Download Exposure to UV light causes increased biotinylation of histones in

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
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0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society
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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
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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
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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
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(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-
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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.
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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).
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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
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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.
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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).
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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
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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
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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
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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. Althaus F. Poly ADP-ribosylation: a histone shuttle mechanism
in DNA excision repair. J Cell Sci 102: 663–670, 1992.
2. Bird A. Methylation talk between histones and DNA. Science
294: 2113–2115, 2001.
3. Cole H, Reynolds TR, Lockyer JM, Buck GA, Denson T,
Spence JE, Hymes J, and Wolf B. Human serum biotinidase
cDNA cloning, sequence, and characterization. J Biol Chem 269:
6566–6570, 1994.
4. Durkacz BW, Omidiji O, Gray DA, and Shall S. (ADPribose)n participates in DNA excision repair. Nature 283: 593–
596, 1980.
5. Erden IA and Kahraman A. The protective effect of flavonol
quercetin against ultraviolet a induced oxidative stress in rats.
Toxicology 154: 21–29, 2000.
6. Garrett RH and Grisham CM. Biochemistry. Forth Worth, TX:
Saunders College, 1995.
7. Hymes J, Fleischhauer K, and Wolf B. Biotinylation of biotinidase following incubation with biocytin. Clin Chim Acta 233:
39–45, 1995.
AJP-Cell Physiol • VOL
8. Hymes J, Fleischhauer K, and Wolf B. Biotinylation of histones by human serum biotinidase: assessment of biotinyl-transferase activity in sera from normal individuals and children with
biotinidase deficiency. Biochem Mol Med 56: 76–83, 1995.
9. Hymes J and Wolf B. Human biotinidase isn’t just for recycling
biotin. J Nutr 129: 485S–489S, 1999.
11. Jenuwein T and Allis CD. Translating the histone code. Science 293: 1074–1080, 2001.
12. Juarez-Salinas H, Sims JL, and Jacobson MK. Poly(ADPribose) levels in carcinogen-treated cells. Nature 282: 740–741,
1979.
13. Klotz JL, Minami RM, and Teplitz RL. An enzyme-linked
immunosorbent assay for antibodies to native and denatured
DNA. J Immunol Methods 29: 155–165, 1979.
14. Leadon SA and Hanawalt PC. Monoclonal antibody to DNA
damage containing thymine glycol. Mutation Res 112: 191–200,
1983.
15. Lewis B, Rathman S, and McMahon R. Dietary biotin intake
modulates the pool of free and protein-bound biotin in rat liver.
J Nutr 131: 2310–2315, 2001.
16. Nickel BE, Allis CD, and Davie JR. Ubiquitinated histone
H2B is preferentially located in transcriptionally active chromatin. Biochemistry 28: 958–963, 1989.
17. Pispa J. Animal biotinidase. Ann Med Exp Biol Fenniae 43:
4–39, 1965.
18. Ramanathan B and Smerdon MJ. Enhanced DNA repair
synthesis in hyperacetylated nucleosomes. J Biol Chem 264:
11026–11034, 1989.
19. Ridsdale JA and Davie JR. Chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength and
contain linker histones are highly enriched in ␤-globin sequences. Nucleic Acids Res 15: 1081–1096, 1987.
20. Rocha E, Davie JR, van Holde KE, and Weintraub H.
Differential salt fractionation of active and inactive genomic
domains in chicken erythrocyte. J Biol Chem 259: 8558–8563,
1984.
20a.SAS Institute. StatView Reference. Cary, NC: SAS, 1999.
21. Sauberlich HE, Judd JH, Nichoalds GE, Broquist HP, and
Darby WJ. Application of the erythrocyte glutathione reductase
assay in evaluating riboflavin nutritional status in a high school
student population. Am J Clin Nutr 25: 756–762, 1972.
22. Stanley JS, Griffin JB, and Zempleni J. Biotinylation of
histones in human cells: effects of cell proliferation. Eur J Biochem 268: 5424–5429, 2001.
23. Wang H, Huang Z-Q, Xia L, Feng Q, Erdjument-Bromage
H, Strahl BD, Briggs SD, Allis CD, Wong J, Tempst P, and
Zhang T. Methylation of histone H4 at arginine 3 facilitating
transcriptional activation by nuclear hormone receptor. Science
293: 853–857, 2001.
24. Wolf B and Heard GS. Biotinidase deficiency. In: Advances in
Pediatrics, edited by Barness L and Oski F. Chicago, IL: Medical
Book, 1991, p. 1–21.
25. Wolffe A. Chromatin. San Diego, CA: Academic, 1998.
26. Yoon YS, Kim JW, Kang KW, Kim YS, Choi KH, and Joe
CO. Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis. J Biol Chem
271: 9129–9134, 1996.
27. 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.
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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.