From the Institute of Medical Biochemistry, Division of Molecular Biology, University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria
Received for publication, May 16, 2002, and in revised form, October 8, 2002
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ABSTRACT |
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Thymidine kinase (TK) is a growth
factor-inducible enzyme that is highly expressed in proliferating
mammalian cells. Expression of mouse TK mRNA is controlled by
transcriptional and posttranscriptional mechanisms including antisense
transcription. Here we report the identification of a novel gene that
is divergently transcribed from the bidirectional TK promoter. This
gene encodes kynurenine formamidase (KF), an enzyme of the tryptophan
metabolism. Whereas the TK gene is induced upon interleukin-2-mediated
activation of resting T cells, the KF gene becomes simultaneously
repressed. The TK promoter is regulated by E2F, SP1, histone
acetyltransferases, and deacetylases. The binding site for the
growth-regulated transcription factor E2F is beneficial for TK promoter
activity but not required for KF expression. In contrast, the SP1
binding site is crucial for transcription in both directions.
Inhibition of histone deacetylases by trichostatin A leads to increased
histone acetylation at the TK/KF promoter and thereby to selective
activation of the TK promoter and simultaneous shut-off of KF
expression. Similarly, TK gene activation by interleukin-2 is linked to
histone hyperacetylation, whereas KF expression correlates with reduced
histone acetylation. The KF gene is the rare example of a mammalian
gene whose expression is linked to histone hypoacetylation at its promoter.
Thymidine kinase (TK)1
belongs to a group of enzymes including dihydrofolate reductase,
thymidylate synthase, DNA polymerase The identification of growth arrest-specific antisense transcription of
the TK locus in mouse fibroblasts provided more evidence for the
complex regulation of the TK gene during growth stimulation (2).
Earlier, Weichselbraun et al. (13) showed that the sequence of about 500 bp upstream of the TK initiation codon contains a bidirectional promoter active in both directions, raising the possibility of a divergently expressed gene.
In the present study, we identified a novel gene encoding for mouse
kynurenine formamidase (KF) that is transcribed in antisense to the TK
gene. The translational start codons of the TK and KF gene are
separated by only 172 bp, suggesting a coordinated transcriptional regulation of these genes. Primer extension and 5'-rapid amplification of cDNA ends (5'-RACE) analysis showed that transcription of the KF
gene was mainly initiated within the bidirectional promoter region but
also revealed a minor transcription start site within intron 2 of the
TK gene, which was infrequently used in liver. Interestingly, we found
that transcription of the KF gene in T cells was inversely regulated
compared with the TK gene. The KF gene was only transcribed in resting
cells, whereas transcription of the TK gene was activated in
proliferating T cells. Finally, by chromatin immunoprecipitation
experiments, we showed that activation of the TK gene involves
hyperacetylation of histone H4 associated with the bidirectional
promoter region. In contrast, transcription of the KF gene can occur in
the presence of hypoacetylated histones.
Cell Culture and Transfection--
Cell lines Swiss 3T3, HeLa,
and TIB73 were cultured in Dulbecco's modified Eagle's medium
containing 10% (v/v) fetal calf serum. B6.1 T cells were cultured in
Dulbecco's modified Eagle's medium containing 5% (v/v)
heat-inactivated fetal calf serum, 50 mM
Genomic Sequence Analysis--
The KF cDNA sequence was
determined by standard methods. Data base searching and alignments were
performed at NCBI using the Basic Local Alignment Search Tool Algorithm
(BLAST). The nonredundant and the expressed sequence tag (EST) data
bases were sourced. Protein homology searches were performed by
comparing the amino acid query sequence against the SWISSPROT data base
at NCBI. The full-length mouse KF cDNA sequence has been deposited
in the GenBankTM data base (accession number
AY099479).
Retroviral Infection and Transfection of Mammalian
Cells--
The BamHI-EcoRI fragment of the mouse
KF cDNA was cloned in frame into the
BamHI/EcoRI-digested retroviral pBabe-His2myc
vector, and the stop codon of the mouse KF cDNA was removed. High
titer retroviral supernatants were generated by transient transfection of BOSC23 cells and used to infect Swiss 3T3 cells as described previously (15). Infected cells were selected for 2 weeks in culture
medium supplemented with 2.5 µg of puromycin per ml and analyzed for
expression of epitope-tagged KF by immunofluorescence and immunoblotting.
Indirect Immunofluorescence--
Infected Swiss 3T3 cells
expressing epitope-tagged KF were fixed with 3% paraformaldehyde and
permeabilized with 0.5% Triton X-100. The Myc-tagged proteins were
detected with monoclonal antibody 9E10 and visualized with a
Cy3-conjugated anti-mouse IgG secondary antibody (Accu-Specs) in a
Zeiss Axiovert 135TV microscope.
