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Am J Physiol Cell Physiol 283: C878–C884, 2002. First published June 13, 2002; 10.1152/ajpcell.00107.2002. Exposure to UV light causes increased biotinylation of histones in Jurkat cells DOROTHEA M. PETERS,1 JACOB B. GRIFFIN,1 J. STEVEN STANLEY,2 MARY M. BECK,3 AND JANOS ZEMPLENI1,4 Departments of 1Nutritional Science and Dietetics, 3Animal Science, and 4 Biochemistry, University of Nebraska at Lincoln, Lincoln, Nebraska 68583; and 2Arkansas Children’s Hospital Research Institute, Little Rock, Arkansas 72202 Received 18 March 2002; accepted in final form 14 May 2002 IN MAMMALS, BIOTIN SERVES as a covalently bound coenzyme for four biotin-dependent carboxylases: acetylCoA carboxylase (EC 6.4.1.2), pyruvate carboxylase (EC 6.4.1.1), propionyl-CoA carboxylase (PCC; EC 6.4.1.3), and 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) (27). Degradation of holocarboxylases by proteases leads to the formation of biotinylated peptides. These peptides are further degraded by biotinidase (EC 3.5.1.12) to release free biotin, which can then be used for the synthesis of new holocarboxylases (24). Recently, Hymes et al. (8) proposed an enzymatic mechanism in which biotinidase acts as biotinyl trans- ferase: breakdown products of carboxylases serve as biotinyl donors, and histones act as biotinyl acceptors. Covalent binding of biotin to histones suggests that biotin might play a role in transcription, replication, or repair of DNA. On the basis of these pioneering studies, our laboratory has demonstrated that human cells indeed biotinylate histones (22). Histones are the primary proteins that mediate the folding of DNA into chromatin (25). Chromatin consists of a repetitive nucleoprotein complex, the nucleosome. Each (mono)nucleosome consists of DNA that is wrapped around an octamer of core histones (2 molecules each of histones H2A, H2B, H3, and H4); nucleosome assembly is completed by incorporation of a linker histone such as histone H1. In vivo, histones are modified posttranslationally by acetylation, methylation, phosphorylation, ubiquitination, poly(ADP-ribosylation), and biotinylation (22, 25). Evidence has been provided that enzymatic poly(ADP-ribosylation) of histones is linked to DNA repair mechanisms (1, 4). Induction of DNA damage in mammalian cells by chemical treatment or UV irradiation causes a dramatic increase of poly(ADPribosylation) of histones (12). Poly(ADP-ribosylation) of histones catalyzes a transient dissociation from and reassociation of histones with DNA (1). It has been proposed that this mechanism may guide enzymes to sites of DNA repair (1). In addition, poly(ADP-ribosylation) might be involved in nucleosomal unfolding of chromatin in DNA excision repair (1). Also, it has been proposed that poly(ADP-ribosylation) of histone H1 might be involved in early apoptotic events (26). In the present study, we determined whether biotinylation of histones might play a role similar to poly(ADP-ribosylation) of histones in the repair of damaged DNA or in apoptosis. A human T cell line (Jurkat cells) was used to determine whether UV-induced oxidative stress and DNA damage cause breakdown of biotindependent carboxylases and increased biotinylation of histones. Address for reprint requests and other correspondence: J. Zempleni, Dept. of Nutritional Sciences and Dietetics, Univ. of Nebraska at Lincoln, 316 Ruth Leverton Hall, Lincoln, NE 68583-0806 (E-mail: [email protected]). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. apoptosis; biotin; carboxylases; DNA damage C878 0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society http://www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on June 18, 2017 Peters, Dorothea M., Jacob B. Griffin, J. Steven Stanley, Mary M. Beck, and Janos Zempleni. Exposure to UV light causes increased biotinylation of histones in Jurkat cells. Am J Physiol Cell Physiol 283: C878–C884, 2002. First published June 13, 2002; 10.1152/ajpcell.00107.2002.