From the Tsukuba Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan, the § Department of
Medical Genetics, China Medical University, Shenyang 110001, China, the
¶ Institute of Applied Biochemistry, University of Tsukuba,
Tsukuba, Ibaraki 305, Japan, the
Department of Molecular
Genetics, Beckman Research Institute of The City of Hope, Duarte,
California 91010, and the ** Department of Neurology, Institute of Brain
Research, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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ABSTRACT |
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We have cloned and characterized the genomic
structure of the human gene for Myc-associated zinc finger protein
(MAZ), which is located on chromosome 16p11.2. This gene is transcribed
as an mRNA of 2.7 kilobases (kb) that encodes a 60-kDa MAZ protein. A 40-kb cosmid clone was isolated that includes the promoter, five
exons, four introns, and one 3'-untranslated region. All exon-intron
junction sequences conform to the GT/AG rule. The promoter region has
features typical of a housekeeping gene: a high G + C content (88.4%);
a high frequency of CpG dinucleotides, in particular within the region
0.5 kb upstream of the site of initiation of translation; and the
absence of canonical TATA and CAAT boxes. An S1 nuclease protection
assay demonstrated the presence of multiple sites for initiation of
transcription around a site 174 nucleotides (nt) upstream of the ATG
codon and such expression was reflected by the promoter activity of a
MAZ promoter/CAT (chloramphenicol acetyltransferase) reporter gene.
Cis-acting positive and negative elements controlling basal
transcription of the human MAZ gene were found from
nucleotides (nt) 383 to
248 and nt
2500 to
948. Moreover,
positive and negative autoregulatory elements were also identified in
the regions from nt
248 to
189 and from nt
383 to
248 after
co-transfection of HeLa cells with plasmids that carried the
MAZ promoter/CAT construct and the MAZ-expression vector.
Our results indicate that the 5'-end flanking sequences are responsible
for the promoter activities of the MAZ gene.
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INTRODUCTION |
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The c-myc protooncogene is a member of a family of genes that encode DNA sequence-specific transcription factors with basic, helix-loop-helix, and leucine zipper domains. The Myc protein binds to DNA as heterodimers with a related polypeptide, Max (1-4). Appropriate regulation of expression of the human c-myc gene is necessary for the proliferation and differentiation of cells and for progression of the cell cycle, and deregulation of the expression of c-myc is associated with tumorigenesis and apoptosis (1, 4). Regulation of the expression of the human c-myc gene occurs at multiple levels, which include the initiation, the termination, and the attenuation of transcription (2, 4). In proliferating cells, the initiation of transcription of the c-myc gene is controlled by two major promoters, P1 and P2, and the RNA initiated from the P2 promoter accounts for 80-90% of the total RNA initiated from the P0, P1, and P2 promoters (4, 5). Initiation of transcription from the P2 promoter requires at least three cis-elements: ME1a2, E2F, and ME1a1 (6, 7). Several transcription factors, including Sp1 (5, 8), the Myc-associated zinc finger protein (MAZ)1 (9, 10), Pur-1 (11), and E2F (12) bind to these elements in vitro and in vivo.
The MAZ protein was identified as a transcription factor that binds to
a GA box (GGGAGGG) at the ME1a1 site, to the attenuator region of P2
within the first exon of the c-myc gene, and to a related
sequence that is involved in the termination of transcription of the
gene for complement 2 (C2) (5, 9). Kennedy and Rutter (11) identified
the Pur-1 protein as a GAGA box binding factor that binds to rat genes
for insulin I and II and to the human gene for islet amyloid
polypeptide (11). We recently reported the isolation of a cDNA
clone for a member of the family of MAZ proteins in human islet cells
(13). MAZ protein plays a role in the control of the initiation of
transcription of genes for the adenovirus major late protein (14), CD4
(15), the serotonin receptor (16), and hematopoietic transcription
factor (17), as well as in the termination of transcription between the
closely spaced human genes for complement (8) and in the termination of
transcription of the introns of the mouse gene for IgM-D (8). Therefore, MAZ appears to be a transcription factor with a dual role in
the initiation and termination of transcription. We showed previously
that MAZ is essential for the ME1a1-mediated expression of the
c-myc gene during the neuroectodermal differentiation of P19
cells (18) and for the nuclease-hypersensitive element-mediated transcription of the c-myc gene in islet -cells (13).