Primer Extension--
Total RNA was isolated from mouse liver,
and poly(A)+ mRNA was purified using the Oligotex
mRNA midi kit from Qiagen according to the manufacturer's
instructions. 10 pmol of [ 5'-RACE--
The 5'-RACE procedure was carried out on 1 µg of poly(A)+ RNA isolated from a mouse liver using the
Marathon cDNA amplification kit (Clontech)
according to the manufacturer's instructions. The following primers
have been used: KF2ext as GSP1 (first-round PCR nested primer) and
KFext or KFXhoI as GSP2 (second-round nested PCR primer). Adapter
primers AP1 and AP2 were provided by the kit. All PCRs were performed
with the Advantage-GC cDNA polymerase mix from
Clontech using a Biometra trio thermoblock. 5'-RACE
reaction products were cloned into a pT7Blue-3 vector using the
Perfectly blunt cloning kit (Novagen). Clones were sequenced from both
ends using primers T7 and U-19mer, and the sequence was aligned with the genomic region of the KF gene.
Southern Blot Analysis--
Analysis of genomic DNA was
performed as described (16). Blots were hybridized by the sandwich
method (17) and washed under high stringency conditions.
RNA Isolation and Northern Blot Analysis--
Isolation of total
RNA was done with TRIZOL® reagent (Invitrogen) as specified by
the manufacturer. Northern blot hybridization was performed by the
sandwich method (17).
Western Blot Analysis--
Western blot analyses of Myc-tagged
KF and HDAC1 was performed as previously described (11).
Nuclear Run-on Transcription Assay--
Transcription reactions
were performed for 20 min in the presence of [ Chromatin Immunoprecipitation--
Chromatin immunoprecipitation
assays were carried out as described previously (18) with a few
modifications. Chromatin was cross-linked for 10 min using
formaldehyde. The resulting chromatin solution was diluted 1:10 and
precipitated with 10 µl of acetyl-specific histone antibodies
(Upstate Biotechnology, Inc., Lake Placid, NY). The following day,
chromatin-antibody complexes were isolated from the solution by
incubation with 30 µl of protein A-Sepharose beads (50% slurry, 100 µg/ml salmon sperm DNA, 500 µg/ml bovine serum albumin) while
rocking at 4 °C for 2 h. The beads were harvested and washed as
described (18). Chromatin-antibody complexes were eluted from the
A-Sepharose beads by the addition of 2% SDS, 0.1 M
NaHCO3, and 10 mM dithiothreitol to the pellet.
Cross-linking was reversed by the addition of 0.05 volume of 4 M NaCl and incubation of the eluted samples for 6 h at
65 °C. The DNA was extracted with phenol/chloroform, precipitated
with ethanol, and dissolved in water. Immunoprecipitated DNA was
analyzed for KF and histone H4 gene sequences by PCR.
PCR Analysis of Immunoprecipitated DNA--
All PCRs were
performed on a Biometra D3 thermocycler using Promega PCR Master Mix.
Primer sequences are available upon request. The linear range for each
primer pair was determined empirically using different amounts of B6.1
genomic DNA. PCRs with increasing amounts of genomic DNA were carried
out along with the immunoprecipitated DNA. PCR products were resolved
on 2% agarose-TAE gels and quantified using the ImageQuant
program (Amersham Biosciences).
Identification of a Novel Gene Transcribed from the Bidirectional
TK Promoter--
Previous findings (2, 13) suggested the presence of
an antisense orientated gene within the murine TK gene locus. In order
to identify the corresponding antisense transcript, we performed a DNA
data base search with the sequence corresponding to the 1-kb upstream
region of the TK promoter (Fig.
1A). Data base searches were
carried out with the BLAST search algorithm at the NCBI. We detected a
significant homology between a sequence within the murine TK promoter
and an EST clone derived from mouse liver (GenBankTM
accession number AA245789). The homologous sequence began 170 bp
upstream of the TK gene and extended for 83 bp to a 5' splice
consensus, in agreement with the presence of an exon of an antisense
gene in the TK upstream region.
Interestingly, the 83-bp sequence identity started exactly at the
5'-end of the EST clone. Further, this putative exon sequence included
a potential start codon. The surrounding sequence had a reasonable
match to the Kozak translation initiation consensus (19), consistent
with it being the first exon of a putative gene. Additional data base
searches revealed homologous EST clones in humans (BG118921), rats
(BI295156), and hamsters (X99216). The 5'-end of these cDNAs showed
significant homology with corresponding sequences within the upstream
region of the respective TK genes (data not shown). A previously
identified 18-mer sequence, which is conserved in the TK promoter from
mice, humans, and hamsters and was originally thought to have a
potential regulatory function (16), is located at the end of exon 1 of
the newly identified gene.
To obtain more information about the full-length cDNA of the newly
identified gene, cloning in silico was applied. Successive BLAST searches with the EST clone AA245789 as query sequence were used
to identify overlapping sequences in the EST data base at the NCBI
(Fig. 1C). Each of these matches was, in turn, searched against the EST data base. Using this approach, we were able to identify several EST clones extending the 3' part of the putative cDNA. No 5'-overlapping EST clones were found, suggesting that the
5'-end of the EST clone AA245789 corresponded to the 5'-end of the
mature mRNA. The most 3'-located EST clone (GenBankTM
accession number AA920084) contained a putative polyadenylation signal,
consistent with being the true 3'-end of the cDNA. The complete
cDNA was about 2.3 kb in size with a 3'-untranslated region
consisting of 1.4 kb.
