- Email: [email protected]
Functional characterization of the promoter for the gene encoding murine CD34
Biochimica et Biophysica Acta 1350 Ž1997. 141–146
Promoter paper
Functional characterization of the promoter for the gene encoding murine CD34 Yuji Yamaguchia a
a,)
, Daniel G. Tenen b, Toshio Suda
a
Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto UniÕersity School of Medicine, Kumamoto 860, Japan b Department of Hematologyr Oncology, Beth Israel Hospital, HarÕard Medical School, Kumamoto 860, Japan Received 16 October 1996; accepted 22 October 1996
Abstract Since CD34 expression is restricted to the hematopoietic stem cells and decreases in differentiating cells, the analysis of the CD34 promoter is of interest to understand regulation of gene expression in stem cells. To characterize the cis-acting elements which control murine CD34 ŽmCD34. gene expression, we sought to clone, sequence, and functionally analyze the mCD34 promoter. An 80% decrease in promoter activity was obtained when sequences between y119 bp and y59 bp upstream of the transcriptional start site were deleted. We identified several DNA-protein complexes which correspond to functional segments defined by the linker-scanning mutants. These findings indicated the presence of the important regulatory element between bp y119 and y100, GGTTAAAAGTGAAGTAGGAA. Furthermore, from the result of functional promoter analysis in the DNase I hypersensitive site ŽHS. located within 5 kb upstream of the mCD34 gene, the presence of the enhancer region in the NcoIrPstI fragment of 5X upstream, y2.8 kb to y1.9 kb, has been identified. These data will provide useful information on the gene transfer using the CD34 promoter and enhancer. Keywords: CD34; Promoter region; Enhancer region; M1 cell; Luciferase; Transcription initiation; ŽMurine.
The CD34 antigen is selectively expressed on hematopoietic stem cells w1,2x. Cells expressing CD34 constitute approx. 1% of the population of human bone marrow ŽhBM. cells and include colony-forming cells w3x. It has been shown that the CD34 antigen is a 105–120 kDa type I transmembrane glycoprotein and is also expressed on vascular endothelial cells w4x. The human CD34 ŽhCD34. gene belongs to a cluster of genes on chromosome lq which encode adhesion molecules. Recently,
)
Corresponding author. Fax: q81 96 3735332. E-mail: [email protected]
Baumhueter et al. showed that L-selectin expressed in lymphocytes binds to the sialomucin of CD34 which is expressed in endothelial venules in lymph nodes w5x. This suggests a possibility that the CD34 antigen functions as an adhesion molecule. However, the function of CD34 has remained to be elucidated. We are interested in understanding the mechanism of transcriptional regulation of CD34 gene to elucidate the molecular basis for commitment in early hematopoiesis, and the possibility of clinical applications in stem cell gene therapy. The murine CD34 ŽmCD34. gene is expressed in a wider variety of tissues than the human homologue, including brain and several fibroblast cell lines w4x. In particular, we
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have shown that the alternative splicing of mCD34 results in two types of CD34 mRNA w6x. The truncated form of CD34 mRNA is expressed at relatively higher levels in brain than in other tissues w6x. Thus, the regulation of CD34 gene expression may differ from that in each tissue. Although several groups have studied human CD34 promoter regulation, they failed to detect the core CD34 promoter which is specific for human CD34q cells w7–9x. The homology with the human sequence is highest in the intracellular domain Ž90% amino acid identity. and lowest in the N-terminal region Ž43% amino acid identity. w10x. While there is a relatively high homology between human and murine CD34 cDNAs, the sequence upstream of the mCD34 gene is entirely different from that of the hCD34 gene. This suggests that gene regulation of mCD34 may be different from that of the hCD34. Accordingly, to identify transcription factors involved in regulating mCD34 gene expression, we isolated the 5X flanking region of the mCD34 gene and analyzed the functional characteristics of its promoter region. Transcriptional start sites of the mCD34 gene. To define the transcriptional start site of the mCD34 gene, the S1 nuclease protection assay was performed ŽFig. 1.. A 20 bp oligonucleotide complementary to the coding strand of the 3X end of exon 1 was 5X end-labeled and used to generate a probe for this experiment, as shown in the upper part of Fig. 1. This 400-nucleotide probe was hybridized to 10 m g of polyŽ A.q RNA derived from M1 cells. After annealing and digestion with nuclease S1, a protected fragment of 160 nucleotides in length was detected ŽFig. 1.. From the result of this nuclease S1 protection