A Splice Variant of E2–2 Basic Helix-Loop-Helix Protein Represses the Brain-specific Fibroblast Growth Factor 1 Promoter through the Binding to an Imperfect E-box

Abstract

We previously demonstrated that acis-element (−489 to −467) in the brain-specific fibroblast growth factor (FGF)-1 promoter (FGF-1.B) binds multiple nuclear factors, and this binding enhances transcriptional activity of this promoter. Here we report the isolation of three cDNA clones, VL1, VL2 and VL3, from a human brain stem cDNA expression library using four tandem repeats of the 26-base pair sequence (−492 to −467) as the probe. These cDNA clones represent the variant of bHLH protein E2–2/SEF2–1 in having 12 additional nucleotides encoding the amino acids RSRS. The glutathione S-transferase (GST) fusion proteins of VLl, VL2, and VL3 immunologically react with anti-E2–2 antibody and anti-GST-VL2 antibody. Electrophoretic mobility shift assay and methylation interference assay revealed that the GST fusion proteins specifically bind to an imperfect E-box sequence (GACCTG) present in the 26-base pair sequence. Transient expression of the full-length E2–2 without RSRS in U1240MG glioblastoma cells resulted in repression of FGF-1.B promoter activity. We further showed a significant repression of promoter activity (>40 fold) by E2–2 (lacking the amino acid sequence RSRS) when the E47 reporter construct, containing a hexameric E-box site, was used. In contrast, the E2–2 variant containing the RSRS sequence has no significant effect on either the FGF-1 promoter or E47 promoter. These results suggest that the relative abundance of the two splice variants of E2–2 in brain could be an important determinant for the expression of FGF-1. We previously demonstrated that acis-element (−489 to −467) in the brain-specific fibroblast growth factor (FGF)-1 promoter (FGF-1.B) binds multiple nuclear factors, and this binding enhances transcriptional activity of this promoter. Here we report the isolation of three cDNA clones, VL1, VL2 and VL3, from a human brain stem cDNA expression library using four tandem repeats of the 26-base pair sequence (−492 to −467) as the probe. These cDNA clones represent the variant of bHLH protein E2–2/SEF2–1 in having 12 additional nucleotides encoding the amino acids RSRS. The glutathione S-transferase (GST) fusion proteins of VLl, VL2, and VL3 immunologically react with anti-E2–2 antibody and anti-GST-VL2 antibody. Electrophoretic mobility shift assay and methylation interference assay revealed that the GST fusion proteins specifically bind to an imperfect E-box sequence (GACCTG) present in the 26-base pair sequence. Transient expression of the full-length E2–2 without RSRS in U1240MG glioblastoma cells resulted in repression of FGF-1.B promoter activity. We further showed a significant repression of promoter activity (>40 fold) by E2–2 (lacking the amino acid sequence RSRS) when the E47 reporter construct, containing a hexameric E-box site, was used. In contrast, the E2–2 variant containing the RSRS sequence has no significant effect on either the FGF-1 promoter or E47 promoter. These results suggest that the relative abundance of the two splice variants of E2–2 in brain could be an important determinant for the expression of FGF-1. A family of proteins that bind to a consensus DNA sequence CANNTG, also known as E-box, has been identified. E-box-binding proteins share a common amino acid sequence motif that is proposed to form two amphipathic helices interrupted by a loop, designated the helix-loop-helix (HLH) 1The abbreviations used are: HLH, helix-loop-helix; bHLH, basic helix-loop-helix; FGF, fibroblast growth factor; bp, base pair(s); EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase. motif. The HLH domain of these proteins mediates the dimerization, and a basic region located N-terminal of the HLH domain is responsible for DNA binding (1Kadesch T. Cell Growth Differ. 1993; 4: 49-55PubMed Google Scholar). HLH proteins are mainly of two types, ubiquitous and tissue-specific. The interplay of these HLH proteins is particularly interesting because it plays a significant role in the regulation of tissue-specific gene expression (1Kadesch T. Cell Growth Differ. 