—Biotin in breakdown products of biotinylated carboxylases serves as substrate for biotinylation of histones by biotinidase. Here we determined whether biotinylation of histones might play a role in repair of damaged DNA and in apoptosis. Jurkat cells were exposed to UV light to induce DNA damage. Abundance of thymine dimers increased about three times in response to UV exposure, consistent with DNA damage. Biotin-containing carboxylases were degraded in response to UV exposure, as judged by Western blot analysis and carboxylase activities. Mitochondrial integrity decreased in response to UV exposure (as judged by confocal microscopy), facilitating the release of breakdown products of carboxylases from mitochondria. Biotinylation of histones increased in response to UV exposure; biotinylation of histones did not occur specifically at sites of newly repaired DNA. UV exposure triggered apoptosis, as judged by caspase-3 activity and analysis by confocal microscopy. In summary, this study provided evidence that increased biotinylation of histones in DNA-damaged cells might either be a side product of carboxylase degradation or a step during apoptosis. BIOTINYLATION OF HISTONES IN JURKAT CELLS MATERIALS AND METHODS AJP-Cell Physiol • VOL vortexing, 50 l of 0.5% Triton X-100 were added to lyse the cells. Complete lysis of cells by this procedure was confirmed by light microscopy. PCC activity was quantified as described previously with minor modifications (28). Briefly, lysed Jurkat cells were incubated with propionyl-CoA, [14C]bicarbonate, and cofactors to allow for covalent binding of [14C]bicarbonate to propionyl-CoA. After incubation, unbound [14C]bicarbonate was volatilized by addition of perchloric acid, and samples were air-dried. Finally, samples were suspended in scintillation fluid, and the bound [14C]bicarbonate was quantified by liquid scintillation counting. Biotinylation of biotin-dependent carboxylases. Holocarboxylases (as opposed to apocarboxylases) contain covalently bound biotin. Biotin in holocarboxylases can be probed with streptavidin peroxidase, using Western blots of cellular protein extracts. In this study, 6 ⫻ 106 Jurkat cells were lysed in 480 l of detergent (BugBuster, Novagen), immediately followed by the addition of 1.6 l of protease inhibitor cocktail (catalog no. P-8340; Sigma) and 0.5 l of DNase (Benzonase, Novagen). Samples were incubated for 3 min at room temperature to allow for hydrolysis of DNA by DNase. Next, 65 l of sample were mixed with 25 l of loading buffer (Nupage 4x; Invitrogen, Carlsbad, CA) and 10 l of 1 M dithiothreitol; samples were heated for 10 min at 70°C and loaded onto a 3–8% Tris-acetate gel (Invitrogen). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes by electroblotting. Biotin in carboxylases was probed by using streptavidin peroxidase in analogy to our previous studies of biotinylated histones (22). Biotinidase activity. Cell suspension containing 10 ⫻ 106 Jurkat cells was centrifuged (250 g for 10 min), and the supernatant was discarded. Cell pellets were suspended in 50 l of Triton X-100 and 180 l of water to cause lysis. Activity of biotinidase in the lysate was determined by measuring the hydrolysis of N-D-biotinyl-p-aminobenzoic acid as described previously (22). Apoptosis. Jurkat cells were monitored for an early marker of apoptosis (activity of caspase-3) by using a commercially available kit (ApoAlert caspase-3 colorimetric assay kit; Clontech). Confocal laser microscopy. Mitochondria in Jurkat cells were stained by using a commercially available kit according to the manufacturer’s instructions (MitoTracker CMXRos; Molecular Probes, Eugene, OR). Images of stained mitochondria were taken by Dr. You Zhou (Beadle Center for Biotechnology, University of Nebraska-Lincoln) by using a confocal laser scanning microscope (Bio-Rad MRC1024ES). Chicken chromatin. Previous studies have revealed that transcriptionally active chromatin has an enhanced sensitivity to DNase compared with transcriptionally inactive chromatin (20); digestion of chromatin by micrococcal nuclease and subsequent salt fractionation of the digest can be used to isolate transcriptionally active and inactive chromatin, respectively. In the present study, nuclei from chicken erythrocytes were used to isolate transcriptionally active chromatin and inactive chromatin by using previously published methods (16, 19) with minor modifications. Briefly, 25 ml of blood from adult White Leghorn chickens were collected in an equal volume of EDTA-containing saline. Erythrocytes were collected by centrifugation. Nuclei were isolated by washing erythrocytes