assay, the cytidine residue located 84 bp 5X from the initiation methionine can be considered as the major transcriptional start site in the mCD34 gene. This evidence is compatible with the suggestion by Brown et al. that the transcriptional start site is probably within 100 bp of the start of the longest cDNA w10x. Functional characterization of mCD34 promoter region. To locate the cis-acting elementŽs. required for mCD34 promoter activity, we analyzed luciferase activity in M1 cells, a murine myeloid leukemia cell line, transiently transfected with mCD34 promoter constructs subcloned into the promoterless pXP2 luciferase expression vector. An mCD34 genomic fragment containing 2.0 kb of sequence upstream of the
Fig. 1. Identification of the transcriptional start site of murine CD34. A 20 bp oligonucleotide complementary to the coding strand of the 3X end of exon 1 was 5X end-labeled as shown. The kinased oligonucleotide primer was extended with Klenow fragment and was digested with Sau3A I. This 400-nucleotide probe was annealed with 10 m g of polyŽA.q RNA from M1 cells and then digested with nuclease S1. Samples were electrophoresed on a 6% denaturing polyacrylamide gel next to sequencing ladder generated with the same 1st exon 20 bp oligonucleotide that was used for this study, and M13mp 8 single-stranded DNA.
transcriptional start site was subcloned into the promoterless pXP2 luciferase vector. When transiently transfected into M1 cells, the y2.0 kbrmCD34luciferase Žluc. promoter construct reproducibly ex-
Y. Yamaguchia et al.r Biochimica et Biophysica Acta 1350 (1997) 141–146
Fig. 2. Functional activity of mcD34 deletion constructs in M1 cells. 10 m g of pBluescript II KSŽy. was transfected as carrier DNA with 15 m g of each mCD34-pXP2 construct into M1 cells. Luciferase activities were normalized for the amount of hGH produced by the co-transfected control CMV-hGH plasmid. Corrected RLU for each construct are shown as the percent activity relative to the mean activity of the wild-type ŽWT. mCD34 promoter construct. Values are shown as means"S.E. for three independent experiments.
pressed ) 50-fold more luciferase activity than did the promoterless pXP2 control. To localize regulatory elements in the mCD34 promoter, a series of 12 deletion routants ŽM1–M12. were produced from the y2.0-kbrmCD34-luc construct using exonuclease III and the polymerase chain reaction ŽPCR.. No significant decrease in promoter activity was observed when sequences between y2.0 kb and y119 bp were deleted ŽFig. 2.. Further deletion to y59 and y21 bp
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produced decreases in luciferase activity of more than 6- and 17-fold Ž94%., respectively, when compared with the y2.0-kb construct Žwild-type; WT.. The cell type-specificity of the mCD34 promoter for CD34 expression was assessed by transfecting the wild-type construct into M1 cells, P815 cells, and WEHI-3 cells. CD34 mRNA expression was observed in M1 cells, but not in P815 cells or WEHI-3 cells Ž data not shown.. The activity of the mCD34 promoter was approx. 10-fold to 20-fold lower in P815 and WEHI-3 cells than in M1 cells Ždata not shown.. To more precisely locate the functional sequences required for mCD34 promoter activity in the region between y119 bp and y60 bp upstream of the transcriptional start site, in which the presence of positively acting regions was anticipated, six linkerscanning mutants were constructed by inserting a 10-bp oligonucleotide consecutively every 10 bp in place of the wild-type promoter sequence ŽFig. 3.. A 5-fold decrease in mCD34 promoter activity relatively to either the wild-type or the M10 deletion mutant was observed when the sequence between bp y119ry110 or bp y109ry100 was replaced by the linker and a 4-fold decrease in activity resulted when the sequence between bp y69 and y60 was replaced. To investigate DNArprotein interactions in the mCD34 promoter, electrophoretic mobility shift assays ŽEMSAs. were carried out. The probes for the
Fig. 3. Linker-scanning analysis of the 119 bp mCD34 promoter in M1 cells. Comparative promoter activity in M1 transiently transfected with the y2 kb promoter ŽWT., y119 bp promoter ŽM10., and six linker scanning mutants of the mCD34 promoter in the pXP2-luciferase vector are shown. The diagonally striped rectangular boxes represent the 10 bp sequences replaced by the linker oligonucleotide. Three independent transfection experiments were performed; the average activities Ž"S.E.. relative to the activity of the M10 Žy119 bp. promoter Ž100%. are shown. Luciferase activity, measured as RLU, was first normalized for transfection efficiency based on the amount of growth hormone produced and were indicated as corrected RLU. The data represents the mean " S.E. of three independent experiments.
