Transcription of BRCA1 Is Dependent on the Formation of a Specific Protein-DNA Complex on the Minimal BRCA1 Bi-directional Promoter
1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês
10.1074/jbc.274.44.31297
ISSN1083-351X
AutoresTing-Chung Suen, Paul E. Goss,
Tópico(s)Gene expression and cancer classification
ResumoBRCA1 is the first tumor suppressor gene linked to hereditary breast and ovarian cancers. Its involvement in sporadic breast cancer, however, remains unclear. Recent studies showed that a loss or lowered expression of BRCA1 is not uncommon in nonfamilial breast cancers. In addition, there have been cases of inherited BRCA1-linked breast cancer with as yet unidentified mutation. Misregulation of BRCA1 at the transcription level is a possible mechanism for loss of BRCA1 expression. To understand transcriptional regulation of the BRCA1 gene, we cloned and examined the BRCA1 promoter, by both functional reporter gene analyses and protein-DNA complex formation electrophorectic mobility shift assays. A bi-directional promoter could be located within a 229-base pair (bp) intergenic region between BRCA1and its neighboring gene, NBR2. Deletion analyses further delineated a minimal 56-bp EcoRI-HaeIII fragment, which could drive transcription in the NBR2 gene direction 2–4-fold higher than in the BRCA1 direction in all cell lines tested. Furthermore, transcriptional activity in theBRCA1 direction was undetectable in the muscle cell line C2C12, whereas activity in the NBR2 direction was maintained. These results were consistent with the expression pattern of the respective genes. A specific protein-DNA complex was detected when nuclear extracts from HeLa cells and Caco2, a colon cell line, were incubated with the 56-bp minimal promoter. This protein binding activity was further localized to an 18-bp fragment and might involve a tissue-specific factor, because binding was not detected in the C2C12 cell line. The correlation of the detection of this protein-DNA complex only in those cell lines that expressed the chloramphenicol acetyltransferase reporter gene in the BRCA1 direction suggests a significant positive role of this complex in the transcription of the BRCA1 gene. BRCA1 is the first tumor suppressor gene linked to hereditary breast and ovarian cancers. Its involvement in sporadic breast cancer, however, remains unclear. Recent studies showed that a loss or lowered expression of BRCA1 is not uncommon in nonfamilial breast cancers. In addition, there have been cases of inherited BRCA1-linked breast cancer with as yet unidentified mutation. Misregulation of BRCA1 at the transcription level is a possible mechanism for loss of BRCA1 expression. To understand transcriptional regulation of the BRCA1 gene, we cloned and examined the BRCA1 promoter, by both functional reporter gene analyses and protein-DNA complex formation electrophorectic mobility shift assays. A bi-directional promoter could be located within a 229-base pair (bp) intergenic region between BRCA1and its neighboring gene, NBR2. Deletion analyses further delineated a minimal 56-bp EcoRI-HaeIII fragment, which could drive transcription in the NBR2 gene direction 2–4-fold higher than in the BRCA1 direction in all cell lines tested. Furthermore, transcriptional activity in theBRCA1 direction was undetectable in the muscle cell line C2C12, whereas activity in the NBR2 direction was maintained. These results were consistent with the expression pattern of the respective genes. A specific protein-DNA complex was detected when nuclear extracts from HeLa cells and Caco2, a colon cell line, were incubated with the 56-bp minimal promoter. This protein binding activity was further localized to an 18-bp fragment and might involve a tissue-specific factor, because