Alternate Activation of Two Divergently Transcribed Mouse Genes from a Bidirectional Promoter Is Linked to Changes in Histone Modification*,

Bernd Schuettengruber, Angelika DoetzlhoferDagger, Karin Kroboth, Erhard Wintersberger, and Christian Seiser§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thymidine kinase (TK)1 belongs to a group of enzymes including dihydrofolate reductase, thymidylate synthase, DNA polymerase alpha , 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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta -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 beta -galactosidase activities were measured.

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 [gamma -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.

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 [alpha -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.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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.

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.

                              
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Table I
Exam-intron boundaries of the mouse KF gene on the TKcos A clone

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).


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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).

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.


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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.

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.


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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 beta -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.

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.


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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.

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 -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 pCMVbeta 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 beta -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.

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.


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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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    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.

    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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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