1993; 4: 49-55PubMed Google Scholar). Fibroblast growth factor (FGF)-1 is a prototype member of the structurally related FGF family, which comprises 14 proteins (2Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1057) Google Scholar, 3Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar). It is found primarily in brain and retina, although low levels of its mRNA have been demonstrated in other tissues (4Chotani M.A. Payson R.A. Winkles J.A. Chiu I.-M. Nucleic Acids Res. 1995; 23: 434-441Crossref PubMed Scopus (30) Google Scholar, 5Myers R.L. Chedid M. Tronick S.R. Chiu I.-M. Oncogene. 1995; 11: 785-789PubMed Google Scholar, 6Weiner H.L. Swain J.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2683-2687Crossref PubMed Scopus (123) Google Scholar). However, the role of FGF-1 in brain is not yet well understood. By in situ hybridization and immunohistochemical analysis, it has been shown that the expression of this growth factor in brain is exclusively in neuronal cells but not in glial cells (7Alam K.Y. Frostholm A. Hackshaw K.V. Evans J.E. Rotter A. Chiu I.-M. J. Biol. Chem. 1996; 271: 30263-30271Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Eckenstein F.P. Kuzis K. Nishi R. Woodward W.R. Meshul C. Sherman L. Ciment G. Biochem. Pharmacol. 1994; 47: 103-110Crossref PubMed Scopus (36) Google Scholar, 9Stock A. Kuzis K. Woodward W.R. Nishi R. Eckenstein F.P. J. Neurosci. 1992; 12: 4688-4700Crossref PubMed Google Scholar). It was also shown that the expression of FGF-1 mRNA in neurons is correlated with specific developmental events (10Wilcox B.J. Unnerstall J.R. Neuron. 1991; 6: 397-409Abstract Full Text PDF PubMed Scopus (100) Google Scholar) and that FGF-1 coexists with tyrosine hydroxylase in neuronal cells (11Bean A.J. Elde R. Cao Y. Oellig C. Tamminga C. Goldstein M. Pattersson R.F. Hökfelt T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10237-10241Crossref PubMed Scopus (136) Google Scholar). Importantly, transcription of tyrosine hydroxylase, a key enzyme for catecholamine synthesis, requires both FGF-1 and an activator (12Du X. Stull N.D. Iacovitti L. J. Neurosci. 1994; 14: 7688-7694Crossref PubMed Google Scholar). FGF-1 mRNA and protein levels were also reported to elevate in lesioned rat brain (13Hara Y. Tooyama I. Yasuhara O. Akiyama H. McGeer P.L. Handa J. Kimura H. Brain Res. 1994; 664: 101-107Crossref PubMed Scopus (16) Google Scholar, 14Logan A. Mol. Cell. Endocrinol. 1988; 58: 275-278Crossref PubMed Scopus (41) Google Scholar). Interestingly, FGF-1 mRNA expression was reported to increase significantly in glioblastoma, the major human intracranial tumor (5Myers R.L. Chedid M. Tronick S.R. Chiu I.-M. Oncogene. 1995; 11: 785-789PubMed Google Scholar,15Takahashi J.A. Mori H. Fukumoto M. Igarashi K. Jaye M. Oda Y. Kikuchi H. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5710-5714Crossref PubMed Scopus (250) Google Scholar). However, the regulatory mechanism of FGF-1 gene expression is only beginning to be understood. The study of tissue- and cell-specific distribution of FGF-1 transcripts has led to the identification of four different transcripts having the same protein coding exons but different 5′-untranslated exons (16Chiu I.-M. Wang W.-P. Lehtoma K. Oncogene. 1990; 5: 755-762PubMed Google Scholar, 17Payson R.A. Canatan H. Chotani M.A. Wang W.P. Harris S.E. Myers R.L. Chiu I.-M. Nucleic Acids Res. 1993; 21: 489-495Crossref PubMed Scopus (40) Google Scholar, 18Wang W.-P. Lehtoma K. Varban M.L. Krishnan I. Chiu I.-M. Mol. Cell. Biol. 1989; 9: 2387-2395Crossref PubMed Scopus (48) Google Scholar, 19Wang W.-P. Myers R.L. Chiu I.-M. DNA Cell Biol. 1991; 10: 771-777Crossref PubMed Scopus (17) Google Scholar, 20Wang W.-P. Quick D. Balcerzak S.P. Needleman S.W. Chiu I.-M. Oncogene. 1991; 6: 1521-1529PubMed Google Scholar). FGF-1.B is the predominant transcript in brain, gliomas, and some glioblastoma cell lines (e.g. U1240MG and U251MG) (5Myers R.L. Chedid M. Tronick S.R. Chiu I.-M. Oncogene. 1995; 11: 785-789PubMed Google Scholar, 21Myers R.L. Payson R.A. Chotani M.A. Deaven L.L. Chiu I.-M. Oncogene. 