enzymatic ubiquitinating and deubiquitinating systems for histone H2A [41]. From our results, it looks like diubiquitinated H2A is a pretty stable component of chromatin, insensitive to changes in the levels of ubiquitin and Rad6 induced by Ni(II), except for harsher conditions of a high dose combined with longer exposure time. Even in this context, however, it is not clear which group of enzymes is responsible for the eventual decrease. Unveiling the exact mechanisms underlying the present results will require more experiments aimed at identification of Ni(II) effects not only

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on the expression and function of the individual components of the very complex ubiquitination/deubiquitination machinery (which is still least understood among the histonemodifying enzymatic and signaling systems [16]), but also Ni (II) effects on proper assembly and function of this machinery. Covalent modifications of core histones in chromatin, such as acetylation, methylation, phosphorylation, ribosylation, ubiquitination, sumoylation, and possibly others (e.g., deimination and biotinylation) serve as regulatory mechanisms of gene transcription [7,16,38,42–45]. Many experimental data indicate that removal of a histone modification opposes the effect of its addition. Thus, for example, increased ubiquitination of histone H2B was associated with gene silencing [46], while a decrease in ubiquitination of histones H2B and H2A was associated with gene activation [47]. Generally, however, the function of the ubiquitin adduct is more complex since increase and decrease in ubiquitination of histone H2B were both found to be involved in transcriptional activation, depending on gene location [7,16,38]. These conflicting results are currently explained by the impact of H2B ubiquitination on modifications of other histones especially through methylation and acetylation [16,24]. As reported by Sun and Allis [24], in the yeast Saccharomyces cerevisiae, conjugation of histone H2B with ubiquitin is a prerequisite for methylation of histone H3 at K4 residue. And, vice versa, deubiquitination of H2B has been known to cause reduction of K4 methylation in this histone [38]. In turn, high level of K4/H3 methylation is indicative of active transcription, whereas for low level the opposite is true [48]. A similar dependence, but also some significant differences in transcriptional activity of the K4/H3 methylated regions, has been reported for chicken chromatin [49]. The present results reveal that a decrease in H2B ubiquitination at high-dose Ni(II) does, indeed, concur with reduction in K4/H3 methylation. In the lower Ni(II) concentrations region, however, the changes in methylation are not fully synchronized with the corresponding changes in ubiquitination. This indicates that the correlation between H2B ubiquitination and K4/H3 methylation, and thus transcriptional activity of genes, may be disturbed by Ni(II) exposure. Since the ubiquitin-conjugating enzyme Rad6 is involved in ubiquitination of not only histones but also of other nuclear proteins, to which it is directed by particular E3-type enzymes (referenced in [34] and [33]), our finding of suppression of its nuclear level by Ni(II) may have broader biological significance. Rad6 is highly conserved among eukaryotic cells and is considered an integral part of the post-replication DNA repair systems [50]. In cultured mammalian cells, imbalances in the expression of Rad6 homologues were found to compromise genomic integrity and lead to chromosomal instability and malignant transformation [26,50,51]. In human lung cancer, the expression of Rad6 homologue Hrad6B was only 40% that found in non-malignant lung tissue; and, interestingly, tobacco smoking tended to lower that expression even more, indicating that dysfunction of Hrad6B might have a role in lung carcinogenesis in smokers [52]. Thus, other chemical lung carcinogens, including nickel, may act in a similar way. For Ni(II), known to induce promutagenic DNA damage and at the same time inhibit its repair [53–55] (reviewed in [5]), the observed lowering of nuclear Rad6 level in general, and

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dysregulation of the H2B ubiquitination-associated signaling of the damage in particular [56], could very likely contribute to the failure of the DNA repair systems and thus assist in malignant transformation of cells.

Acknowledgments The authors wish to thank Dr. Y.-H. Shiao for critical comments on the manuscript. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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