in detergent-containing buffer. Nuclei were suspended in 20 ml of digestion buffer and incubated with 1,000 units of micrococcal nuclease (Takara, Shiga, Japan) at 37°C for 20 min; digestion was terminated by addition of 2 ml of 10 mM EGTA, and nuclei were collected by centrifugation. By using digested nuclei, chromatin was extracted sequentially with 5 ml of extraction buffer (10 mM Tris, pH 7.0, 2 mM 283 • SEPTEMBER 2002 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on June 18, 2017 Cells and culture conditions. Jurkat cells (clone E6-1) were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in humidified atmosphere (5% CO2) at 37°C. Culture medium consisted of 90% RPMI 1640 (Hyclone, Logan, UT) and 10% fetal bovine serum (Hyclone), containing 100 IU/ml penicillin and 100 g/ml streptomycin. Unless otherwise noted, cells were exposed to 79 mW of UV light (254 nm) per 121 cm2 of surface area over a period of 1 h to induce DNA damage; at timed intervals, aliquots were collected before, during, and after UV irradiation for the analyses described below. Glutathione reductase. Cellular activity of glutathione reductase may decrease moderately in response to oxidative stress. Activity of glutathione reductase was measured in Jurkat cells before, during, and after exposure to UV light to confirm oxidative stress. UV-irradiated Jurkat cells and controls (5 ⫻ 106 cells) were centrifuged (250 g for 10 min), and the supernatant was discarded. The cell pellet was suspended in a mixture of 250 l of distilled water, 250 l of detergent (BugBuster, Novagen, Madison, WI), 1 l of protease inhibitor cocktail (catalog no. P-8340; Sigma, St. Louis, MO), and 1 l of DNase (Benzonase, Novagen). Samples were incubated for 3 min at room temperature to allow for hydrolysis of DNA by DNase. Activity of glutathione reductase in the lysate was measured as described previously (21). The assay is based on monitoring the oxidation of NADPH (decrease of absorbance at 340 nm) by glutathione reductase over a period of 15 min. DNA damage. UV light causes DNA damage, e.g., formation of thymine dimers. Here, ELISA was developed on the basis of previously published procedures (13, 14) to quantify thymine dimers in DNA. Briefly, 96-well plates (Nunc Maxisorb surface; Fisher, Pittsburgh, PA) were coated with 16 g of protamine sulfate (in 100 l of water) per well for 90 min at room temperature; the plates were washed three times with water and allowed to dry (stable for up to 3 mo). DNA was isolated from 8 ⫻ 106 UV-exposed Jurkat cells (79 mW over 1 h) and normal controls by using a commercially available kit (DNeasy tissue kit; Qiagen, Valencia, CA). DNA was quantified spectrophotometrically at 260 nm. DNA (50 ng) in 50 l of phosphate-buffered saline was added per protamine sulfate-coated well; plates were dried at 37°C for 16 h to adsorb DNA to the wells. The plates were washed, and nonspecific binding sites were blocked as described previously (14). Mouse anti-thymine dimer monoclonal antibody (0.1 ml) was added to each well (clone H3, Sigma; final dilution 1:10,000); plates were incubated at 37°C for 90 min. Plates were washed three times (14), and 0.1 ml of peroxidase-conjugated goat anti-mouse IgG (Sigma; final dilution 1:10,000) was added to each well; plates were incubated at 37°C for 90 min. Plates were washed three times. For color development, 100 l of 3,3⬘,5,5⬘-tetramethylbenzidine onecomponent microwell substrate (catalog no. 50-76-04; Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added to each well; plates were incubated for 0.5 h at room temperature. Reaction was terminated by addition of 100 l of stop solution (catalog no. 50-85-04; Kirkegaard and Perry Laboratories), and the absorbance was read at 450 nm in an Emax microwell plate reader (Molecular Devices, Sunnyvale, CA). PCC activity. This assay quantifies the binding rate of radioactive bicarbonate to propionyl-CoA, catalyzed by PCC in samples of lysed cells. Aliquots of cell suspension (8 ⫻ 106 cells) were centrifuged for 10 min at 2260 g; supernatants were discarded, and the cell pellets were suspended in 180 l of homogenization buffer as described previously (28). After C879 C880 BIOTINYLATION OF HISTONES IN JURKAT CELLS AJP-Cell Physiol • VOL (20a). StatView 5.0.1 (SAS Institute, Cary, NC) was used to perform