1993; 8: 341-349PubMed Google Scholar). We have identified a 23-bp cis-element (−489 to −467) in FGF-1.B promoter that binds to a 37-kDa protein, p37brn, and this binding is linked to the enhanced functional activity of the promoter (22Myers R.L. Ray S.K. Eldridge R. Chotani M.A. Chiu I.-M. J. Biol. Chem. 1995; 270: 8257-8266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Here we screened a human brain stem cDNA expression library for DNA-binding proteins using a labeled oligonucleotide probe made of four tandem repeats of the sequence −492 to −467. We have isolated and expressed three overlapping cDNA clones. All three clones represent a splice variant of the E2–2 gene product (23Henthron P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar, 24Corneliussen A. T. J. 1991; PubMed Google and the protein binds to an imperfect E-box present in the probe. We further showed that of an E2–2 variant RSRS the transcriptional of both the FGF-1.B promoter and the E47 reporter containing a hexameric E-box A expression made from human brain stem as previously (16Chiu I.-M. Wang W.-P. Lehtoma K. Oncogene. 1990; 5: 755-762PubMed Google was screened for expression of DNA-binding proteins using the by H. Cell. 1988; Full Text PDF PubMed Scopus Google Scholar). with four tandem repeats of the sequence −492 to the was labeled with by the and used as a probe. clones, VL1, VL2, and VL3, were using the same DNA probe. J. T. Scholar) were used to the and the cDNA from the clones were the of The cDNA clones were using DNA sequence was using The VL2 cDNA was used as a probe to to human The of from different human The was to using J. T. Scholar). were in a containing for The were in for The cDNA of and by of VL2, VL3, and clones, were and in with the glutathione S-transferase of The of the was by The proteins were expressed and using as by and 1988; PubMed Scopus Google Scholar). The protein was used as the for antibody in The were by protein as by and D. A Scholar). and were from GST fusion proteins of the E2–2 gene and and nuclear were to in proteins were to and by in containing The were with either E2–2 antibody or anti-GST-VL2 antibody and three for with the The were with either the anti-E2–2 or the anti-GST-VL2 a of the was by using The (−492 to −467) oligonucleotide was with using the The sequence was S.K. Chiu I.-M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The binding the binding and with DNA probe of the GST fusion protein and a when in a of The brain nuclear was also in in the of of GST or that of was used. of the when was further with antibody for The was in a in the was and The human brain nuclear was made in with the by K. M. U. Cell. 47: Full Text PDF PubMed Scopus Google Scholar). from (22Myers R.L. Ray S.K. Eldridge R. Chotani M.A. Chiu I.-M. J. Biol. Chem. 1995; 270: 8257-8266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) was with and used as a probe in binding containing were from the DNA by EMSA, from the with and in as (22Myers R.L. Ray S.K. Eldridge R. Chotani M.A. Chiu I.-M. J. Biol. Chem. 1995; 270: 8257-8266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). is a full-length E2–2 expression that was by in of A. T. J. 1991; PubMed Google Scholar) with the from the to full-length E2–2/SEF2–1 with four additional amino acids as shown in The and E47 reporter M. Y. J. A. T. 1994; PubMed Scopus Google Scholar) were by and previously designated as was in the expression A. T. J. 1991; PubMed Google Scholar). an expression for the bHLH protein E47 G. S. C. Mol. Cell. Biol. 1993; PubMed Scopus Google was by the gene by the promoter. The were as previously (22Myers R.L. Ray S.K. Eldridge R. Chotani M.A. Chiu I.-M. J. Biol. Chem. 1995; 270: 8257-8266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). U1240MG cells were in to by cells were with the reporter and expression DNA using the DNA was using the In were in The results the of two The activity was by the protein as (22Myers R.L. Ray S.K. Eldridge R. Chotani M.A. Chiu I.-M. J. Biol. Chem. 1995; 270: 8257-8266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) or by the activity using the as by the both functional and we previously identified a region (−492 to −467) of the brain-specific FGF-1.B promoter (22Myers R.L. Ray S.K. Eldridge R. Chotani M.A. Chiu I.