binding was not detected in the C2C12 cell line. The correlation of the detection of this protein-DNA complex only in those cell lines that expressed the chloramphenicol acetyltransferase reporter gene in the BRCA1 direction suggests a significant positive role of this complex in the transcription of the BRCA1 gene. base pair(s) polymerase chain reaction chloramphenicol acetyltransferase kilobase (pair) 1,4-piperazinediethanesulfonic acid electrophorectic mobility shift assay cAMP-responsive element binding site specific complex BRCA1 is the first breast cancer susceptibility gene isolated (1Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigan S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. 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A. 1997; 94: 5605-5610Crossref PubMed Scopus (422) Google Scholar). Its ability to stimulate p21 expression provides direct evidence of its role as a transcription factor (19Somasundaram K. Zhang H. Zeng Y.-X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar). BRCA1 interacts with Rad51, a protein that is known to be involved in DNA repair. The co-localization of both proteins on asynapsed elements of human synaptonemal complexes during meiosis suggests a role of BRCA1 in the control of genome integrity (20Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1326) Google Scholar). The involvement of BRCA1 in cell cycle check point control is also apparent because its expression (21Vaughn J.P. Davis P.L. Jarboe M.D. Huper G. Evans A.C. Wiseman R.W. Berchuck A. Igelehart J.D. Futreal P.A. Marks J.R. Cell Growth & Differ. 1996; 7: 711-715PubMed Google Scholar) and phosphorylation (22Thomas J.E. Smith M. Tonkinson J.L. Rubinfeld B. Polakis P. Cell Growth & Differ. 1997; 8: 801-809PubMed Google Scholar) are induced before DNA synthesis. Since the cloning of the BRCA1 gene, more than a hundred mutations have been found throughout the entire coding sequence (2Futreal P.A. Liu Q. Shattuck-Eidens D. Cochran C. Harshman K. Tavtigan S. Bennett L.M. Haugen-Strano A. Swensen J. Miki Y. Eddington K. McClure M. Frye C. Weaver-Feldhaus J. Ding W. Gholami Z. Soderkvist P. Terry L. Jhanwar S. Berchuck A. Iglehart J.D. Marks J. Ballinger D.G. Barrett J.C. Skolnick M.H. Kamb A. Wiseman R. Science. 1994; 266: 120-122Crossref PubMed Scopus (1139) Google Scholar, 3Gayther S.A. Warren W. Mazoyer S. Russell P.A. Harrington P.A. Chiano M. Seal S. Hamoudi R. van Rensburg E.J. Dunnuing A.M. Love R. Evans G. Easton D. Clayton D. Stratton M.R. Ponder B.A.J. Nat. Genet. 1995; 11: 428-433Crossref PubMed Scopus (465) Google Scholar, 4Feunteun J. Lenoir G.M. Biochim. Biophys. Acta. 1996; 1242: 177-180Crossref PubMed Scopus (24) Google Scholar,10Bertwistle D. Ashworth A. Curr. Opin. Genet. & Dev. 1998; 8: 14-20Crossref PubMed Scopus (91) Google Scholar, 23Xu C.-F. Solomon E. Semin. Cancer Biol. 1996; 7: 33-40Crossref PubMed Scopus (49) Google Scholar). However, there remain cases of breast and ovarian cancer families with unknown mutations. Some of these patient samples exhibit a specific allelic loss of transcripts and are thought to have a regulatory mutation (1Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigan S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. 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Harshman K. Tavtigan S. Bennett L.M. Haugen-Strano A. Swensen J. Miki Y. Eddington K. McClure M. Frye C. Weaver-Feldhaus J. Ding W. Gholami Z. Soderkvist P. Terry L. Jhanwar S. Berchuck A. Iglehart J.D. Marks J. Ballinger D.G. Barrett J.C. Skolnick M.H. Kamb A. Wiseman R. Science. 1994; 266: 120-122Crossref PubMed Scopus (1139) Google Scholar). It is hypothesized that mutations in BRCA1 might affect the process of growth regulation at an early stage of development, as is demonstrated by mouse knock-out experiments (27Gowen L.C. Johnson B.L. Latour A.M. Sulik K.K. Koller B.H. Nat. Genet. 