To gain a better understanding of the regulation of expression of MAZ, of the splicing mechanism, of the differential polyadenylation and of the potential interactions of MAZ with other factors, we isolated a human genomic gene for MAZ from cosmid and YAC libraries. We characterized the genomic structure of the gene for MAZ protein and identified regulatory elements in 5'-end flanking sequences that are involved in basal transcription and in the autoregulation of the gene for MAZ by the MAZ protein itself.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- pCMV-MAZ was constructed as described previously (13). MAZ-CAT reporter plasmids, namely pMAZCAT0, pMAZCAT1, pMAZCAT2, pMAZCAT3, pMAZCAT4, pMAZCAT5, and pMAZCAT6 were constructed by digesting the DNA of the promoter region with HindIII/XmnI, StuI/XmnI, MscI/XmnI, DpnI/XmnI, PvuII/XmnI, BssHII/XmnI, and HgaI/XmnI, respectively, and then subcloning, via the pHindIII linker, into the HindIII site of pSV00CAT (19). Similarly, deletion mutants, namely, pMAZCAT2-d and pMAZCAT3-d, were constructed by digesting the insert DNA of pMAZCAT2 or pMAZCAT3 with DpnI/PvuII or PvuII/BssHII, respectively, and subcloning into the HindIII site of the pSV00CAT vector.
Screening of a Library of Human Genomic DNA-- A cosmid library (20) was constructed from the genomic DNA of the HLA-homologous B-lymphoblastoid cell line AKIBA (A24, Bw52, Dw12, DQw1, and Cp63), which had been partially digested with Sau3AI, with subsequent ligation of fragments to the cosmid vector pWE15 (Stratagene, La Jolla, CA). The library was screened by colony hybridization with 1.8- and 0.7-kb EcoRI fragments of MAZ cDNA from pCMVMAZ (13) as probes (21). Filters were prehybridized at 68 °C for 30 min in a solution that contained 6 × SSC (1 × SSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0), 5 × Denhardt's solution (0.1% Ficoll (type 400; Pharmacia LKB, Uppsala, Sweden), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin (fraction V; Sigma)), 0.1% SDS, and 0.1 mg/ml denatured calf-thymus DNA and then hybridized in the same buffer supplemented with 1 × 106 cpm/ml of radioactive probe at 68 °C for 20 h. The hybridization probe for MAZ was prepared by the random primed labeling method (22) using a Random Primed DNA Labeling Kit (Boehringer Mannheim, Mannheim, Germany). Filters were washed three times at room temperature for 20 min in 2 × SSC, 0.1% SDS and once at 65 °C for 30 min in 0.2 × SSC, 0.1% SDS and then exposed to XAR-5 film (Eastman Kodak Co., Rochester, NY) with an intensifying screen. A 40-kb DNA insert of a cosmid clone was digested with EcoRI and then subcloned into the pBluescriptII SK+ vector (Stratagene) for further studies. The human MAZ-yeast artificial chromosome (YAC) recombinant clones, y645D4 and y976H4 were isolated from a library of CGM1 DNA (CHEF, Paris, France) by PCR-mediated methods according to the protocol provided by CHEF. The primers for YAC screening were described elsewhere (13, 18).
Nucleotide Sequencing-- DNA sequencing was carried out by the dideoxy chain termination method (23) with an automated DNA sequencer (ABI 373A; Applied Biosystems Inc., Foster City, CA) using a DNA Sequencing Kit (Ref. 24; Dye Terminator Cycle Sequencing Ready Reaction; Applied Biosystems Inc.). In the case of DNA fragments with an unusually high G + C content, cycle sequencing was performed according to the protocols provided by the manufacturer (Applied Biosystems Inc.). Nucleotide and deduced amino acid sequences were analyzed with the GCG program (25).