-M. J. Biol. Chem. 1995; 270: 8257-8266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, S.K. Chiu I.-M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The sequence was and a probe with four tandem repeats was used to a human brain stem cDNA expression library in (16Chiu I.-M. Wang W.-P. Lehtoma K. Oncogene. 1990; 5: 755-762PubMed Google Scholar). clones and were isolated the and an additional was isolated the The cDNA from three clones were showed that the three clones have an sequence with also known as and a basic HLH protein which was previously isolated using E-box (23Henthron P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar, 24Corneliussen A. T. J. 1991; PubMed Google Scholar). E2–2 (23Henthron P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar) and of the four A. T. J. 1991; PubMed Google Scholar) four amino acids of the bHLH sequence and we showed that three of cDNA clones the RSRS sequence The resulted from of the same E2–2/SEF2–1 gene (23Henthron P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar, 24Corneliussen A. T. J. 1991; PubMed Google Scholar). the distribution of E2–2 we containing an of from different human tissues with VL2 cDNA probe. shown in the pair human E2–2 transcript is in brain and to a The expression of the transcript in other tissues (e.g. and was the It is also that the expression of E2–2 mRNA is in the In to the protein by cDNA clones VL2, and we expressed these as GST fusion proteins and antibody in of the fusion proteins The fusion proteins were for the with the E2–2 antibody or anti-GST-VL2 antibody. In the same we also nuclear from cell which is known to E2–2 protein G. S. C. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar). It is from the results that anti-E2–2 antibody and but it not with GST with protein were to with anti-E2–2 antibody in the of or could be to of the fusion proteins or its of The of the major GST fusion proteins that were by anti-E2–2 antibody for and for are to the on the sequence major proteins of and in by the in nuclear proteins from cells also with the same antibody The sequence showed that the three clones reported in this are the splice variant of the E2–2 The that was not by anti-E2–2 is to the of in this protein that be by the anti-E2–2 antibody. we the same with anti-GST-VL2 antibody antibody the GST fusion proteins and GST The same two major and were found to be present in cells that could with both anti-E2–2 and anti-GST-VL2 It is to that G. S. C. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar) a protein in cells with anti-E2–2 antibody. it is that the determinant for anti-GST-VL2 antibody both the GST and E2–2 of the fusion We not the protein is a E2–2 protein or a related protein having the common These results suggest that the for the anti-E2–2 antibody in the acid domain by nucleotides and that VL1, VL2, and VL3 represent the splice variant of E2–2 gene the we using the GST fusion protein and labeled oligonucleotide probe containing the sequence −492 to in the or of a of A (−492 to −467) or to −467) It is that and the of the labeled oligonucleotide and the of was on the of the binding It is also that these are could be by the A and The of by the same of and that the these proteins bind the −492 to Interestingly, a of this binding revealed a sequence S.K. Chiu I.-M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that an imperfect E-box site, (23Henthron P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar). further the of these we anti-E2–2 or antibody the shown in anti-E2–2 antibody the either by or These are also specific for the E2–2 was to the and However, the by was not in the same assay by anti-E2–2 antibody. to the which showed that the to be by anti-E2–2 antibody. However, anti-GST-VL2 could the with The same antibody was also shown to The antibody from the same used to to or antibody the with or in of or protein was with the labeled oligonucleotide A in the and or and of a of A as in The of protein was by an antibody assay using anti-E2–2 antibody and anti-GST-VL2 antibody and or antibody the in the DNA these fusion proteins we the methylation interference assay using and the probe as and A that the in the