all calculations. Differences were considered significant if P ⬍ 0.05. Data are expressed as means ⫾ SD. RESULTS UV-induced oxidative stress and DNA damage. Studies were conducted to confirm that UV exposure of Jurkat cells caused oxidative stress and DNA damage. Previous studies have provided evidence that oxidative stress might be associated with a moderate decrease of glutathione reductase activities (5) and that UV exposure causes the formation of thymine dimers in DNA (6). In the present study, cells were exposed to UV light for 1 h; samples were collected at timed intervals before, during, and after UV exposure. Cells responded to UV exposure with a 15% decrease of activities of glutathione reductase compared with cells before UV exposure (data not shown). This is consistent with the hypothesis that UV exposure of Jurkat cell caused oxidative stress. Note that the coenzyme of glutathione reductase (flavin adenine dinucleotide) is light sensitive; thus photodegradation of flavin adenine dinucleotide could have contributed to decreased activity of glutathione reductase in response to UV exposure. Next, concentrations of thymine dimers were measured as a marker for UV-induced DNA damage. Thymine dimers were quantified immediately after UV exposure, i.e., when dimer concentrations reach a maximum. The abundance of thymine dimers was significantly greater in UV-exposed cells compared with normal controls [0.32 ⫾ 0.05 vs. 0.09 ⫾ 0.03 units of absorbance at 450 nm per 50 ng of DNA (P ⬍ 0.01)], suggesting that UV exposure caused DNA damage. Biotin-dependent carboxylases. Previous studies have suggested that biotin in breakdown products of biotinylated carboxylases serve as substrate for biotinylation of histones (8). We determined whether Jurkat cells respond to UV exposure with increased degradation of biotin-dependent carboxylases, providing substrate for biotinylation of histones. Cells were exposed to UV light for 1 h; cells were collected at timed intervals before, during, and after UV exposure. Cells responded to UV exposure with a progressive degradation of holoenzymes of pyruvate carboxylase, PCC (␣-chain), and 3-methylcrotonyl-CoA carboxylase (␣chain; Fig. 1). Nineteen hours after UV exposure, holocarboxylases were barely detectable in cell extracts. Note that the fourth carboxylase, acetyl-CoA carboxylase, was not detectable in Jurkat cells. The biotinylated ␣-chains of PCC and 3-methylcrotonyl-CoA carboxylase have similar molecular masses (80 and 83 kDa, respectively) and migrate as one single band on polyacrylamide gels (Fig. 1). Next, activities of PCC were measured in Jurkat cells at timed intervals before, during, and after exposure to UV light. PCC activities decreased by about 40% during UV exposure and then transiently returned to normal levels before decreasing again by ⬃35% compared with cells before UV exposure (Fig. 2). This decrease of PCC activity is somewhat less than what was expected based on staining of holocarboxy- 283 • SEPTEMBER 2002 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on June 18, 2017 MgCl2, 1% thiodiglycol, 25 mM KCl, 10 mM EGTA, and 30 mM sodium butyrate) containing increasing concentrations of NaCl (0.1 and 0.6 M) in analogy to Nickel et al. (16). Previous studies have provided evidence that transcriptionally active chromatin is enriched in the fraction extracted with 0.1 M NaCl; inactive chromatin is enriched in the fraction extracted with 0.6 M NaCl (16, 19, 20). Finally, histones in chromatin extracts were isolated by using 1 M HCl (final concentration) as described in our previous studies (22). Note that this procedure of chromatin fractionation has the following limitations. 1) Partitioning of transcriptionally active and inactive chromatin is not complete; i.e., the fraction extracted with 0.1 M NaCl may contain some transcriptionally inactive chromatin, and the fraction extracted with 0.6 M NaCl may contain some transcriptionally inactive chromatin (16). 