Southern and Northern Blotting Analysis--
High-molecular
weight DNA from human cells was extracted, digested, fractionated on a
1% gel, and transferred onto a nylon membrane as described previously
(21). The DNA on the membrane was allowed to hybridize with the probe,
and the membrane was washed and exposed to Kodak XAR-5 film with an
intensifying screen as described elsewhere (21). Multiple tissue
Northern blots were obtained from CLONTECH (human
MTN II, 7759-1, and human MTN III, 7767-1;
CLONTECH, Palo Alto, CA). The blots were hybridized and washed before autoradiography as described elsewhere (21). A 1.8-kb
EcoRI fragment of cDNA for MAZ and a 0.6-kb
EcoRI/BamHI fragment of the human DNA (1.0 kb
to
0.4 kb relative to the major site of initiation of transcription)
were radiolabeled for use as DNA probes for further hybridization.
S1 Nuclease Assay--
Total RNA from HeLa cells or human
peripheral blood lymphocytes (PBL) was prepared by the guanidine
thiocyanate method, as described elsewhere (21). S1 nuclease protection
was conducted essentially as described previously (26) using a 208-nt
probe (nt 98 to 106) generated by the digestion of the promoter
region of MAZ DNA with SmaI. The sizes of
protected fragments were determined from comparisons with nucleotide
sequencing ladders prepared from the protected DNA fragments with a
Sequence 7-deaza-D-GTP kit (version 2.0) from Amersham.
Chromosome Mapping-- Fluorescent in situ hybridization was performed as described by Ozawa et al. (27). Metaphase preparations were obtained from phytohemagglutinin-stimulated normal human male lymphocytes after synchronization with 5-bromodeoxyuridine (Sigma). The cosmid DNA was labeled with biotin-14-dATP (Life Technologies, Inc., Gaithersburg, MD) by nick-translation as described elsewhere (28). After hybridization, slides were washed, blocked, and incubated with rabbit antibodies against biotin (Enzo Diagnostic, Inc., New York) Slides were then incubated with second and third antibodies (goat antibodies against rabbit IgG and rabbit antibodies against goat IgG) conjugated with fluorescein isothiocyanate. After washing and drying, slides were counterstained with propidium iodide in anti-bleach mounting medium. Slides were examined under an Optiphot microscope (UFX-IIA; Nikon, Tokyo, Japan), and photographs were taken on Ektachrome 400 film (Kodak). Then, chromosomes were subjected to Q-band staining. The localization of fluorescent signals on chromosomes was determined under a fluoresence microscope and photographed under the bright field of a light microscope with Minicopy HR II film (Fuji-Film Co., Kanagawa, Japan).
Culture and Transfection of Cells and Assay of CAT
Activity--
HeLa cells were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Life
Technologies, Inc.) and 60 µg/ml kanamycin (Sigma). Transfections for
assays of short-term expression of chloramphenicol acetyltransferase (CAT) were performed as described elsewhere (29, 30) with one single
change. After sonication, each crude extract was incubated for 10 min
at 60 °C to denature proteinases (30) Each thin-layer chromatography
plate was exposed to RX film (Fuji, Tokyo, Japan). The extent of
conversion of chloramphenicol to its acetylated form was determined
with a Bio-Image analyzer (model BAS 2000; Fuji). -Galactosidase
activity was assayed as described by Katoh et al. (31). The
ratio of CAT activity to that of
-galactosidase was used for
normalization of results (31).