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Fig. 4. Identification of sequence-specific DNA-binding proteins by electrophoretic mobility shift assays ŽEMSAs.. ŽA. 32 P-end-labeled oligomer A Žlanes 1–3., oligomer B Žlanes 4–6., oligomer C Žlanes 7–9., and oligomer D Žlanes 10–12. were incubated in the absence Žlanes 1, 4, 7, and 10. or in the presence Žlanes 2, 3, 5, 6, 8, 9, 11, and 12. of nuclear extracts from M1 cells. A 200-fold molar excess of each unlabeled oligonucleotide Žcompetitors., identical to the probe in the binding reaction, were added Žlanes 3, 6, 9, and 12.. ŽB. Cross-competition assay. Unlabeled competitors Žoligomer A, B, C, and D. were included in the binding reactions containing 32 P-labeled oligomer A probe.
EMSAs, oligomers A, B, C, and D, were 30-bp double-stranded oligonucleotides corresponding to sequences extending from bp y129 to bp y40 of the mCD34 gene. As shown in Fig. 4A, the nuclear
protein from M1 cells specifically bound to oligomers A and C, spanning sequences y129 to y100 bp and y89 to y60 bp, respectively. However, the bands of DNA binding protein formed with oligomer C were
Fig. 5. Functional analysis of the mCD34 5X DNase I HS in M1 cells. M1 cells were transiently transfected with the luciferase ŽLuc. reporter constructs containing the minimal promoter, y119 bprluc, shown schematically. RV, RI, N, and P represent EcoRV, EcoRI, NcoI, and PstI, respectively. 10 m g of pBluescript II KSŽy. was transfected as carrier DNA with 15 m g of each construct into M1 cells. Luciferase activities were normalized for the amount of hGH produced by a co-transfected control CMV-hGH plasmid. Corrected RLU for each construct are shown as the percent activity relative to the mean activity of the minimal promoter construct, y119rluc. Values are shown as means" S.E. for three independent experiments.
Y. Yamaguchia et al.r Biochimica et Biophysica Acta 1350 (1997) 141–146
relatively faint compared to those formed with oligomer A. Each unlabeled DNA probe, in 200 molar excess, was added and the bands were completely competed out. A cross-competition assay of EMSAs was performed to confirm whether the bands of DNA binding protein formed with oligomer A were specific for this sequence. As shown in Fig. 4B, the bands observed in the presence of oligomer A probe were competed out by the addition of the excess non-radioactive self competitor, but not other competitors, i.e., oligomers B, C, and D. These results suggest that the DNA sequence between position y129 and y100 bp specifically binds to the nuclear protein derived from M1 cells. An enhancer is located approximately 3 kb upstream of the transcriptional start site in the mcD34 gene. An enhancer in the DNase I hypersensitive site ŽHS. of the mCD34 gene has been revealed by May and Enver w11x. To maximize mCD34 promoter activity, we examined the enhancer region of the 5X DNase I HS. Each fragment of DNase I HS located within 5 kb upstream of the mCD34 gene Ž EcoRVrEcoRI fragment, EcoRIrEcoRI fragment, EcoRIrNcoI fragment, and NcoIrPstI fragment., was subcloned into the mCD34 minimal promoter, the y119 bprCD34-luc construct ŽFig. 5.. The activity of mCD34-luc promoter construct containing the 5.0 kb upstream of mCD34 gene, y5 krluc, was approx. 4-fold higher compared to that of minimal promoter construct, y119rluc. In promoter constructs containing each region of 5X DNase I HS with the minimal promoter, the promoter activity of the region containing the NcoIrPstI fragment, 0.9 k enhrluc, was approx. 7-fold greater when compared with the minimal promoter alone, y119rluc. This evidence that the region located 3 kb upstream of the transcriptional start site acts as an enhancer is compatible with the data reported by May and Enver w11x. From the results of the functional promoter assays as shown in Fig. 2 and Fig. 3 in addition to the EMSAs performed by mutational analysis, our analyses of the mCD34 promoter regions revealed the presence of the important regulatory element between bp y119 and y100. These coincident regions, the sequence between bp y119 and y100, contain potential binding sites for several transcription factors including IRF-1rIRF-2 w12x and ATF w13x. IRF-1 and IRF-2 are structurally related, and bind specifically to