1996; 12: 191-194Crossref PubMed Scopus (396) Google Scholar, 28Hakem R. de La Pompa J.L. Sirard C. Mo R. Woo M. Hakem A. Wakeham A. Potter J. Reitmair A. Billia F. Firpo E. Hui C.C. Roberts J. Rossant J. Mak T.W. Cell. 1996; 85: 1009-1023Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 29Liu C.-Y. Lesken-Nikitin A. Li S. Zeng Y. Lee W.-H. 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Genet. 1997; 16: 298-302Crossref PubMed Scopus (227) Google Scholar, 34Ludwig T. Chapman D.L. Papaioannou V.E. Efstratiadis A. Genes & Dev. 1997; 11: 1226-1241Crossref PubMed Scopus (462) Google Scholar).BRCA1 has been shown to physically associate withp53 and co-activate p53-responsive genes (35Ouchi T. Monteiro A.N.A. August A. Aaronson S.A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2302-2306Crossref PubMed Scopus (333) Google Scholar,36Zhang P. Somasundaram K. Peng Y. Tian H. Zhang H. Bi D. Weber B.I. El-Deiry W.S. Oncogene. 1998; 16: 1713-1721Crossref PubMed Scopus (426) Google Scholar). Interestingly, p53 mutations seem to occur at a very high frequency in BRCA1-associated familial breast cancer (37Crook T. Crossland S. Crompton M.R. Osin P. Gusterson B.A. Lancet. 1997; 350: 638-639Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Taken together, these studies suggest that p53 might be an important checkpoint for BRCA1-associated tumorigenesis. It is possible that in most sporadic breast cancers,BRCA1 mutations were not being searched for because those samples might have p53 mutations or other oncogenes or tumor suppressor genes identified. On the other hand, a disruption in regulatory sequences could also be the cause for a decrease in BRCA1 expression during sporadic breast cancer progression (38Thompson M.E. Jensen R.A. Obermiller P.S. Page D.L. Holt J.T. Nat. Genet. 1995; 9: 444-450Crossref PubMed Scopus (544) Google Scholar). The analysis of the promoter or the identification of important regulatory sequences will therefore provide information on where to search for these regulatory mutations. We report here the isolation and functional analyses of the human BRCA1 promoter. A 229-base pair (bp)1 intergenic region could serve as a promoter for both the BRCA1 and its neighboring gene, NBR2. Deletion analyses delimited a minimal 56-bp fragment within the intergenic region, which retained the bi-directional promoter activity. A specific protein-DNA complex was detected with an 18-bp element within the minimal promoter. The existence of this complex only in BRCA1-expressing cells correlated with the ability of the minimal promoter to drive transcription in theBRCA1 direction. It is conceivable that mutations found within either the cis-acting element or thetrans-acting transcription factor described in this report could be a target of mutagenesis in BRCA1-linked cancers with regulatory mutations or even in sporadic breast cancer where no mutation of BRCA1 has been found (2Futreal P.A. Liu Q. Shattuck-Eidens D. Cochran C. Harshman K. Tavtigan S. Bennett L.M. Haugen-Strano A. Swensen J. Miki Y. Eddington K. McClure M. Frye C. Weaver-Feldhaus J. Ding W. Gholami Z. Soderkvist P. Terry L. Jhanwar S. Berchuck A. Iglehart J.D. Marks J. Ballinger D.G. Barrett J.C. Skolnick M.H. Kamb A. Wiseman R. Science. 