Polymerase Chain Reaction--
Each polymerase chain reaction
(PCR) was carrried out in a total volume of 20 µl that contained 10 ng of each primer, 10 ng of DNA, 200 µM each dNTP, Tth
polymerase buffer (Toyobo, Kyoto, Japan), and 2.5 units of
Tth
polymerase (Toyobo). Samples were heated at 96 °C for 2 min to
denature the template DNA and then subjected to 30 cycles of 95 °C
for 40 s, 60 °C for 30 s, and 72 °C for 30 s in a
DNA thermal cycler (9600 model; Perkin-Elmer, Foster City, CA). The
products of PCR were separated by electrophoresis on a 8% acrylamide
gel.
Reverse Transcription (RT)-Polymerase Chain Reaction-- The reaction mixture for RT-PCR containing cDNA (equivalent to 20 or 25 ng of total RNA), specific primer sets (5 pmol each), 50 µM dNTPs, 1 × buffer for KOD Dash and 0.5 units of KOD Dash (Toyobo, Osaka, Japan) was subjected to a DNA thermal cycler. Samples were heated at 95 °C for 2 min to denature the tenmplate DNA and then subjected to 25 cycles of 95 °C for 40 s, 60 °C for 40 s and 72 °C for 40 s. Sequences of primers for MAZ and glyceraldehyde-3-phosphate dehydrogenase were as follows: sense primer, 5'-TTCCTTGCACGCTGCTG-3'; antisense primer, 5'-CCTGGAATGGGACTCTGG-3' and sense primer, 5'-TCCACCACCCTGCTGCTGTA-3'; antisense primer, 5'-ACCACAGTCCATGCCATCAC-3', respectively. The products were resolved on a polyacrylamide gel (8%) and stained with VistraGreen (Amersham, Buckinghamshire, United Kingdom).
Synchronization of the Cell Cycle and Western Blotting
Analysis--
Human normal diploid cells (WI-38) were grown in DMEM
supplemented with 10% FBS and 60 µg/ml kanamycin. To induce the
arrest of WI-38 cells in the G0 phase, cells were allowed
to grow exponentially in monolayer and maintained for 30 h in DMEM
supplemented with 0.1% FBS. Arrested cells were switched to incubation
in DMEM supplemented with 10% FBS and then harvested at 6-h intervals
as indicated. G0-arrested cells were also maintained in
DMEM supplemented with 10% FBS in the presence of 1 mM
hydroxyurea (Sigma) for 24 h for resynchronization in early S
phase. The cells were then incubated in DMEM supplemented with 10% FBS
and 7.5 µg/ml Hoechst 33342 (Sigma) for another 12 h for
accumulation of cells in the G2 phase. Cells were lysed and
boiled for 3 min at 90 °C and then lysates were sonicated for
10 s. The sonicates were fractionated by SDS-polyacrylamidg gel
electophoresis (8% polyacrylamide) and then proteins were electroblotted on a polyvinylidene difluoride-nylon membrane (Daiichi Pure Chemicals Co., Tokyo, Japan). The membrane was blocked with 5%
nonfat milk in PBS-T (0.2% Tween 20 in PBS) for 1 h at 25 °C and then washed three times with PBS-T. The membrane was incubated with
1000-fold diluted polyclonal antibodies against MAZ (13, 18) or against
human 2-microglobulin (BM-63; Sigma) in PBS-T for 2 h at 25 °C and then washed three times with PBS-T. Antibodies that
had bound to the membrane were detected with horseradish peroxidase-conjugated antibodies against mouse immunoglobin G (Zymed
Laboratories Inc., South San Francisco, CA) and ECL detection reagents
(Amersham Japan).