Sequence analysis showed that the EST clone contained an open reading
frame of 918 nucleotides, which encoded a putative 305-residue protein
(Fig. 1B). This protein was very recently identified as KF, an enzyme involved in tryptophan degradation (20). In
agreement with the size of the purified mouse KF enzyme (20), in
vitro transcription-translation experiments confirmed the
molecular mass of 34 kDa predicted from the amino acid sequence (data
not shown).
Organization of the KF Gene--
The previously described cosmid
TKcosA carrying the entire mouse TK gene and additional 10-kb upstream
sequences (15) was analyzed to map putative KF exon sequences. The EST
clone AA245789 was subjected to detailed restriction analysis, and
three subfragments (E/S-200, S-700, and S/N-540) were used for Southern
blot analysis (Fig. 2A).
Hybridization of these fragments to a Southern blot of
AccI-, EcoRI-, or HindIII-digested
TKcosA DNA revealed the presence of multiple, putative exon sequences
located within the upstream region of the TK gene (Fig. 2B).
Each fragment produced a characteristic pattern of signals, which
resulted from the presence of KF exons. With the S/N-540 fragment, only
a very weak hybridization signal was observed, indicating the presence
of only short homologous DNA sequences on the TKcosA clone.
Further detailed restriction and sequence analysis of the cosmid clone
TKcosA allowed us to position 10 exons and the corresponding introns to
set up the KF gene structure (Fig. 2C). The first 10 KF
exons representing the first 910 bp of the EST clone AA245789 included
the complete open reading frame. The sequence of these exons showed
100% homology to the sequence of the KF mRNA. Based on sequence
information, the exon-intron boundaries and the exact size of each exon
were determined (Table I). The size of
exons ranged from 49 to 105 bp, whereas the size of introns fluctuated widely from 76 bp to 4.3 kb. The DNA sequences of all splice donor and
acceptor sites complied with the invariant GT and AG rule.
KF Is a Conserved Protein with Homology to the Esterase
Superfamily--
Data base searching yielded significant homology of
the KF protein to known and hypothetical esterases and revealed an
active site serine motif GXSXG (21) within the KF
sequence (Fig. 1B). The presence of related but yet
uncharacterized proteins in bacteria, plants, worms, and flies
indicates that the KF protein is conserved throughout evolution (Fig.
3A).
To investigate the intracellular localization of the mouse KF protein,
the protein was expressed with a Myc epitope at its carboxyl terminus
in Swiss 3T3 fibroblasts. Expression was monitored by Western blot
analysis, which showed a single band with the expected size of about 35 kDa for Myc-tagged KF (Fig. 3B). Indirect immunofluorescence
microscopy revealed that the KF protein is localized predominantly in
the cytoplasm but also to some extent in the nucleus (Fig.
3C). In contrast, vector-transfected control cells gave no
detectable signal. Overexpression of the KF protein had no effect
on proliferation and cell cycle progression of transfected fibroblasts
(data not shown).
Determination of the Transcription Start Sites of the KF
Gene--
5'-RACE analysis was performed with mRNA isolated from
adult mouse liver to determine the transcription initiation site of the
KF gene. The two most pronounced PCR products were subcloned and
subjected to sequence analysis. The results indicated that the mouse KF
gene initiated transcription at multiple sites. The longest 5'-RACE
fragment extended up to intron 2 of the TK gene, 404 bp upstream of the
KF ATG start codon. The shorter 5'-RACE product expanded up to 92 bp
upstream of the KF translational start codon. This suggests that in
liver, transcription of the KF gene is not only initiated within the
promoter region shared with the TK gene but also within intron 2 of the
TK gene.
In order to verify results from 5'-RACE, primer extension analysis was
performed on poly(A)+-selected mRNA from mouse liver
(Fig. 4A). Primers were
designed to span putative transcriptional start sites and used for
reverse transcription. Primers KFXhoI (lane 5,
located in KF exon 1), KFext, and KF2ext (lanes 6 and 7, located in KF exon 2) were used to investigate the
downstream start site within the bidirectional promoter region. Primer
KF552 (lane 8, located in TK exon 1) was used to
examine the upstream start site within TK intron 2. In all cases,
positioning of initiation sites (Fig. 4A, black
arrows) was consistent with 5'-end points determined by
5'-RACE. The occurrence of multiple bands with primer KFXhoI indicated
that transcription within the bidirectional promoter region is
initiated from various sites. The result from primer KF552 revealed a
strong signal indicating a major transcription start site within TK
intron 2. Alignment with a dideoxynucleotide sequence ladder from the
same primer indicated that the strong band corresponds to an A that is
located 404 bp upstream of the translational start codon. In Fig.
4B, the sequence of the KF/TK gene locus and transcription
start sites of each gene are shown.