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the same virus-inducible cis-elements of the IFN-a 1 and IFN-b gene promoters as well as to the interferon response sequence of MHC class I gene promoters w14x. It has been shown that IRF-1 is a primary response gene, and IFN-b is a secondary response gene in the developmental program of terminal myeloid differentiation induced by IL-6 or LIF w15x. However, neither IRF-1 nor IRF-2 showed any significant effect on the promoter activity of the mCD34 gene Ždata not shown. . The adenovirus transcription factor ATF stimulates transcription from the adenovirus early region 4 ŽE4. promoter w13x. It has been shown that cyclic AMP response element-binding protein Ž CREB. and ATF are identical w16x. The mRNA of HB16 transcription factor which recognizes the same motif as CREB, with which it also displays structural similarity, is most abundant in brain w17x. The truncated form of CD34 mRNA is also expressed at relatively higher level in the brain than in other tissues w6x. Although no complete matches to the ATF or CREB motif ŽTGACGT. appeared to be present in the mCD34 promoter ŽTGAAGT., CREB might play a role in CD34 gene expression in the brain. Since the region between bp y107 and bp y100 matches the MZF-1 first domain-binding site at 6 of 8 bases, we determine the transactivation activity of MZF-1 on the promoter activity of mCD34 gene. No significant enhancement of the minimal promoter, y119rCD34-luc, or the promoter of the y5 kb upstream, -SkbrCD34-luc, was observed when the MZF-1 expression vector was cotransfected into M1 cells Ždata not shown. . Morris et al. have reported that MZF-1 transactivated the human CD34 promoter containing 1.1 kb upstream of transcriptional start site in hematopoietic cells but not in non-hematopoietic cells w18x. The discrepancy of the result on the transactivation of the MZF-1 between murine and human CD34 promoter activity may suggest differences between murine and human CD34 gene regulation. We wish to thank Dr. S.J. Ackerman for searching the Ghosh transcription factor data base. We also thank Dr. M. Ishihara and Dr. T. Taniguchi for generously providing us with IRF-1 and IRF-2 expression vectors, and Dr. R. Hromas for providing us with MZF-1 expression vectors. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.
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References w1x Civin, C.I., Strauss, L.C., Brovall, C., Frackler, M.J., Schwartz, J.F. and Shaper, J.H. Ž1984. J. Immunol. 133, 157–165. w2x Andrews, R.G., Singer, J.W. and Bernstein, I.D. Ž1986. Blood 67, 842–845. w3x Yamaguchi, Y., Gunji, Y., Nakamura, M., Hayakawa, K., Maeda, M., Osawa, H., Nagayoshi, K., Kasahara, T. and Suda, T. Ž1993. Exp. Hematol. 21, 1233–1238. w4x Fina, L., Molgaard, H.V., Robertson, D., Bradley, N.J., Monaghan, P., Delia, D., Sutherland, D.R., Baker, M.A. and Greaves, M.F. Ž1990. Blood 75, 2417–2426. w5x Baumhueter, S., Singer, M.S., Henzel, W., Hemmerich, S., Renz, M., Rosen, S.D., Lasky, L.A. Ž1993. Science 262, 436–438. w6x Suda, J., Sudo, T., Ito, M., Ohno, N., Yamaguchi, Y. and Suda, T., Ž1992. Blood 79, 2288–2295. w7x He, X.Y., Antao, V.P., Basila, D., Marx, J.C. and Davis R.B. Ž1992. Blood 79, 2296–2302. w8x Burn, T.C., Satterthwaite, A.B. and Tenen, D.G. Ž1992. Blood 80, 3051–3059.