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RESULTS |
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Cloning and Determination of the Genomic Structure of the Human Gene for MAZ-- Screening of a cosmid library of the genomic DNA from human B-lymphoblastoid AKIBA cells with the 1.8- and 0.7-kb EcoRI fragment of the coding region and the untranslated region of pMAZi cDNA, respectively (13), as probe yielded seven clones. DNA from the seven cosmid clones was digested with EcoRI and then allowed to hybridize with the same 1.8-kb DNA probe. A single 9.0-kb EcoRI fragment was detected in all seven cosmid clones and subcloned into BluescriptII SK+. The complete restriction maps of the seven subclones were identical. From one of the seven subclones, pJSMAZ9.0E, we further subcloned the 1.3-kb PstI-PstI fragment and the 3.5-kb PstI-EcoRI fragment into pBluescriptII SK+ to generate pJSMAZ1.3P/P and pJSMAZ3.5P/E, respectively. A 1.1-kb EcoRI fragment adjacent to the inserted DNA of pJSMAZ9.0E was also isolated and further subcloned into pBluescriptII SK+ to generate pJSMAZ1.1E. These plasmids, pJSMAZ1.3P/P, pJSMAZ3.5P/E, and pJSMAZ1.1E were fully sequenced and characterized (Fig. 1).
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Sequence of the 5'-Flanking Region of the MAZ Gene-- We compared the genomic MAZ sequence with corresponding sequences of cDNAs for members of the MAZ family that had been reported previously (9-11). We found relatively limited homology in the 5'-flanking region even though the sequences of the coding regions of these genomic and cDNA clones were identical (Fig. 3).
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Characterization of the Site of Initiation of Transcription-- The 5'-boundary of exon 1 was determined by S1 nuclease protection analysis of the SmaI/SmaI fragment (208 nt) of the promoter region of the MAZ genomic clone (Fig. 5). A strong signal corresponding to 106 bp was detected by the S1 protection analysis in the presence of the total RNA from HeLa cells. In addition, five weak signals corresponding to 100, 98, 97, 93, and 92 bp were also detected (Fig. 5). These results indicated that transcription of the MAZ gene started at multiple sites, namely, at positions +1, +7, +9, +10, +14, and +15. All of these sites are located within a CT repeated sequence (see Fig. 4).
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MAZ is a Single-copy Gene Located on Chromosome 16p11.2-- Southern hybridization analysis using DNA from HeLa cells and human PBL showed that a single 9.0-kb EcoRI fragment hybridized with the MAZ-specific cDNA probe (Fig. 6). When DNA was digested with BamHI, we detected three major MAZ fragments, of 4.4, 2.1, and 1.6 kb, respectively, using human PBL and HeLa cells DNAs. These results suggested that MAZ might be encoded by a single, unique gene. Digestion with BamHI revealed common 4.4-, 2.1-, and 1.6-kb fragments in WI-38, MKN7, MKN28, HGC27, GCY1, MKN45, PBL, and HeLa cells. The weak band of the EcoRI-generated 7.2-kb fragment might represent cross-hybridization with the DP-1 gene, which exhibits weak sequence homology to the gene for MAZ (Ref. 40; data not shown). The reciprocal hybridization of human PBL DNAs with the genomic BamHI fragments of MAZ DNA as probes yielded distinct fragments of 4.4, 2.1, and 1.6 kb (Fig. 6c). These results suggest that MAZ is encoded by a single, unique gene. The physical mapping of human-YAC recombinant clones confirmed the presence of a single, unique gene for MAZ (data not shown).
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Characterization of the Three Major Transcripts-- We examined the tissue distribution of MAZ transcripts using human multitissue Northern blots (Fig. 8). Transcripts were identified in all the tissues examined, albeit at different levels. We detected three mRNAs, of 1.6, 2.7, and 4.6 kb, respectively, with the latter two transcripts being major species. The transcripts of 2.7 and 4.6 kb were present in all tissues examined but in the liver, in particular, the level of the 2.7-kb transcript was very low. The levels of the 2.7- and 4.6-kb transcripts in the heart, placenta, pancreas, thymus, prostate, testis, colon, peripheral blood leukocytes, thyroid, and adrenal gland were higher than those in other tissues. The 1.6-kb transcript was detected mainly in the heart, placenta, pancreas, spleen, prostate, colon, thyroid, spinal cord, trachea, and adrenal gland.
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Expression of MAZ during the Cell Cycle--
As shown in Fig.