1994; 266: 120-122Crossref PubMed Scopus (1139) Google Scholar, 39Vogelstein B. Kinzler K.W. Cell. 1994; 79: 1-3Abstract Full Text PDF PubMed Scopus (58) Google Scholar). Restriction enzymes and other DNA-modifying enzymes such as T4 kinase, T4 polymerase, T4 ligase, Klenow fragment, calf intestinal phosphatase, and S1 nuclease were purchased from Life Technologies Inc., New England Biolabs (Mississauga, Ontario, Canada), Roche Molecular Biochemicals, or Amersham Pharmacia Biotech. All isotopes were products from Amersham Pharmacia Biotech. Chemicals used for the S1 nuclease protection, CAT, and β-galactosidase assays were purchased from Sigma. Thin layer chromatography (TLC) plates were products of Kodak (Rochester, NY). Cell culture media and reagents were obtained from Life Technologies, Inc. The plasmid pCR2.1 (Invitrogen, Carlsbad, CA) was used for cloning PCR-amplified products. The plasmid pBluescript(IIKS) (Stratagene, La Jolla, CA) was used for general subcloning purposes. pMT.IC3 is a plasmid containing multiple cloning sites placed upstream of the chloramphenicol acetyltransferase (CAT) gene (40Suen T.-C. Hung M.-C. Mol. Cell. Biol. 1990; 10: 6306-6315Crossref PubMed Scopus (41) Google Scholar). Most of the BRCA1 DNA restriction fragments were cloned into pBluescript(IIKS) and were then shuffled into the matching unique restriction sites on the polylinker of pMT.IC3. DNA fragments were blunt-ended with Klenow fragment or T4 polymerase when no appropriate restriction enzymes could be used for directional cloning. In addition, reversed orientation of a subcloned fragment in the pMT.IC3 plasmid could easily be obtained by cutting with HindIII, which flank the polylinker, followed by religation. All of the CAT constructs were named according to the direction of transcription. Therefore the pBR and pNB series of CAT plasmids indicate promoters transcribing toward the BRCA1and the NBR2 gene directions, respectively. pCMVβ (CLONTECH, Palo Alto, CA), a plasmid that contains the lacZ gene driven by the cytomegalovirus enhancer (41MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 17: 2365Crossref PubMed Scopus (440) Google Scholar), was used for monitoring transfection efficiency. Detailed maps of all of the plasmids used in this study will be distributed along with the materials upon request. The T7 polymerase sequencing kit was purchased from Amersham Pharmacia Biotech. Dideoxy sequencing of double-stranded plasmids with [α-35S]dATP or [α-35S]dCTP (Amersham Pharmacia Biotech) was performed according to the manufacturer. Most of the BRCA1 promoter subclones, particularly those with DNA inserts of less than 300-bp in size, were confirmed by sequencing. Oligonucleotides, obtained from the Hospital for Sick Children Biotechnology Center, Toronto, were as follows: PCR primer, BRCA1 + 89 (5′-GCAGAGGGTGAAGGCCTCCTG-3′) and BRCA1 + 2 (5′-GCTCGCTGAGACTTCCTGGAC-3′); sequencing primer, pMTIC3 (5′-TAGGCGTATCACGAGGCCC-3′) and pBluescript(IIKS) (the reverse primer or universal primer was used (Stratagene)). Several cell lines representing different tissues of origin were used in this study and are all available from American Type Culture Collection (ATCC, Manassas, VA). This includes HeLa, a human cervical carcinoma cell line; Caco2, a human colon carcinoma cell line; MCF7, a human mammary carcinoma cell line; and C2C12, a mouse myoblast cell line. All cell lines were cultured in Dulbecco's modified Eagle's-F12 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and kept in a humidified, 37 °C, 5% CO2 incubator. A pair of DNA primers located in exon 1 of the BRCA1 cDNA were synthesized (Fig.1 B), and PCR was performed on human lymphocyte DNA. Amplified DNA product of the expected size was gel purified and subcloned into the plasmid pCR2.1. After sequence confirmation, the exon 1 DNA was labeled as a probe to screen a phage artificial chromosome (PAC) library of human genomic DNA. Three clones were obtained, one of which, P103014, has been described by others (42Brown M.A. Xu C.-F. Nicolai H. Griffiths B. Chambers J.A. Black D. Solomon E. Oncogene. 