2) The fraction denoted “transcriptionally active chromatin” may be enriched for both genes that are transcriptionally active and genes that are no longer transcriptionally active but that have been transcribed at earlier states of cellular differentiation. Thus Rocha et al. (20) have proposed to refer to these regions as “transcriptionally competent chromatin.” Biotinylation of histones in UV-treated Jurkat cells. Histones were extracted from Jurkat cell nuclei (4 ⫻ 106 cells) by using HCl as described previously (22). Equimolar amounts of histones (as judged by gel densitometry after staining with Coomassie blue) were electrophoresed by using 16% Tris glycine gels (Invitrogen); biotin in histones was probed by using streptavidin peroxidase as described previously (22). Alternatively, biotin in histones was probed by using an anti-biotin antibody (22). Previous studies have provided evidence that newly repaired regions of DNA are initially more sensitive than bulk DNA to digestion by nuclease (18). In the present study, experiments were conducted to provide evidence as to whether biotinylation of histones occurs specifically at sites of newly repaired DNA. In these experiments, Jurkat cells were exposed to UV light for 1 h as described in DNA damage; controls were not exposed to UV light. One hour after UV exposure, 96 ⫻ 106 cells were harvested by centrifugation (250 g for 10 min) and nuclei were isolated as described previously (22). Nuclei were digested with micrococcal nuclease as described in Chicken chromatin for nuclei from chicken erythrocytes with the following minor modification: nuclei from Jurkat cells were suspended in a volume of 1 ml of digestion buffer, and 50 units of micrococcal nuclease were added. Next, nuclei were collected by centrifugation, and chromatin was extracted by using 0.25 ml of extraction buffer containing 0.6 M sodium chloride at 4°C for 20 min. Extract (16 l) was mixed with 4 l of 5⫻ Trisborate-EDTA (TBE) high-density loading buffer (Invitrogen) and electrophoresed under nondenaturing conditions on a 6% TBE DNA retardation gel (Invitrogen). Gels were stained with 0.1 g/ml ethidium bromide (final concentration) for 1 h, followed by visual inspection with a Kodak EDAS 290 documentation and analysis system (Rochester, NY), to confirm digestion of DNA by nuclease. A second set of gels was used to electroblot histones in chromatin onto polyvinylidene difluoride membranes; biotin in histones was probed with streptavidin peroxidase as described previously (22). Statistics. Homogeneity of variances among groups was tested using Bartlett’s test (20a). Variances were homogenous; therefore, data were not transformed before further statistical testing. Significance of differences among groups was tested by one-way ANOVA. Fisher’s protected least significant difference procedure was used for post hoc testing BIOTINYLATION OF HISTONES IN JURKAT CELLS Fig. 1. UV exposure affects levels of holocarboxylases in Jurkat cells. Cells were exposed to UV light (254 nm) for 1 h. Aliquots were collected before (0 h), during (hours of UV), and after (hours after UV) exposure to UV light, and the following holocarboxylases were quantified by Western blot analysis: pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). The biotinylated ␣-chains of MCC and PCC have similar molecular masses (83 and 80 kDa, respectively) and migrate as one single band. Fig. 2. UV exposure affects activities of PCC in Jurkat cells. Cells were exposed to UV light (254 nm) for 1 h. Aliquots were collected before, during, and after exposure to UV light, and PCC activity was measured. *P ⬍ 0.05; **P ⬍ 0.01, significantly different from controls; n ⫽ 8. AJP-Cell Physiol • VOL Fig. 3. UV exposure affects biotinylation of histones in Jurkat cells. Cells were exposed to UV light (254 nm) for 1 h. Aliquots were collected before, during, and after exposure to UV light, and biotinylation of histones (H1, H2A, H2B, H3, and H4) as quantified by probing with streptavidin. histones, providing further evidence that histones were biotinylated (data not shown). Previous studies have suggested that biotinylation of histones is catalyzed by biotinidase (8). In the present study, biotinidase activity was quantified in Jurkat cells at timed intervals before, during, and after UV irradiation. Biotinidase activity was similar for all samples: ⬃40 pmol of p-aminobenzoic acid released per 106 cells over 30 min. Possible explanations for increased biotinylation of histones despite of constant biotinidase activity are provided in DISCUSSION. Previous studies have provided evidence that newly repaired regions of DNA are initially more