9a, the level of expression of
MAZ protein was modulated by the cell cycle. The 60-kDa MAZ protein was
produced during the G0 and G2 phases, but it
was not detected during early S phase (Fig. 9a) When
serum-starved normal diploid WI-38 cells were stimulated by addition to
the medium of a high concentration of serum, the level of expression of
MAZ appeared to be reduced at 12 h; the protein disappeared at
30 h and then it reappeared at 36 to 48 h (Fig.
9a). The level of expression of human HLA-associated 2-microglobulin was not changed significantly during the
cell cycle (Fig. 9a). These results indicated that the level
of MAZ protein was modulated in a cell cycle-dependent
manner. At early S phase, in particular, we were unable to detect the
expression of MAZ protein. We next examined the level of MAZ mRNA
by RT-PCR during cell cycle and found the similar changes as that of
MAZ protein. By contrast, the level of expression of
glyceraldehyde-3-phosphate dehydrogenase was unaltered (Fig.
9b). The CAT activities of WI-38 cells transformed with the
pMAZCAT0 reporter construct reflected the variations in the level of
the MAZ protein during the cell cycle (see Fig. 9c).
Furthermore, the introduction of a MAZ expression vector into HeLa
cells significantly and dose dependently enhanced the promoter activity
of the pMAZCAT0 reporter plasmid (Fig. 9d). Thus, expression
of the MAZ gene appears to be controlled during the cell
cycle and to be regulated by the MAZ protein itself.
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Characterization of the Promoter--
To examine whether the
5'-flanking region of the human MAZ gene contained a
functional promoter, we generated a series of chimeric MAZ
promoter/CAT gene reporter constructs, as depicted schematically in
Fig. 10. These constructs were used to
transfect HeLa cells to characterize the regulatory elements of the
human MAZ promoter. Promoter activity was retained after
deletion to position 383 relative to the major site of initiation of
transcription (pMAZCAT3; Fig. 10a). Further deletion to
positions
248,
189, and
40 (pMAZCAT4, pMAZCAT5, and pMAZCAT6)
resulted in significant decreases in promoter activity. Moreover, the
internal deletion mutant pMAZCAT2-d (with a deletion from nt
383 to
248) had diminished promoter activity. Therefore, DNA sequences
between nt
383 and
248 appeared to be required for high basal
promoter activity in HeLa cells. Negative elements might be present
between nt
948 and
2500 because the activity due to pMAZCAT0 was
significantly repressed as compared with that due to pMAZCAT1. These
results clearly demonstrated that the 5'-end flanking region contained the functional promoter of the human gene for MAZ.
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DISCUSSION |
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The present study revealed the exon-intron structure of the human gene for MAZ that spans approximately 6.0 kb and consists of promoters, five exons, four introns, and a 3'-untranslated region. In addition, physical mapping studies of MAZ-YAC clones demonstrated that the MAZ gene is a single and unique gene. All exon-intron boundaries begin with GT at the 5'-end and terminate with AG at the 3' end, conforming to the GT-AG rule (42). When compared with three reported cDNAs for human MAZ, the insert in our clone was most similar to clone 33819 from HeLa cells (the coding region was the same as that of clone 33819). The major differences were found in the 5'-end promoter region (see Fig. 3). The corresponding region of the gene for MAZi from human islets is missing about 35 nucleotides (G + C; from nt +69 to +128). From the results of Southern blotting and RT-PCR with cosmids and YAC recombinant clones, it appeared that MAZ might be encoded by a single gene (Figs. 3 and 6). The heterogeneity of the 5'-end promoter regions of cDNA sequences for MAZ might be due to artifacts that arose because of the high G + C content. Alternatively, differential splicing might have occurred in the case of the MAZi gene from human islets.