Expression Patterns of KF and TK in Different Mouse
Tissues--
Results obtained from 5'-RACE and primer extension
analysis indicated that two regions separated by 300 bp were used as
transcription initiation sites in liver. Initiation of transcription
within the bidirectional promoter region should give rise to a 2.6-kb transcript. On the contrary, transcription initiation in intron 2 of
the TK gene should result in a 2.9-kb KF mRNA with an extended 5'-untranslated region. However, both putative KF transcripts comprise
the same open reading frame encoding for the 305-residue KF protein.
To analyze the actual size of KF mRNAs and tissue-specific
expression of the KF gene, Northern blot analysis of different mouse
tissues was performed (Fig. 5).
Hybridization with a 700-bp fragment of the KF cDNA (KF 700)
revealed a 2.6-kb transcript expressed in particular tissues. The
highest KF mRNA levels were seen in liver. Significant expression
was detected in kidney, whereas only very low KF mRNA levels were
found in heart and spleen. Interestingly, both KF and TK were
expressed in liver and spleen.
To examine the expression of the longer KF transcript, Northern blot
analysis was performed with a genomic DNA fragment encompassing the
first two TK exons (Fig. 2C,
KF5'RACE). A signal corresponding to the longer
2.9-kb KF transcript was only detected in the liver (Fig. 5,
black arrow). However, the band was very weak,
indicating that the upstream transcription initiation site is utilized
infrequently in liver and not at any detectable levels in other
tissues. A second transcript detected by the same genomic probe
corresponded to the TK mRNA (Fig. 5, gray
arrow). The detection of smaller transcripts by the 5'-RACE
probe was most likely due to cross-hybridization.
Growth-dependent Regulation of the KF Gene--
As
previously reported, TK gene expression is strongly regulated during
growth induction and cell cycle progression. To examine a potential
growth factor-dependent regulation of the KF gene, we
utilized the IL-2-dependent mouse T cell line B6.1 (22). Exponentially growing B6.1 cells were deprived of IL-2 for various periods of time, and levels of KF mRNA and TK mRNA were
determined by Northern blot analysis (Fig.
6A). Growth arrest was
monitored by fluorescence-activated cell sorting analysis. TK mRNA
expression was high in dividing cells but rapidly diminished when cells
arrested in G0 phase. In contrast, KF mRNA was absent
in proliferating cells and was found to be increased in resting B6.1
cells.
To examine whether induction of the KF gene during cell cycle arrest
occurred at the transcriptional level, we performed run-on transcription assays of nuclei isolated from resting or IL-2-induced B6.1 cells (Fig. 6B). Consistent with arrest-specific KF
mRNA expression, the KF transcription rate was high in
IL-2-deprived cells and strongly decreased when cells were restimulated
to cycle. In contrast, TK transcription was very low in resting B6.1
cells but increased during IL-2 stimulation as previously reported (1). Our results indicate that increased KF expression in resting B6.1 cells
is at least in part due to transcriptional activation of the KF gene.
Functional Analysis of the KF Promoter--
Previously, it has
been shown that the 490-bp upstream sequence of the TK gene exhibit
bidirectional promoter activity (13). To examine whether upstream
sequences of the KF gene enhance promoter activity in the opposite
direction of the TK gene, a series of 5' promoter deletion constructs
including introns 1 and 2 of the TK gene were generated (Fig.
7A). PCR-amplified fragments
were inserted into the luciferase reporter vector pGL2neo, and promoter activities were assessed by transient transfection into HeLa cells and
into mouse TIB73 liver cells (Fig. 7B). Deletion analysis indicated that basal transcription is mediated primarily by elements residing between +1 and
We have previously shown that the mouse TK gene is transcriptionally
regulated by E2F and SP1 (4). To determine whether the same DNA
sequences are important for the activation of the KF promoter, the
effects of point mutations in SP1 and E2F binding sites were tested by
transient transfection assays with HeLa cells or TIB73 cells. The
binding sites for SP1 and E2F were individually mutated in the shortest
(Fig. 7C) and the longest promoter construct (Fig.
7D).
In agreement with previously published data (4), mutation of the E2F
binding site in the context of the TK promoter led to a slight decrease
in promoter activity in logarithmically growing cells, whereas mutation
of the SP1 site resulted in significant loss in luciferase activity
(Fig. 7C). In the case of the KF promoter, mutation of the
E2F site had no significant effect, whereas mutation of the SP1 site
significantly reduced promoter activity. The effect of mutated binding
sites for E2F and SP1 on KF promoter activity did not significantly
differ when examined in the shorter or longer promoter construct (Fig.
7, B and C). Moreover, we tested the importance
of the SP1 site in a genomic context by establishing stably transfected
TIB 73 cell lines carrying either the wild-type KF promoter or the SP1
mutant KF promoter. Luciferase reporter analysis of these cell lines
confirmed the data from transient transfection assays (data not shown).
These results indicate that the SP1 binding site is crucial for both TK
and KF transcription, whereas E2F binding is only important for
efficient TK expression.