sensitive than bulk DNA to digestion by nuclease (18); thus newly repaired DNA is enriched in the mononucleosomal fraction of nuclease-digested chromatin. Theoretically, if biotinylation of histones occurs specifically at sites of DNA damage, the abundance of biotinylated histones should be greater in mononucleosomes compared with large fragments of chromatin. In the present study, biotinylation of histones was determined in chromatin from UV-irradiated Jurkat cells and normal controls after digestion with micrococcal nuclease. First, digestion of chromatin by nuclease was confirmed by staining of DNA gels with ethidium bromide (Fig. 4). Next, biotinylated histones in digested chromatin were probed with streptavidin. In both UVirradiated cells and normal controls, biotinylated histones were located in large fragments of chromatin (barely visible in Fig. 4) rather than in mononucleosomes. This finding suggests that biotinylation of histones did not occur specifically at sites of DNA damage or newly repaired DNA. Nevertheless, biotinylation of histones in UV-damaged chromatin might be important for unfolding of higher-order chromatin structures. In analogy, regions of chicken chromatin that contain biotinylated histones are relatively resistant to digestion by micrococcal nuclease. Transcriptionally active chromatin and transcriptionally inactive chromatin were isolated from nuclei in chicken erythrocytes by nuclease digestion and salt fractionation; biotinylated histones were detected exclusively in the fraction containing transcriptionally inactive chromatin (Fig. 5). There is precedence for transcriptional silencing of chromatin by covalent modification of histones, e.g., methylation of lysine-9 in histone H3 (2). 283 • SEPTEMBER 2002 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on June 18, 2017 lases with streptavidin peroxidase (Fig. 1); within-day variations may account for the difference between these two experiments. In summary, the findings presented here are consistent with the hypothesis that UV irradiation causes breakdown of holocarboxylases, thereby providing substrate for biotinylation of histones. Also, studies outlined below provided evidence for decreased integrity of mitochondria in response to UV exposure, suggesting that breakdown products of holocarboxylases may leak out of mitochondria into cytoplasm. Biotinylation of histones. Jurkat cells were exposed to UV light for 1 h; cells were collected at timed intervals before, during, and after UV exposure. Biotinylation of histones increased in response to UV irradiation (Fig. 3). Apparently, the increased biotinylation of histones was not restricted to a single class of histones but was seen in classes H1, H2A, H2B, H3, and H4. Histones H2A and H2B electrophoresed as one single band. Thus it remains uncertain whether the probe streptavidin bound to histone H2A, H2B, or both. In parallel experiments, biotin in histones was probed with an antibody to biotin. The antibody bound to C881 C882 BIOTINYLATION OF HISTONES IN JURKAT CELLS Taken together, the findings in UV-damaged Jurkat cells and in normal chicken erythrocytes suggest that biotinylation of histones occurs at sites of chromatin that do not contain newly repaired DNA and do not contain transcriptionally active DNA. Apoptosis. The present study is consistent with the hypothesis that UV irradiation caused apoptosis. For Fig. 5. Biotinylated histones are enriched in transcriptionally inactive chromatin. Transcriptionally active chromatin and transcriptionally inactive chromatin were isolated from nuclei in chicken erythrocytes. Histones were acid extracted, and biotin in histones was probed with streptavidin by Western blot analysis. AJP-Cell Physiol • VOL DISCUSSION This study is consistent with the hypothesis that exposure of Jurkat cells to UV light (254 nm) is associated with increased biotinylation of histones. Evidence has been provided that biotinylation of histones is caused by the following mechanistic sequence. 1) UV irradiation causes oxidative stress and DNA damage, as judged by activity of glutathione reductase and formation of thymine dimers. 2) Biotinylated carboxylases are degraded, as judged by Western blot analysis of holocarboxylases and cellular activity of PCC. 3) Breakdown products of carboxylases leak from mitochondria into cytoplasm, due to decreased integrity of mitochondria in response to UV irradiation. 