w9x He, X.Y., Antao, V.P., Basila, D., Marx, J.C. and Davis R.B. Ž1992. Blood 79, 2296–2302. w10x Brown, J., Greaves, M.F. and Molgaard, H.V. Ž1991. Int. Immunol. 3, 175–184. w11x May, G. and Enver, T. Ž1995. EMBO J. 14, 564–574. w12x Fujita, T., Shibuya, H., Hotta, H., Yamanishi, K. and Taniguchi, T. Ž1987. Cell 49, 357–367. w13x Lin, Y-S. and Green, M.R. Ž1988. Proc. Natl. Acad. Sci. USA 85, 3396–3400. w14x Harada, H., Willison, K., Sakakibara, J., Miyamoto, M., Fujita, T. and Taniguchi, T. Ž1990. Cell 63, 303–312. w15x Abdollahi, A., Lord, K.A., Hoffman-Lieberman, B., Lieberman, D.A. Ž1991. Cell Growth Differ. 2, 401–407. w16x Hurst, H.C., Masson, N., Jones, N.C. and Lee K.A.W. Ž1990. Mol. Cell Biol. 10, 6192–6203. w17x Kara, C., Hou, H,-C., Ivashkiv, L.B. and Glimcher, L.H. Ž1990. Mol. Cell Biol. 10, 1347–1357. w18x Morris, J.F., Rauscher III, F.J., Davis, B., Klemsz, M., Xu, D., Tenen, D.G. and Hromas, R. Ž1995. Blood 86, 3640– 3647.
Promoter paper
Functional characterization of the promoter for the gene encoding murine CD34 Yuji Yamaguchia a
a,)
, Daniel G. Tenen b, Toshio Suda
a
Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto UniÕersity School of Medicine, Kumamoto 860, Japan b Department of Hematologyr Oncology, Beth Israel Hospital, HarÕard Medical School, Kumamoto 860, Japan Received 16 October 1996; accepted 22 October 1996
Abstract Since CD34 expression is restricted to the hematopoietic stem cells and decreases in differentiating cells, the analysis of the CD34 promoter is of interest to understand regulation of gene expression in stem cells. To characterize the cis-acting elements which control murine CD34 ŽmCD34. gene expression, we sought to clone, sequence, and functionally analyze the mCD34 promoter. An 80% decrease in promoter activity was obtained when sequences between y119 bp and y59 bp upstream of the transcriptional start site were deleted. We identified several DNA-protein complexes which correspond to functional segments defined by the linker-scanning mutants. These findings indicated the presence of the important regulatory element between bp y119 and y100, GGTTAAAAGTGAAGTAGGAA. Furthermore, from the result of functional promoter analysis in the DNase I hypersensitive site ŽHS. located within 5 kb upstream of the mCD34 gene, the presence of the enhancer region in the NcoIrPstI fragment of 5X upstream, y2.8 kb to y1.9 kb, has been identified. These data will provide useful information on the gene transfer using the CD34 promoter and enhancer. Keywords: CD34; Promoter region; Enhancer region; M1 cell; Luciferase; Transcription initiation; ŽMurine.