Acetylation Status of Core Histones at the KF/TK Gene
Locus during Gene Activation--
The previously observed repressive
effect of histone deacetylase 1 on the murine TK promoter (11)
suggested that chromatin remodeling via reversible histone acetylation
is involved in the regulation of the mouse TK gene. The deacetylase
inhibitor trichostatin A (TSA) was shown to activate the TK promoter in
resting fibroblasts. The identification of a gene transcribed from the
bidirectional TK promoter caused us to examine the effect of TSA on KF
gene expression. Resting B6.1 cells were treated with TSA, and KF
mRNA levels were examined by Northern blot analysis. As shown in
Fig. 8A, inhibition of
deacetylase activity by TSA led to a rapid and efficient block of KF
expression. In contrast to the significant activation of the TK
promoter by TSA (11), expression of mature TK mRNA in resting cells
was only slightly and transiently induced by the deacetylase inhibitor
(Fig. 8A). This is most probably due to the fact that in
resting cells repression of the TK gene is mediated by a TSA-sensitive
transcriptional mechanism and a TSA-insensitive post-transcriptional
mechanism. In agreement with this idea, TK mRNA expression in
cycling cells was shown to be induced by the deacetylase inhibitor (6).
To directly link histone hyperacetylation with the regulation of the
TK/KF locus, we investigated the acetylation status of core histones
associated with the bidirectional TK/KF promoter. First, we performed
immunoprecipitation experiments with chromatin isolated from resting
and TSA treated B6.1 cells under conditions that have been used to
demonstrate activation of the TK promoter by TSA (11). After
cross-linking of cells with formaldehyde, sonicated chromatin was
immunoprecipitated with acetyl-specific histone antibodies. In
parallel, chromatin immunoprecipitation assays were performed in the
absence of antibodies to control the specificity of the
immunoprecipitation. Primers specific for the mouse histone H4 gene
were used as control for DNA input and amplification efficiency.
Further PCRs were carried out with standards comprising fixed amounts
of genomic DNA as template to ensure that amplification reactions
remain in the linear range. As shown in Fig. 8B, acetylation
of both histone H3 and histone H4 was increased at the bidirectional
promoter (Fig. 8C, primer pair 1-2) upon TSA treatment. In
contrast, histone acetylation at the control gene (H4) was not induced
by the deacetylase inhibitor, proving the specificity of the changes in
histone acetylation detected at the TK promoter. No signal was observed
for chromatin immunoprecipitations in the absence of specific
antibodies. Taken together, these data show that activation of the TK
promoter by TSA is linked to histone hyperacetylation and seems to
interfere with KF transcription.
Next, we analyzed the acetylation state of histones H3 and H4 on the
TK/KF locus during G1/S phase transition. To this end, immunoprecipitation assays were performed with chromatin prepared from
resting or IL-2-stimulated B6.1 cells as described above. Immunoprecipitation of equivalent amounts of chromatin from
IL-2-deprived (Fig. 8D, rest) and IL-2-induced
cells (Fig. 8D, ind) with the acetyl-H4 antibody
revealed a 2.5-fold increase in the association of acetylated histone
H4 with the KF/TK intragenic region and a 4.4-fold increase within
intron 2 of the KF gene. Whereas histone H4 acetylation levels at
intron 2 of the TK gene were only slightly (1.6-fold) affected,
acetylation levels of histone H3 at the TK/KF locus remained unchanged
during growth stimulation. Again, histone acetylation at the histone H4
gene was not induced in IL-2-stimulated B6.1 cells, indicating that the
increased association of acetylated histone H4 with the TK promoter
region is significant. Thus, TK gene expression in proliferating cells
is linked to histone H4 hyperacetylation, whereas KF gene expression
correlates with reduced histone acetylation.
Regulation of the TK/KF Locus--
Thymidine kinase is
a member of the family of nucleotide salvage pathway enzymes. These
enzymes are involved in the fine tuning of dNTP pools and in the
reutilization of nucleosides originating from the degradation of DNA
(for review, see Ref. 23). Tight regulation of TK expression is thought
to be necessary to avoid increased mutation rates due to loss of the
fine tuning of thymidine nucleotide precursors. Transcriptional
activation of the TK gene is in part responsible for induced TK
expression at the reentry into the cell cycle (23). Further, different
transcription factors including SP1, E2F, and NF-Y were implicated in
the regulation of the TK promoter in humans, mice, and hamsters (4,
24-27). It was also noted that despite the overall diversity of the
upstream regulatory regions, TK promoters from different species
contain a conserved 18-mer motif (16, 23). Here, we show that this sequence is part of the gene encoding the enzyme kynurenine
formamidase. The KF protein was recently isolated and
characterized (20). Nearly nothing is known about the regulation of KF
expression in mammalian cells. The KF gene and the TK gene are located
in a head-to-head orientation and share a bidirectional promoter. The
organization of TK/KF locus is highly conserved in humans, mice, rats,
and hamsters. Whereas TK and KF are co-expressed in some mouse tissues
including liver and spleen, we did not detect simultaneous expression
of the two genes in a particular cell type or cell line, suggesting
that expression of TK and KF is mutually
exclusive.2
Recently, the mouse TK gene was disrupted to study the biological
function of TK in more detail (28, 29). The resulting TK null allele
lacks the bi-directional TK/KF promoter. Therefore, it might be
necessary to reexamine TK-deficient mice for lack of kynurenine
formamidase and its potential consequences.