1996; 12: 2507-2513PubMed Google Scholar). Repeated rounds of restriction mapping, subcloning, and exon 1 probing allowed the isolation of a 3.8-kb PstI fragment. Sequencing analyses of various subcloned fragments confirmed their identity to the BRCA1 genomic sequence that has been deposited in the GenBankTM (accession no. U37574) and not to a duplicated pseudogene located at the same region (42Brown M.A. Xu C.-F. Nicolai H. Griffiths B. Chambers J.A. Black D. Solomon E. Oncogene. 1996; 12: 2507-2513PubMed Google Scholar, 43Barker D.F. Liu X. Almeida R.A. Genomics. 1996; 38: 215-222Crossref PubMed Scopus (27) Google Scholar). A 2.7-kbPstI-XbaI fragment containing intron 1 and upstream sequences of the BRCA1 gene is shown schematically with respect to the genomic organization (Fig. 1 A). The sequence was numbered as in U37574. The sequence of the untranslated exon 1 and the upstream region of BRCA1, including the complete intergenic region between BRCA1 and the neighboring gene NBR2 (44Xu C.-F. Brown M.A. Nicolai H. Chambers J.A. Griffiths B.L. Solomon E. Hum. Mol. Genet. 1997; 6: 1057-1062Crossref PubMed Scopus (75) Google Scholar), is shown in Fig. 1 B. The focus of this report is restricted to the intergenic region between theEcoRI and the SstI sites (shown inbold). The S1 nuclease protection method was used to determine the transcription initiation sites (40Suen T.-C. Hung M.-C. Mol. Cell. Biol. 1990; 10: 6306-6315Crossref PubMed Scopus (41) Google Scholar). RNAs were isolated from different cell lines with Triazol Reagent (Life Technologies, Inc.). A 728-bpStuI-StuI fragment was dephosphorylated and labeled with [γ-32P]ATP by T4 kinase. 20 μg of RNAs from various cell lines, or yeast tRNA (as a control), was co-precipitated with 50,000 cpm of probe in ethanol with the addition of lithium acetate. The precipitate was washed with 70% ethanol, dried, and resuspended in 50 μl of S1 hybridization buffer (80% deionized formamide, 40 mm PIPES, pH 6.8, 400 mm NaCl, 1 mm EDTA), after which it was submerged at 60 °C overnight. 300 μl of S1 digestion buffer (280 mm NaCl, 50 mm sodium acetate, pH4.6, 4.5 mm ZnSO4, 20 μg/ml ssDNA) containing 200 units/ml S1 nuclease was added to the hybridized mix and incubated at 37 °C for 30 min. 50 μl of stop buffer (4 m ammonium acetate, 0.1 mm EDTA) was added, which was followed by phenol/chloroform extraction. Two volumes of ethanol was added to precipitate the DNA, which was then dried in a SpeedVac. The sample was resuspended in loading dye (48% urea, 1 mm EDTA, 0.1m NaOH, 0.01% bromophenol blue) and run on a 7.5% denaturing gel. A sequencing ladder of a known plasmid DNA was run alongside as a size marker. The gel was dried and exposed to Kodak XAR-5 film at −80 °C. A calcium phosphate precipitation method (45Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar) was used for transfection as modified and described previously (46Suen T.-C. Hung M.-C. Mol. Cell. Biol. 1991; 11: 354-362Crossref PubMed Scopus (56) Google Scholar). Briefly, cells were split at a predetermined ratio into 100-mm tissue culture dishes (Falcon) the day before transfection. Unless otherwise indicated, 1 μg of pCMVβ and 10 μg of a CAT reporter DNA were co-precipitated in the buffer at room temperature for 25 min, before it was added directly to the cells. Precipitate was left incubated with the cells for 16–20 h, after which the cells were washed three times with phosphate-buffered saline, re-fed with fresh medium, and returned to the 37 °C incubator. Cells were washed and harvested after 20–24 h, and the freeze/thaw cycle method was used to lyse the cells. One-fifth of the cell lysate was used for the β-galactosidase assay usingO-nitrophenyl-β-d-galactopyranoside as substrate, and the results were used to adjust the amount of lysate for the CAT assay. The TLC method of CAT assays was performed as described previously (40Suen T.