The CD34 antigen is selectively expressed on hematopoietic stem cells w1,2x. Cells expressing CD34 constitute approx. 1% of the population of human bone marrow ŽhBM. cells and include colony-forming cells w3x. It has been shown that the CD34 antigen is a 105–120 kDa type I transmembrane glycoprotein and is also expressed on vascular endothelial cells w4x. The human CD34 ŽhCD34. gene belongs to a cluster of genes on chromosome lq which encode adhesion molecules. Recently,
)
Corresponding author. Fax: q81 96 3735332. E-mail: [email protected]
Baumhueter et al. showed that L-selectin expressed in lymphocytes binds to the sialomucin of CD34 which is expressed in endothelial venules in lymph nodes w5x. This suggests a possibility that the CD34 antigen functions as an adhesion molecule. However, the function of CD34 has remained to be elucidated. We are interested in understanding the mechanism of transcriptional regulation of CD34 gene to elucidate the molecular basis for commitment in early hematopoiesis, and the possibility of clinical applications in stem cell gene therapy. The murine CD34 ŽmCD34. gene is expressed in a wider variety of tissues than the human homologue, including brain and several fibroblast cell lines w4x. In particular, we
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have shown that the alternative splicing of mCD34 results in two types of CD34 mRNA w6x. The truncated form of CD34 mRNA is expressed at relatively higher levels in brain than in other tissues w6x. Thus, the regulation of CD34 gene expression may differ from that in each tissue. Although several groups have studied human CD34 promoter regulation, they failed to detect the core CD34 promoter which is specific for human CD34q cells w7–9x. The homology with the human sequence is highest in the intracellular domain Ž90% amino acid identity. and lowest in the N-terminal region Ž43% amino acid identity. w10x. While there is a relatively high homology between human and murine CD34 cDNAs, the sequence upstream of the mCD34 gene is entirely different from that of the hCD34 gene. This suggests that gene regulation of mCD34 may be different from that of the hCD34. Accordingly, to identify transcription factors involved in regulating mCD34 gene expression, we isolated the 5X flanking region of the mCD34 gene and analyzed the functional characteristics of its promoter region. Transcriptional start sites of the mCD34 gene. To define the transcriptional start site of the mCD34 gene, the S1 nuclease protection assay was performed ŽFig. 1.. A 20 bp oligonucleotide complementary to the coding strand of the 3X end of exon 1 was 5X end-labeled and used to generate a probe for this experiment, as shown in the upper part of Fig. 1. This 400-nucleotide probe was hybridized to 10 m g of polyŽ A.q RNA derived from M1 cells. After annealing and digestion with nuclease S1, a protected fragment of 160 nucleotides in length was detected ŽFig. 1.. From the result of this nuclease S1 protection assay, the cytidine residue located 84 bp 5X from the initiation methionine can be considered as the major transcriptional start site in the mCD34 gene. This evidence is compatible with the suggestion by Brown et al. that the transcriptional start site is probably within 100 bp of the start of the longest cDNA w10x. Functional characterization of mCD34 promoter region. To locate the cis-acting elementŽs. required for mCD34 promoter activity, we analyzed luciferase activity in M1 cells, a murine myeloid leukemia cell line, transiently transfected with mCD34 promoter constructs subcloned into the promoterless pXP2 luciferase expression vector. An mCD34 genomic fragment containing 2.0 kb of sequence upstream of the
Fig. 1. Identification of the transcriptional start site of murine CD34. A 20 bp oligonucleotide complementary to the coding strand of the 3X end of exon 1 was 5X end-labeled as shown. The kinased oligonucleotide primer was extended with Klenow fragment and was digested with Sau3A I. This 400-nucleotide probe was annealed with 10 m g of polyŽA.q RNA from M1 cells and then digested with nuclease S1. Samples were electrophoresed on a 6% denaturing polyacrylamide gel next to sequencing ladder generated with the same 1st exon 20 bp oligonucleotide that was used for this study, and M13mp 8 single-stranded DNA.
transcriptional start site was subcloned into the promoterless pXP2 luciferase vector. When transiently transfected into M1 cells, the y2.0 kbrmCD34luciferase Žluc. promoter construct reproducibly ex-
Y. Yamaguchia et al.r Biochimica et Biophysica Acta 1350 (1997) 141–146
Fig. 2. Functional activity of mcD34 deletion constructs in M1 cells. 10 m g of pBluescript II KSŽy. was transfected as carrier DNA with 15 m g of each mCD34-pXP2 construct into M1 cells. Luciferase activities were normalized for the amount of hGH produced by the co-transfected control CMV-hGH plasmid. Corrected RLU for each construct are shown as the percent activity relative to the mean activity of the wild-type ŽWT. mCD34 promoter construct. Values are shown as means"S.E. for three independent experiments.