4) Breakdown products of carboxylases diffuse from cytoplasm into the cell nucleus, where they are used as substrate for biotinylation of histones. 5) Cells increase biotinylation of histones in response to UV irradiation, as judged by Western blot analysis of histones. Fig. 6. UV exposure affects activities of caspase-3 in Jurkat cells. Cells were exposed to UV light (254 nm) for 1 h. Aliquots were collected before, during, and after exposure to UV light, and activity of caspase-3 was measured. *P ⬍ 0.05 vs. time 0 controls (n ⫽ 3). 283 • SEPTEMBER 2002 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on June 18, 2017 Fig. 4. Biotinylation of histones did not occur specifically at sites of newly repaired DNA in Jurkat cells. Cells were exposed to UV light (254 nm) for 1 h; controls were not UV irradiated. One hour after UV exposure, 96 ⫻ 106 cells were harvested and nuclei were digested with micrococcal nuclease. Chromatin was extracted and electrophoresed on a nondenaturing 6% TBE gel. Lane a: representative sample of nuclease-digested chromatin after staining with ethidium bromide to verify digestion of chromatin. Lane b: biotinylated histones in digested chromatin from UV-exposed cells were probed with streptavidin. Lane c: same as lane b, but cells were not UV irradiated. In both lanes b and c, biotin was associated with large fragments of DNA (barely visible on gels) rather than with mononucleosomes. example, peak activity of caspase-3 (an early marker of apoptosis) was approximately three times greater in UV-irradiated cells compared with cells before UV irradiation (Fig. 6). Note that caspase-3 activity increased later than the observed increase of carboxylase degradation and histone biotinylation (Figs. 1–3). Apoptosis in response to UV irradiation was further investigated by using confocal microscopy. Jurkat cells were collected at timed intervals before, during, and after exposure to UV light. Cells were incubated with a dye (MitoTracker) to stain mitochondria; after fixation, cells were evaluated qualitatively by microscopy. UV irradiation caused clustering of mitochondria in cells (Fig. 7B), formation of vacuoles in cells (Fig. 7E), and, eventually, lysis of cells. This decreased mitochondrial and cellular integrity in response to UV exposure parallels the increased biotinylation of histones observed in response to UV treatment (Fig. 3). Theoretically, increased biotinylation of histones might be part of the apoptotic (or necrotic) processes that occur in UVdamaged cells. BIOTINYLATION OF HISTONES IN JURKAT CELLS C883 This mechanistic sequence is consistent with previous studies by Wolf and coworkers in normal cells and in vitro experiments. These investigators have demonstrated that biotin-dependent carboxylases are degraded by proteases, leading to the release of biotinylated peptides (24). These peptides are hydrolyzed by biotinidase to release biocytin (biotinyl lysine) (3, 24). Biocytin is further hydrolyzed by biotinidase, and the biotinyl moiety is transferred to histones for covalent attachment (7–9). Currently, it remains uncertain whether increased biotinylation of histones is 1) a mechanism of DNA repair, 2) a process leading to apoptosis, or 3) a side product of metabolism that does not play a physiological role in UV-damaged cells. This study provided evidence that biotinylation of histones does not occur specifically at sites of newly repaired DNA. Rather, biotinylation of histones occurs at sites that have not been repaired (or damaged) and sites that are transcriptionally inactive. These findings, along with the observation that UV-irradiated cells entered apoptosis, suggest that biotinylation of histones might not be associated with DNA repair. Rather, increased biotinylation of histones is an artifact caused by increased availability of substrate (biocytin), or part of a series of events that lead to cell death. Nevertheless, future studies need to provide conclusive evidence as to AJP-Cell Physiol • VOL whether biotinylation of histones plays a role in DNA repair or apoptosis. Both repair and apoptosis are potentially beneficial processes that may prevent tumor initiation by eliminating abnormal DNA. The magnitude by