Antisense Transcription--
During recent years, naturally
occurring antisense transcription has emerged as an important
post-transcriptional regulatory mechanism for the expression of
mammalian genes (30). We have previously shown that, in addition
to promoter regulation, growth-regulated antisense transcription from
intron 3 controls TK expression in mouse fibroblasts (2). In contrast,
KF transcription does not seem to be directly involved in the
regulation of TK gene expression. Only a minority of KF transcripts was
found to be initiated within the transcribed region of the TK gene. In
fact, our data suggest that expression of the KF gene is dependent on
the absence of TK transcription. This idea is supported by the finding
that activation of the TK promoter by IL-2 or TSA correlates with
simultaneous shut-off of KF expression. Thus, these stimuli seem to
reverse the polarity of the TK/KF promoter. In this context, it is
worth mentioning that the vast majority of bidirectional promoters
known to date regulate the expression of coordinately activated genes. Most of these gene pairs have related functions and might originate from gene duplication. In contrast, the TK/KF promoter is a rare example of an upstream region regulating the alternate expression of
two divergently transcribed genes.
Chromatin Acetylation at the TK/KF
Locus--
Cooperation between SP1 and E2F was previously shown to
be crucial for TK expression in proliferating mouse fibroblasts (4). SP1 seems to function as an anchor for positive and negative
transcriptional regulators such as E2F and HDAC1 (11). We show here
that the SP1 binding site is also necessary for KF expression, whereas the E2F site is dispensable for KF promoter activity.
Histone acetylation is thought to be a prerequisite for initiation of
transcription. Accordingly, TK transcription was found to be activated
by the deacetylase inhibitor TSA (11). Here we show that
transcriptional silencing of the TK gene in resting B6.1 cells
correlates with histone hypoacetylation at the 5' part of the gene.
Together, these findings are in full accordance with the
previously presented model predicting the presence of repressor complexes containing SP1, E2F, pocket proteins, and histone
deacetylases at the TK promoter (11). Growth factor stimulation leads
then to hyperacetylation of the TK promoter and activation of the
TK gene (this report). In contrast, enhanced transcription of the KF
gene in resting cells correlates with reduced histone acetylation at
the bidirectional promoter and within the 5' part of the KF gene.
Similarly, inhibition of deacetylases results in repressed KF
expression. We propose that the default polarization of the bidirectional TK/KF promoter in mouse T cells is in favor of TK transcription. Only when TK transcription is turned off by mechanisms involving local histone deacetylation, KF transcription becomes activated.
To our knowledge, the kynurenine formamidase gene is the first
mammalian gene whose expression was shown to correlate with histone hypoacetylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and proliferating cell
nuclear antigen that are involved in DNA synthesis and precursor
production. The expression of these genes is low in resting cells but
increases to high levels when cells traverse the G1/S
boundary. Multiple and complex regulatory mechanisms at the
transcriptional as well as at the post-transcriptional level ensure the
exclusive expression of the TK gene in replicating cells. As measured
by run-on experiments, TK mRNA levels are regulated by a
growth-dependent rise in transcription rate (1, 2). It was
demonstrated that upstream elements recognized by protein complexes
containing E2F, pocket proteins, and SP1 are crucial for
transcriptional activation of the TK gene (3, 4). Recruitment of HDACs
by pocket proteins has been identified as important mechanism for the
regulation of growth- and cell cycle-regulated genes (5-7) (reviewed
in Refs. 8-10). Recently, we have shown that in addition to its
association with pocket proteins, histone deacetylase 1 (HDAC1) can
repress TK gene expression by targeting SP1 in resting mouse
fibroblasts (11). E2F1 can compete with HDAC1 for binding to SP1,
thereby relieving HDAC1-mediated repression of the TK promoter.
Further, two DNase-hypersensitive sites within intron 2 of the murine
TK gene had been identified, and the presence of these sequences was
shown to positively modulate the activity of the TK promoter
(12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 10 mM L-glutamine, 10 mM HEPES, and 100 units/ml recombinant human interleukin-2
(IL-2) (kind gift of M. Nabholz, ISREC, Lausanne, Switzerland). B6.1
cells were growth-arrested for 34 h in medium without IL-2 and
restimulated to enter the cell cycle by adding fresh medium containing
IL-2 (1). Growth arrest and stimulation were routinely controlled by
fluorescence-activated cell sorting analysis with a Partec PAS-II
sorter. For transient transfection, a series of 5' deletion constructs
were generated spanning from
1 to
1800 bp of the translational
start codon using PCR primers containing XhoI and
KpnI restriction sites and cloned into the pGL2neo vector.