pressed ) 50-fold more luciferase activity than did the promoterless pXP2 control. To localize regulatory elements in the mCD34 promoter, a series of 12 deletion routants ŽM1–M12. were produced from the y2.0-kbrmCD34-luc construct using exonuclease III and the polymerase chain reaction ŽPCR.. No significant decrease in promoter activity was observed when sequences between y2.0 kb and y119 bp were deleted ŽFig. 2.. Further deletion to y59 and y21 bp
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produced decreases in luciferase activity of more than 6- and 17-fold Ž94%., respectively, when compared with the y2.0-kb construct Žwild-type; WT.. The cell type-specificity of the mCD34 promoter for CD34 expression was assessed by transfecting the wild-type construct into M1 cells, P815 cells, and WEHI-3 cells. CD34 mRNA expression was observed in M1 cells, but not in P815 cells or WEHI-3 cells Ž data not shown.. The activity of the mCD34 promoter was approx. 10-fold to 20-fold lower in P815 and WEHI-3 cells than in M1 cells Ždata not shown.. To more precisely locate the functional sequences required for mCD34 promoter activity in the region between y119 bp and y60 bp upstream of the transcriptional start site, in which the presence of positively acting regions was anticipated, six linkerscanning mutants were constructed by inserting a 10-bp oligonucleotide consecutively every 10 bp in place of the wild-type promoter sequence ŽFig. 3.. A 5-fold decrease in mCD34 promoter activity relatively to either the wild-type or the M10 deletion mutant was observed when the sequence between bp y119ry110 or bp y109ry100 was replaced by the linker and a 4-fold decrease in activity resulted when the sequence between bp y69 and y60 was replaced. To investigate DNArprotein interactions in the mCD34 promoter, electrophoretic mobility shift assays ŽEMSAs. were carried out. The probes for the
Fig. 3. Linker-scanning analysis of the 119 bp mCD34 promoter in M1 cells. Comparative promoter activity in M1 transiently transfected with the y2 kb promoter ŽWT., y119 bp promoter ŽM10., and six linker scanning mutants of the mCD34 promoter in the pXP2-luciferase vector are shown. The diagonally striped rectangular boxes represent the 10 bp sequences replaced by the linker oligonucleotide. Three independent transfection experiments were performed; the average activities Ž"S.E.. relative to the activity of the M10 Žy119 bp. promoter Ž100%. are shown. Luciferase activity, measured as RLU, was first normalized for transfection efficiency based on the amount of growth hormone produced and were indicated as corrected RLU. The data represents the mean " S.E. of three independent experiments.
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Fig. 4. Identification of sequence-specific DNA-binding proteins by electrophoretic mobility shift assays ŽEMSAs.. ŽA. 32 P-end-labeled oligomer A Žlanes 1–3., oligomer B Žlanes 4–6., oligomer C Žlanes 7–9., and oligomer D Žlanes 10–12. were incubated in the absence Žlanes 1, 4, 7, and 10. or in the presence Žlanes 2, 3, 5, 6, 8, 9, 11, and 12. of nuclear extracts from M1 cells. A 200-fold molar excess of each unlabeled oligonucleotide Žcompetitors., identical to the probe in the binding reaction, were added Žlanes 3, 6, 9, and 12.. ŽB. Cross-competition assay. Unlabeled competitors Žoligomer A, B, C, and D. were included in the binding reactions containing 32 P-labeled oligomer A probe.
EMSAs, oligomers A, B, C, and D, were 30-bp double-stranded oligonucleotides corresponding to sequences extending from bp y129 to bp y40 of the mCD34 gene. As shown in Fig. 4A, the nuclear
protein from M1 cells specifically bound to oligomers A and C, spanning sequences y129 to y100 bp and y89 to y60 bp, respectively. However, the bands of DNA binding protein formed with oligomer C were
Fig. 5. Functional analysis of the mCD34 5X DNase I HS in M1 cells. M1 cells were transiently transfected with the luciferase ŽLuc. reporter constructs containing the minimal promoter, y119 bprluc, shown schematically. RV, RI, N, and P represent EcoRV, EcoRI, NcoI, and PstI, respectively. 10 m g of pBluescript II KSŽy. was transfected as carrier DNA with 15 m g of each construct into M1 cells. Luciferase activities were normalized for the amount of hGH produced by a co-transfected control CMV-hGH plasmid. Corrected RLU for each construct are shown as the percent activity relative to the mean activity of the minimal promoter construct, y119rluc. Values are shown as means" S.E. for three independent experiments.