which biotinylation of histones increased in response to UV exposure was quantitatively moderate. However, quantitatively small changes in the level of one modification of a given histone may have important effects regarding other modifications in the same histone molecule. For example, methylation of arginine-3 in histone H4 facilitates subsequent acetylation of histone H4 tails, leading to transcriptional activation (23); numerous other examples for synergistic and antagonistic modifications of histones have been identified (11). By analogy, a moderate increase of biotinylation of histones may affect other modifications of histones. Biotinylation of histones is catalyzed by biotinidase (8). In the present study, evidence has been provided that biotinidase activity remains unchanged if cells are exposed to UV light. How can cells increase biotinylation of histones without increasing the activity of the enzyme that mediates biotinylation? In previous studies, we observed that cells responded to proliferation with increased biotinylation of histones despite constant expression of the biotinidase gene (22). In these previous studies, we offered the following explanations 283 • SEPTEMBER 2002 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on June 18, 2017 Fig. 7. Effects of UV exposure on mitochondrial and cellular integrity in Jurkat cells. Cells were exposed to UV light (254 nm) for 1 h. Aliquots were collected before, during, and after exposure to UV light; mitochondria were stained, and cells were evaluated by microscopy. A: nonirradiated controls. Arrow indicates mitochondria that were evenly distributed. B: cells after 1 h of UV exposure. Arrow indicates clustering of mitochondria in cells. C: cells 1 h after termination of UV exposure. D: cells 3 h after termination of UV exposure. Arrow indicates clustering of mitochondria. E: cells 18 h after termination of UV exposure. Arrow indicates formation of vacuoles in cells and initiation of cell lysis. F: cells in which apoptosis was induced by culturing with 50 M of actinomycin D for 18 h. Arrows indicate clustering of mitochondria and lysis of cell membrane. C884 BIOTINYLATION OF HISTONES IN JURKAT CELLS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-60447 and U.S. Department of Agriculture/National Research Initiative Competitive Grants Program Award 2001-35200-10187. This study is a contribution of the University of Nebraska Agricultural Research Division, Lincoln, NE 68583 (journal series no. 13614). REFERENCES 1. 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Zempleni J and Mock DM. Biotin biochemistry and human requirements. J Nutr Biochem 10: 128–138, 1999. 28. Zempleni J, Trusty TA, and Mock DM. Lipoic acid reduces the activities of biotin-dependent carboxylases in rat liver. J Nutr 127: 1776–1781, 1997. 283 • SEPTEMBER 2002 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on June 18, 2017 for our findings: biotinylation of histones might be regulated by import of biotinidase into the cell nucleus, or biotinylation of histones might be catalyzed by enzymes other than biotinidase. In the present study, we add another possible explanation: biotinylation of histones is regulated by substrate (biocytin) availability. The following observations are consistent with this hypothesis. The Michaelis-Menten constant of biotinidase for biocytin is ⬃10 M (17); the concentration of total biotin metabolites (including biocytin) in tissues such as rat liver is 32 nmol/kg (15), i.e., 300 times less than the Michaelis-Menten constant of biotinidase. Thus small changes of biocytin concentrations in cells may substantially affect rates of biotinidase-catalyzed reactions. The present study provided evidence that concentrations of biotinylated breakdown products of carboxylases (including biocytin) increase in response to UV irradiation. It remains uncertain why biotinylation of histones at transcriptionally inactive sites of chromatin is favored over sites that are transcriptionally active. Many of the known modifications of histones (e.g., acetylation) require interactions of various regulatory elements such as transcription factors with enzymes that catalyze these modifications. Future studies need to determine whether transcription factors also direct biotinidase to discrete regions of chromatin.