is important for the binding of this GST fusion protein with the It is to that this is in to the imperfect E-box the levels by a of the of It is also from this that the in the is significantly in with the same in the the methylation interference we using and the (−492 to −467) oligonucleotide probe having the sequence or having to A and The binding of with the probe was in with that by the probe. the binding of was identified to be in the region −492 to it is that the this of the binds to the sequence from −492 to we for the E2–2 protein in brain nuclear that bind to that we could by the it was not by anti-E2–2 antibody or by However, we could an additional by the having mobility when the brain nuclear was with was not when the nuclear was with GST the same Importantly, this additional was by anti-E2–2 antibody but not by the antibody These results suggest that E2–2 a with proteins present in brain and that the binds to the −492 to of E2–2 with and without are known to be expressed in different cells (23Henthron P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar, 24Corneliussen A. T. J. 1991; PubMed Google Scholar, A. U. J. K. J. R. R. J. 1996; PubMed Scopus Google Scholar, J. P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The three cDNA clones VL2, and isolated from human brain stem cDNA library represent the splice variant of E2–2 containing RSRS However, an E2–2 cDNA that the sequence encoding RSRS was isolated from a brain cDNA library A. U. J. K. J. R. R. J. 1996; PubMed Scopus Google Scholar). the of both of E2–2 in brain is these we the effect of of both and without RSRS) on the FGF-1.B promoter activity in U1240MG cells We that which full-length E2–2 protein without RSRS A. T. J. 1991; PubMed Google significantly the promoter the other splice variant with RSRS not this promoter activity. the same the and bHLH protein J.E. L. H. Science. 1995; PubMed Scopus Google Scholar) not the activity of the FGF-1.B promoter. repression of FGF-1.B promoter activity is we E47 promoter reporter which a hexameric E-box The reporter activity of this promoter was previously shown to increase expression of E47 cDNA (23Henthron P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar, M. Y. J. A. T. 1994; PubMed Scopus Google Scholar, G. S. C. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar). A that the expression encoding E47 protein gene the E47 and this a of a in with that by the same of the These results suggest that the of the promoter the is also in U1240MG the other the same and the which the full-length E2–2 protein without RSRS a of the promoter activity. of the reporter activity was on the of this E2–2 splice variant having the repression when of the was In contrast, with RSRS) not effect on this E47 reporter activity of the Interestingly, the activity of the promoter an imperfect to was also in cells in a the of of FGF-1.B promoter activity was not as as that of E47 it was in in The of on the other not the FGF-1.B promoter activity. These results suggest that the present in both be the of by E2–2 protein the RSRS The present study was to that binds to of FGF-1.B promoter. (22Myers R.L. Ray S.K. Eldridge R. Chotani M.A. Chiu I.-M. J. Biol. Chem. 1995; 270: 8257-8266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, S.K. Chiu I.-M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) showed that the specific in a 23-bp sequence (−489 to −467) of is linked to the enhanced functional activity of the FGF-1.B promoter in the glioblastoma cell The present the identification of three E2–2/SEF2–1 cDNA clones VL2, and from the brain stem cDNA library using four tandem repeats of the sequence −492 to −467. of VL1, VL2, and VL3 with the full-length cDNA A. T. J. 1991; PubMed Google Scholar) showed that clones four additional amino acids N-terminal to the bHLH domain The of GST fusion proteins of VL1, VL2, and VL3 to the anti-E2–2 antibody in and in further that these clones represent a splice variant of the E2–2 gene The of the binding by using of overlapping sequence as well as by methylation interference assay revealed that the protein product of these cDNA clones binds to an imperfect E-box the sequence −492 to we showed that the sequence to −467) of FGF-1.B is to the promoter in U1240MG cells S.K. Chiu I.