-C. Hung M.-C. Mol. Cell. Biol. 1990; 10: 6306-6315Crossref PubMed Scopus (41) Google Scholar), except that the standard [14C]chloramphenicol was replaced with 1-deoxy[dichloro-acetyl-1-14C]chloramphenicol (Amersham Pharmacia Biotech). Because only one acetyl group could be transferred to this substrate, only one product could be seen on the TLC as opposed to three possible products that are generally observed in the literature. This also improves the quantitative aspect of the CAT assays. EMSA was performed as described previously (40Suen T.-C. Hung M.-C. Mol. Cell. Biol. 1990; 10: 6306-6315Crossref PubMed Scopus (41) Google Scholar). Nuclear extract was isolated from the different cell lines by the Dignam method (47Dignam J.D. Leibovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). The DNA fragment was isolated by digesting a plasmid subclone with the appropriate restriction enzymes, gel-purified, and labeled with [α-32P]dATP or [α-32P]dCTP (depending on the restriction site) by Klenow fragment. Reaction mixture was added in the order of H2O, 10× binding buffer (1×, 10 mm Tris, pH 7.5, 50 mm KCl, 1 mmdithiothreitol, 0.1 mm EDTA, 1 mmMgCl2, and 5% glycerol), 3 μg of poly(dI·dC)·poly(dI·dC), 10 μg of nuclear extract, an appropriate amount of unlabeled competitor if desired, and finally, 20,000 cpm of probe. The mixture was incubated at room temperature for 25 min, after which it was loaded onto a 6% native polyacrylamide gel. The gel was dried under vacuum in a gel dryer and exposed to a Kodak BioMAX MR film at −80 °C. Sequence analysis of the intergenic region betweenBRCA1 and its neighboring gene with respect to consensus transcription factor binding sites has been discussed extensively (42Brown M.A. Xu C.-F. Nicolai H. Griffiths B. Chambers J.A. Black D. Solomon E. Oncogene. 1996; 12: 2507-2513PubMed Google Scholar,48Brown M.A. Nicolai H. Xu C.-F. Griffiths B. Jones K.A. Solomon E. Hosking L. Trowsdale J. Black D.M. McFarlane R. Nature. 1994; 372: 733Crossref PubMed Scopus (31) Google Scholar, 49Xu C.-F. Brown M.A. Chambers J.A. Griffiths B. Nicolai H. Solomon E. Hum. Mol. Genet. 1995; 4: 2259-2264Crossref PubMed Scopus (94) Google Scholar). Because promoter function was confined to the 56-bpEcoRI-HaeIII fragment (see below), only potential transcription factor binding sites within this sequence are highlighted (Fig. 1 B). A CT (or AG)-rich sequence, a possible binding site for the Ets family of transcription factors (50Sharrocks A.D. Brown A.L. Ling Y. Yates P.R. Int. J. Biochem. Cell Biol. 1997; 29: 1371-1387Crossref PubMed Scopus (283) Google Scholar), was found between nucleotides 1380 and 1406. A “TTACGTCA” sequence, located between 1405 and 1412, is almost identical to the consensus cAMP-responsive element binding (CREB) site (TGACGTCA) (51Sassone-Corsi P. Annu. Rev. Cell Dev. Biol. 1995; 11: 355-377Crossref PubMed Scopus (337) Google Scholar). A “GGGTGG” sequence located at 1427 to 1432 is identical to the GT box, known to bind the Sp1 family of transcription factors (52Lania L. Majello B. De Luca P. Int. J. Biochem. Cell Biol. 1997; 29: 1313-1323Crossref PubMed Scopus (263) Google Scholar) (Fig. 1 B). Xu and co-workers (49Xu C.-F. Brown M.A. Chambers J.A. Griffiths B. Nicolai H. Solomon E. Hum. Mol. Genet. 1995; 4: 2259-2264Crossref PubMed Scopus (94) Google Scholar) mapped the transcription start site to position 1579, and named this nucleotide +1 in their paper (marked by a closed circle above the nucleotide in Fig. 1 B). However, upon careful inspection and comparison of our sequence with sequences published by the same authors (42Brown M.A. Xu C.-F. Nicolai H. Griffiths B. Chambers J.A. Black D. Solomon E. On
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