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relatively faint compared to those formed with oligomer A. Each unlabeled DNA probe, in 200 molar excess, was added and the bands were completely competed out. A cross-competition assay of EMSAs was performed to confirm whether the bands of DNA binding protein formed with oligomer A were specific for this sequence. As shown in Fig. 4B, the bands observed in the presence of oligomer A probe were competed out by the addition of the excess non-radioactive self competitor, but not other competitors, i.e., oligomers B, C, and D. These results suggest that the DNA sequence between position y129 and y100 bp specifically binds to the nuclear protein derived from M1 cells. An enhancer is located approximately 3 kb upstream of the transcriptional start site in the mcD34 gene. An enhancer in the DNase I hypersensitive site ŽHS. of the mCD34 gene has been revealed by May and Enver w11x. To maximize mCD34 promoter activity, we examined the enhancer region of the 5X DNase I HS. Each fragment of DNase I HS located within 5 kb upstream of the mCD34 gene Ž EcoRVrEcoRI fragment, EcoRIrEcoRI fragment, EcoRIrNcoI fragment, and NcoIrPstI fragment., was subcloned into the mCD34 minimal promoter, the y119 bprCD34-luc construct ŽFig. 5.. The activity of mCD34-luc promoter construct containing the 5.0 kb upstream of mCD34 gene, y5 krluc, was approx. 4-fold higher compared to that of minimal promoter construct, y119rluc. In promoter constructs containing each region of 5X DNase I HS with the minimal promoter, the promoter activity of the region containing the NcoIrPstI fragment, 0.9 k enhrluc, was approx. 7-fold greater when compared with the minimal promoter alone, y119rluc. This evidence that the region located 3 kb upstream of the transcriptional start site acts as an enhancer is compatible with the data reported by May and Enver w11x. From the results of the functional promoter assays as shown in Fig. 2 and Fig. 3 in addition to the EMSAs performed by mutational analysis, our analyses of the mCD34 promoter regions revealed the presence of the important regulatory element between bp y119 and y100. These coincident regions, the sequence between bp y119 and y100, contain potential binding sites for several transcription factors including IRF-1rIRF-2 w12x and ATF w13x. IRF-1 and IRF-2 are structurally related, and bind specifically to
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the same virus-inducible cis-elements of the IFN-a 1 and IFN-b gene promoters as well as to the interferon response sequence of MHC class I gene promoters w14x. It has been shown that IRF-1 is a primary response gene, and IFN-b is a secondary response gene in the developmental program of terminal myeloid differentiation induced by IL-6 or LIF w15x. However, neither IRF-1 nor IRF-2 showed any significant effect on the promoter activity of the mCD34 gene Ždata not shown. . The adenovirus transcription factor ATF stimulates transcription from the adenovirus early region 4 ŽE4. promoter w13x. It has been shown that cyclic AMP response element-binding protein Ž CREB. and ATF are identical w16x. The mRNA of HB16 transcription factor which recognizes the same motif as CREB, with which it also displays structural similarity, is most abundant in brain w17x. The truncated form of CD34 mRNA is also expressed at relatively higher level in the brain than in other tissues w6x. Although no complete matches to the ATF or CREB motif ŽTGACGT. appeared to be present in the mCD34 promoter ŽTGAAGT., CREB might play a role in CD34 gene expression in the brain. Since the region between bp y107 and bp y100 matches the MZF-1 first domain-binding site at 6 of 8 bases, we determine the transactivation activity of MZF-1 on the promoter activity of mCD34 gene. No significant enhancement of the minimal promoter, y119rCD34-luc, or the promoter of the y5 kb upstream, -SkbrCD34-luc, was observed when the MZF-1 expression vector was cotransfected into M1 cells Ždata not shown. . Morris et al. have reported that MZF-1 transactivated the human CD34 promoter containing 1.1 kb upstream of transcriptional start site in hematopoietic cells but not in non-hematopoietic cells w18x. The discrepancy of the result on the transactivation of the MZF-1 between murine and human CD34 promoter activity may suggest differences between murine and human CD34 gene regulation. We wish to thank Dr. S.J. Ackerman for searching the Ghosh transcription factor data base. We also thank Dr. M. Ishihara and Dr. T. Taniguchi for generously providing us with IRF-1 and IRF-2 expression vectors, and Dr. R. Hromas for providing us with MZF-1 expression vectors. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.
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