To obtain pKF170, pKF-SP1mut, and pKF-E2Fmut, the vector pMSGTKCAT and
the corresponding constructs with mutated binding sites for either SP1
or E2F (11) were used as PCR template using primers containing
XhoI and NheI restriction sites. To generate pTK,
pTK-SP1mut and pTK-E2Fmut primers containing KpnI and
NheI restriction sites were used. To obtain pKF1800-SP1mut and pKF1800-E2Fmut, plasmids pKF-SP1mut and pKF-E2Fmut were digested with XmnI and the fragment containing the individual point
mutations were ligated into XmnI-digested pKF1800 vector.
For transfection, 8 × 104 HeLa cells or TIB73 cells
were seeded in 3-cm diameter Petri dishes and transfected the following
day with a total of 2 µg of DNA using polyethyleneimine-assisted gene
transfer (14). 2 µl of polyethyleneimine were diluted in 125 µl
of HEPES-buffered saline and added dropwise to 2 µg of DNA
diluted in 125 µl of HEPES-buffered saline. After a 20-min
incubation at room temperature, the transfection mix was added to the
cells, which, prior to the transfection, had their growth medium
replaced by 800 µl of serum-free medium. The transfection medium was
replaced by fresh medium after 6 h. After 48 h, luciferase
and
-galactosidase activities were measured.
-32P]ATP-labeled
oligonucleotides and 1 µg of poly(A)+-selected RNA were
dissolved in 10 µl of water, heated up to 70 °C for 10 min, and
chilled on ice. Reverse transcription was carried out at 42 °C for
1 h using Superscript Reverse Transcriptase (Invitrogen). The same
oligonucleotides were also used to sequence the mouse genomic regions
cloned into pAT153 vectors. Reaction products were separated on a 6%
polyacrylamide gel and visualized by autoradiography overnight.
-32P]CTP
(800 Ci/mmol) as described previously (1). Hybridization and washing
was done as reported by Sutterluety et al. (2). TK and KF cDNA fragments were subcloned in M13-derived vectors and
used as single-stranded probes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of a novel gene divergently
transcribed from the murine TK promoter region. A,
graphic representation of the EST data base search. The
thick bar shows the 1-kb upstream region, used as
query sequence. The box contains the sequence homologous to
the EST clone AA245789. B, sequence and translated open
reading frame of the EST clone AA245789. The conserved esterase domain
is boxed. C, in silico cloning to
generate a contiguous cDNA of 2333 bp. Each bar
represents an individual EST. The EST clone at the extreme 3'-end of
the cDNA contains a putative polyadenylation signal.
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Fig. 2.
Organization of the mouse KF gene.
A, schematic representation and restriction mapping of the
EST clone AA245789. The thick bar represents the
open reading frame. Subfragments used for Southern blot analysis are
indicated. B, Southern blot analysis of KF-related genomic
structures. The TKcosA clone (16) was digested with HindIII,
EcoRI, and AccI and analyzed on Southern blots by
hybridization with the radiolabeled full-length EST clone AA245789 or
with the indicated subfragments. C, restriction map and
organization of the mouse KF gene on the genomic TKcosA clone. Exons
1-10 of the KF gene are shown as black boxes.
Exon 1 and 2 of the TK gene are illustrated by gray
boxes. Recognition sites for selected restriction enzymes
are indicated.
Exam-intron boundaries of the mouse KF gene on the TKcos A clone
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Fig. 3.
The KF protein. A,
phylogenetic tree. Alignment was performed with homologous
sequences from Arabidopsis thaliana (AC084221),
Pseudomomas sp. (AB047272), Caenorhabditis
elegans (NM_068748), Homo sapiens
(XM_058889), Drosophila melanogaster (NM_135192),
and Clostridium perfringens (AB028629) using the
MegAline program (DNASTAR). B, Western blot analysis of
mouse Myc-tagged KF protein expressed in Swiss 3T3 fibroblasts. Extract
from cells expressing Myc-tagged HDAC1 protein was included as antibody
control. Epitope-tagged proteins were detected with monoclonal antibody
9E10. C, subcellular localization of the KF protein in Swiss
3T3 fibroblasts. Indirect immunofluorescence (IF) microscopy
of Swiss 3T3 cells expressing Myc-tagged KF protein or untransfected
Swiss 3T3 cells (control). Epitope-tagged KF protein was visualized
with 9E10 antibody followed by Cy3-conjugated anti-mouse immunoglobulin
G (red). In parallel, the nuclear DNA was stained with
4',6-diamidino-2-phenylindole (DAPI)
(blue).
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Fig. 4.
Mapping of the mouse KF transcription start
site. A, analysis of KF transcription start sites by
primer extension analysis. Reverse transcription was carried out with
the indicated primers using poly(A)+-selected mRNA
isolated from a mouse liver. In parallel, sequence reactions were
performed using primers KFXhoI (lane 1-4) and
KF552 (lanes 9-12). The resulting ladders were
used to position the primer extension signals. Relevant nucleotide
sequences are shown with the initiation sites indicated by
arrows. B, nucleotide sequence of the genomic DNA
comprising the bidirectional mouse KF/TK promoter. The major
transcription initiation sites of the TK gene (gray
bent arrows) and the KF gene (black
bent arrows) are indicated. Transcription factor
binding sites important for the regulation of the TK gene are shown by
ellipses. Sequences and locations of primers within the
promoter region used for primer extension and 5'-RACE are
underlined.