Multiple sites for initiation of transcription were found within 174 bp upstream of the site for initiation of translation by the S1 nuclease protection assay (Fig. 5). These sites are located in a putative initiator sequence near the major site for initiation of transcription that matches the 5'-YYC(A/T)YYYYY-3' (Y, pyrimidine) consensus sequence (43). Furthermore, the results of a transcription experiment in vitro showed that the sequence in the vicinity of the defined site of initiation of transcription was sufficient to promote faithful transcription (data not shown). We tried to confirm the site of initiation of transcription in a primer extension assay. However, we were unsuccessful since the reverse transcriptase did not read through the 5'-end regions with a high G + C content. Furthermore, the MAZ promoter-CAT construct also demonstrated the significant activity of the promoter (Figs. 9 and 10). Taken together, our results demonstrate that transcription of the MAZ gene originated from a TATA-less promoter in vivo and in vitro and that the 5'-end region really contained the promoter region of the MAZ gene required to generate a 2.7-kb mRNA. The exact nature of the transcription factors and the specificity of expression of the MAZ gene from this promoter region remain to be determined.
Sequence analysis of the first exon and the 5'-upstream region
suggested that this region had an unusually high G + C content. In
particular, the average G + C content of the 500-bp region upstream of
the ATG codon was 88.4%. It is noteworthy that two regions, extending
over 71 and 77 bp (nt 103 to
33 and nt
306 to
230 relative to
the ATG codon, respectively), had extremely high G + C contents of 98.6 and 97.4%, respectively (Fig. 4). The 1.3-kb fragment of the 5'-end
boundary of the MAZ gene contained 171 copies of CpG and 196 copies of GpC islands. Furthermore, G + C-rich sequences were located
before and after the cap site (+1) of the MAZ gene, within
280 bp. These two long G + C-rich sequences might contribute to changes
in DNA conformation and might be modified, for example, by methylation
(34-36). Such changes might explain the strength of the promoter of
the MAZ gene under certain conditions and in different types
of cells.
CT tracts were present in the region upstream of the small 77-bp region with high G + C content. Such CT tracts are strongly reminiscent of structures described in the gene for the receptor of epidermal growth factor (EGFR) (44) and in other promoters, such as those of the ets-2 (45), c-Ki-ras (46), and c-myc (4, 13) genes. Such repeats are thought to form triplex or H-DNA structures that are sensitive to a variety of nucleases (47). In an examination of CT tracts in the gene for epidermal growth factor receptor, Johnson et al. (44) showed that a specific factor (CTF) binds these sequences and that deletion of the CT tracts significantly down-regulates transcription. We reported similarly that a MAZ protein binds the CT tracts of the c-myc promoter region (13).
(CCG)8 repeats were present in the 71-bp GC-rich stretch and several (CCG)3, (GC)4, (GC)5 repeats were also present in the 500-bp long GC-rich region. GC-rich promoters and the absence of a TATA sequence are characteristic of housekeeping genes (34-36, 43). In fact, Northern blotting analysis revealed the expression of MAZ transcripts in a large variety of human tissues. Examination of the DNA sequence upstream of exon 1 failed to reveal any TATA box or CAAT box. Similar findings have been obtained for a variety of oncogenes, such as human ets-1 (48), human ets-2 (45), human fgr (49), human c-src (50) and murine c-Ki-ras (46); for genes for growth factors and their receptors, such as human epidermal growth factor receptor (44) and insulin-like growth factor receptor (51); and for housekeeping genes, such as the gene for adenosine deaminase (52). The promoters of such genes have a number of common characteristics, such as the presence of multiple sites for initiation of transcription, presumably because of the absence of a TATA box and a CAAT box, and they often have an unusually high G + C content. We found 16 consensus binding sites for Sp1 and 26 binding sites for AP-2 that were located close together or as partially overlapping sites in the 5'-end promoter region. There were also several potential binding sites for the epidermal growth factor receptor-specific transcription factor (ETF) in the promoter region. This protein is thought to replace TATA-binding proteins in the control of expression of genes that lack TATA boxes (53).