-M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the the not be in the of FGF-1.B promoter activity in U1240MG the other this are important because FGF-1.B is expressed in neuronal cells (7Alam K.Y. Frostholm A. Hackshaw K.V. Evans J.E. Rotter A. Chiu I.-M. J. Biol. Chem. 1996; 271: 30263-30271Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Eckenstein F.P. Kuzis K. Nishi R. Woodward W.R. Meshul C. Sherman L. Ciment G. Biochem. Pharmacol. 1994; 47: 103-110Crossref PubMed Scopus (36) Google Scholar, B.J. Unnerstall J.R. Neuron. 1991; 6: 397-409Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 14Logan A. Mol. Cell. Endocrinol. 1988; 58: 275-278Crossref PubMed Scopus (41) Google the HLH which bind to E-box as or (1Kadesch T. Cell Growth Differ. 1993; 4: 49-55PubMed Google are also expressed in neuronal cells J.E. L. H. Science. 1995; PubMed Scopus Google Scholar, J.E. D.J. 1990; PubMed Scopus Google the expression of these HLH in neurons also the of the J.E. L. H. Science. 1995; PubMed Scopus Google Scholar, J.E. D.J. 1990; PubMed Scopus Google Scholar, C. M. C. S. R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, S. Cell Growth Differ. 1992; Google Scholar, Y. Cell. 1993; Full Text PDF PubMed Scopus Google and the expression of FGF-1 gene in brain is also (7Alam K.Y. Frostholm A. Hackshaw K.V. Evans J.E. Rotter A. Chiu I.-M. J. Biol. Chem. 1996; 271: 30263-30271Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, B.J. Unnerstall J.R. Neuron. 1991; 6: 397-409Abstract Full Text PDF PubMed Scopus (100) Google Scholar). We have shown that the specific binding of these E2–2 proteins and with the oligonucleotide probe (−492 to −467) was the imperfect E-box However, we were to by EMSA, E2–2 either in U1240MG cells not or in human brain nuclear that bind to the imperfect The showed that the levels of mRNA is in brain in other tissues it is that the of this protein in the brain is low to be However, it is to that protein binding to the has not been in nuclear M. D. Science. PubMed Scopus Google Scholar, R. D. Cell. Full Text PDF PubMed Scopus Google Scholar). We not this to the is to the low of the E2–2 proteins for the imperfect or to the that the of this protein in nuclear in a form to bind to this is low to be The is because the fusion of cDNA with the domain could in the 1993; PubMed Scopus Google the promoter containing four tandem repeats of the same DNA binding sequence (−492 to −467) not the of protein in the brain nuclear a a which be by the E2–2 antibody the that the to −467) is to the FGF-1.B promoter in U1240MG cells S.K. Chiu I.-M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the regulation of FGF-1.B promoter activity in other (e.g. in neuronal cells different of further In the in U1240MG cells showed that the of the full-length E2–2 protein the RSRS domain the activity of an promoter or of FGF-1.B promoter containing the The of the four amino is in cDNA from a human cDNA library A. T. J. 1991; PubMed Google Scholar) but is present in three cDNA clones isolated from a human brain cDNA that the brain E2–2 with RSRS without RSRS. E2–2 has been shown to promoter activity by with J. P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google the report that a splice variant of E2–2 RSRS) has activity. The mechanism that could the two splice variants of E2–2 protein with or without RSRS) in of effect on the promoter activity the is not The of effect RSRS) also has not been However, it is interesting to that RSRS is a of amino In the of transcriptional are M. 1988; PubMed Scopus Google Scholar). the or of RSRS of the domain in the E2–2/SEF2–1 protein by of the splice could be to the different transcriptional of these It is that the relative abundance and of the two splice variants in the brain could be an important determinant for the expression of we showed that in brain nuclear a E2–2 binds to the imperfect of the FGF-1.B promoter It be of to the brain-specific bHLH transcription factor that with E2–2 or with other bHLH proteins to the FGF-1.B gene expression in the We and for and the E47 reporter for expression and for expression