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Fig. 5.
Northern blot analysis of TK and KF
transcripts in mouse tissues. Northern blot with
poly(A)+ RNA isolated from different mouse tissues was
analyzed by subsequential hybridization with the
32P-labeled KF cDNA fragment (KF 700), the TK cDNA
(TK), and the extreme 5' part of the KF gene obtained from primer
extension and 5'-RACE (KF 5'RACE). As
control, the blot was hybridized with a -actin probe. The
black arrow marks the 2.9-kb KF transcript,
observed only in liver. Gray arrows show the TK
mRNA. The asterisk indicates presumptive
cross-hybridization signals.
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Fig. 6.
Growth-regulated expression of mouse KF and
TK gene in B6.1 cells. A, exponentially growing B6.1
cells were cultured in complete medium without IL-2 for various times
(indicated in hours). Total RNA was isolated and expression of KF
mRNA and TK mRNA was analyzed by Northern blot hybridization.
18 S rRNA was visualized by staining of the nylon membrane with
methylene blue. Cell cycle arrest was monitored by
fluorescence-activated cell sorting analysis. B, in
vitro run-on transcription assays. Nuclei of B6.1 cells deprived
for IL-2 (rest) or induced with IL-2 for 16 h
(ind) were used to determine the transcription rate of the
KF gene and the TK gene, respectively. Equal amounts of radiolabeled
newly synthesized RNA were hybridized to single-stranded DNA probes. As
control, empty vector DNA (M13) was used.
370. Protein binding sites at the beginning and at the end of intron 2 of the TK gene have been shown to be important to enhance TK promoter activity (12) (see Fig.
7A). In HeLa cells, luciferase activity did not change
significantly by including TK introns 1 and 2 in the reporter gene
(KF1190), whereas in TIB73 cells KF1190 promoter activity was
moderately increased. Moreover, KF promoter activity in TIB73 liver
cells seemed to be slightly higher than in HeLa cells. These data
suggest that protein binding sites in the bidirectional promoter region and within intron 2 most probably have a tissue-specific function in
the activation of the KF promoter.
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Fig. 7.
Functional analysis of the KF promoter
region. A, schematic drawing of the bidirectional
promoter region of the KF/TK gene locus. Promoter constructs used for
luciferase assays are shown. Transcription factor binding sites and
DNase-hypersensitive sites (DHS), are indicated. Mutated
binding sites are marked by crossed boxes.
B, expression of luciferase reporter constructs driven by
the KF promoter. Reporter plasmid together with the control vector
pCMV Gal was transiently transfected into HeLa cells and into
TIB73 cells, harvested after 48 h, and assayed for luciferase
activity. Enzymatic activity was normalized to
-galactosidase
activity. Data are depicted as mean values obtained from triplicate
transfections. C and D, effect of mutated binding
sites for SP1 and E2F on luciferase reporters driven by the TK or the
KF promoter. Transfection experiments were performed as described
above.
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Fig. 8.
Acetylation of histone H3 and H4 on
the KF and TK chromatin during growth induction. A,
Northern blot analysis of RNA isolated from resting B6.1 cells treated
with 50 ng/ml TSA for various times (indicated in hours).
B, formaldehyde cross-linked chromatin was prepared from
resting (rest) and TSA-treated B6.1 cells and
immunoprecipitated with either acetyl-H3 antibodies (AcH3)
or acetyl-H4 antibodies (AcH4). DNA from the antibody-bound
fraction and DNA isolated from total input chromatin (1×, 0.25×, and
0.0625×) was analyzed by quantitative PCR. PCR products were
quantified using the ImageQuant program, and relative signal
intensities are indicated. Results of three representative experiments
are shown. C, schematic representation of the KF/TK gene
locus. Primers used for analysis of the precipitated chromatin are
indicated. D, formaldehyde cross-linked chromatin was
prepared from resting (rest) and IL-2-induced B6.1 cells
(ind), and immunoprecipitated with either acetyl-H3
antibodies or acetyl-H4 antibodies. Precipitated DNA was analyzed as
described above.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank I. Mudrak and E. Ogris for providing the TKcosA subclones, M. Nabholz for the B6.1 cell line, T. Sauer, S. Keusch, and E. Simboeck for valuable help, and H. Rotheneder and N. Foeger for critical reading of the manuscript.
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FOOTNOTES |
---|
* This project was supported by the Herzfelder Family Foundation and Austrian Science Fund Grant P14909-GEN.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.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental figure.
Present address: House Ear Institute, 2100 W 3rd St., Los Angeles,
CA 90057-1922.
§ To whom correspondence should be addressed. Tel.: 431-4277-61770; Fax: 431-4277-9617; E-mail: cs@mol.univie.ac.at.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M204843200
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY099479.
2 B. Schuettengruber and C. Seiser, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: TK, thymidine kinase; KF, kynurenine formamidase; IL-2, interleukin-2; HDAC, histone deacetylase; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; TSA, trichostatin A.
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