We identified mRNAs of 1.6, 2.7, and 4.6 kb, respectively. It is of
interest that only the 4.6-kb mRNA, was detected when the
EcoRI/BamHI DNA fragment that corresponded to the
promoter region of the MAZ gene (1.0 to
0.4 kb relative
to the site of initiation of transcription) was used as DNA probe (data
not shown). Thus, it is likely that the transcription of the 4.6-kb
mRNA is initiated from the far upstream promoter. Studies of
transcription and promoter activities in vitro and
nucleotide sequencing indicated that each transcript, namely, the 4.6, 2.7, and 1.6 kb transcripts, might have been generated by corresponding
independent promoters, which the possibility of differential splicing
or differentiated polyadenylation seems less likely. However, we failed
to isolate a full-length cDNA clone that corresponded to the 4.6-kb
mRNA by molecular cloning, probably because of the unusual high G + C content of this region, as described above.
The 60-kDa MAZ protein was detected during the G0 and G2 phases but not during the early S phase. When the cells were stimulated to reinitiate the cell cycle from the G0 phase by addition of a high concentration of serum, the expression of MAZ was down-regulated at 12 h, was lowest at 30 h and increased from 36 to 48 h (Fig. 9a). The similar changes in the levels of MAZ mRNA were obtained during the cell cycle (Fig. 9b). These data indicated that expression of the MAZ gene was controlled by the cell cycle, as was the activity of the MAZ promoter (Fig. 9c).
In order to identify the elements that regulate the basal transcription
of the MAZ gene, we constructed a series of deletion mutants
of a MAZ-CAT fusion gene and transfected HeLa cells with them (Fig. 10). We identified a positive control element from nt 383
to
248. This region was required for maximal promoter activity, containing multiple consensus binding sites for Sp1 and AP-2, as well
as two (TCCC)2 elements. It remains to be determined
whether these or other factors are really involved in the regulation of expression of the human MAZ gene. Negative regulatory
elements might be localized in the far upstream region between nt
2500 and
948.
We also showed that the expression of the MAZ gene is
controlled by its own product, the MAZ protein. Positive elements for autoregulation by the MAZ protein were putatively identified in the
proximal region from nt 248 to
189 and in the distal region from nt
2500 to
948. The former proximal region contains two consensus
(TCCC) elements and Sp1- and AP-2-binding sites. Negative autoregulatory elements were found in the region between nt
383 and
248 that is adjacent to the positive element. The regulatory elements
for autoregulation by the MAZ protein had a reciprocal relationship to
the regulatory elements for basal transcription of the MAZ
gene: the enhancer region for basal transcription was "shut off"
and the region for negative control of basal transcription was
"turned on" by the product of the MAZ gene. This
scenario is supported by the observation that forced expression of the MAZ gene resulted in significant and
dose-dependent enhancement of the promoter-CAT activities
of the pMAZCAT0 construct (Fig. 9d). We do not yet know the
functional significance of these elements in autoregulation. We are
currently trying to identify the transcriptional factors that are
involved in autoregulation of the MAZ gene.
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ACKNOWLEDGEMENTS |
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We thank Drs. C. Geltinger, T. Murata, N. Adachi, T. Koga, and H. Ugai for many helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Special Coordination Funds of the Science and Technology Agency of Japan, the Life Science Projects of RIKEN, and grants from the Ministry of Education, Science, Sports and Culture of Japan (to K. K. Y).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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D89880.
To whom correspondence should be addressed: Tsukuba Life
Science Center, The Institute of Physical and Chemical Research
(RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. Tel:
81-298-36-3612; Fax: 81-298-36-9120; E-mail:
kazunari{at}rtc.riken.go.jp.
The abbreviations used are: MAZ, Myc-associated zinc finger protein; kb, kilobase pair(s); YAC, yeast artificial chromosome; PCR, polymerase chain reaction; RT, reverse transcriptase; PBL, peripheral blood lymphocytes; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; bp, base pair(s); nt, nucleotides.
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Appendix |
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The figure summarizes the CAT fusion genes with the gene for human MAZ (Fig. s1).
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REFERENCES |
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