Stra13 Homodimers Repress Transcription through Class B E-box Elements

A mammalian basic helix-loop-helix protein known variably as Stra13, Sharp2, and Dec1 has been implicated in cell activation, proliferation, and differentiation. Indeed,Stra13 null mice develop age-induced autoimmunity as a result of impaired T-lymphocyte activation, leading ultimately to the accumulation of autoreactive T-cells and B-cells. Stra13 is expressed in embryonic as well as adult tissues derived from neuroectoderm, mesoderm, and endoderm and has been associated with response to hypoxia, suggesting a complex role for this protein and the highly related Sharp1/Dec2 protein in homeostatic regulation. Whereas Stra13 is known to regulate many important cellular functions and is known to cross-regulate biological responses to other basic helix-loop-helix containing transcription factors, including c-Myc and USF, it is unclear if this protein binds directly to DNA. Indeed, the basic domain of Stra13 contains a proline residue at an unprecedented position. Herein, we have determined that Stra13 binds with high affinity to CACGTG class B E-box elements as a homodimer with preference for elements preceded by T and/or followed by A residues. In addition, transient transfection experiments reveal that Stra13 represses transcription when bound to these and related sites. Our data suggest that Stra13 regulates cellular functions through antagonism of E-box activator proteins and also through active repression from E-box elements. A mammalian basic helix-loop-helix protein known variably as Stra13, Sharp2, and Dec1 has been implicated in cell activation, proliferation, and differentiation. Indeed,Stra13 null mice develop age-induced autoimmunity as a result of impaired T-lymphocyte activation, leading ultimately to the accumulation of autoreactive T-cells and B-cells. Stra13 is expressed in embryonic as well as adult tissues derived from neuroectoderm, mesoderm, and endoderm and has been associated with response to hypoxia, suggesting a complex role for this protein and the highly related Sharp1/Dec2 protein in homeostatic regulation. Whereas Stra13 is known to regulate many important cellular functions and is known to cross-regulate biological responses to other basic helix-loop-helix containing transcription factors, including c-Myc and USF, it is unclear if this protein binds directly to DNA. Indeed, the basic domain of Stra13 contains a proline residue at an unprecedented position. Herein, we have determined that Stra13 binds with high affinity to CACGTG class B E-box elements as a homodimer with preference for elements preceded by T and/or followed by A residues. In addition, transient transfection experiments reveal that Stra13 represses transcription when bound to these and related sites. Our data suggest that Stra13 regulates cellular functions through antagonism of E-box activator proteins and also through active repression from E-box elements. Basic helix-loop-helix proteins represent a large and diverse class of transcription factors implicated in cell fate specification, cell proliferation, apoptosis, metabolism, and cell activation (1Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1392) Google Scholar). The Stra13, Sharp2, Dec1 basic helix-loop-helix transcription factor has been identified in a number of biological contexts (for simplicity we will refer to this protein as Stra13). For example, Stra13was identified as a retinoic acid-inducible gene that promotes neuronal differentiation in P19 embryonal carcinoma cells (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar). In addition, Stra13 and a related protein, Sharp1/Dec2, were identified in a degenerate PCR screen for bHLH 1The abbreviations used for: bHLH, basic helix-loop-helix; EMSA, electrophoretic mobility shift assay; BSA, bovine serum albumin; wt, wild type; PBS, phosphate-buffered saline. proteins expressed in the adult rat brain (3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell Neurosci. 1997; 9: 460-475Crossref PubMed Scopus (109) Google Scholar). Interestingly, Stra13 andSharp1/Dec2 were both induced as immediate early genes in cultured PC12 pheochromocytoma cells treated with nerve growth factor, and Stra13 was rapidly induced by glutamatergic stimulation throughout the rat cerebral cortex (3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell Neurosci. 1997; 9: 460-475Crossref PubMed Scopus (109) Google Scholar). This gene was also identified as a cAMP-inducible transcript in differentiating chondrocytes (4Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Crossref PubMed Scopus (145) Google Scholar) and was later found to be cAMP-inducible in many cell types (5Shen M. Kawamoto T. Teramoto M. Makihira S. Fujimoto K. Yan W. Noshiro M. Kato Y. Eur. J. Cell Biol. 2001; 80: 329-334Crossref PubMed Scopus (38) Google Scholar). More recent work has described Stra13 induction following T-cell activation (6Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (151) Google Scholar), tyrosine kinase receptor signaling (7Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), hypoxia (8Wykoff C.C. Pugh C.W. Maxwell P.H. Harris A.L. Ratcliffe P.J. Oncogene. 2000; 19: 6297-6305Crossref PubMed Scopus (239) Google Scholar, 9Ivanova A.V. Ivanov S.V. Danilkovitch-Miagkova A. Lerman M.I. J. Biol. Chem. 2001; 276: 15306-15315Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 10Yoon D.Y. Buchler P. Saarikoski S.T. Hines O.J. Reber H.A. Hankinson O. Biochem. Biophys. Res. Commun. 2001; 288: 882-886Crossref PubMed Scopus (73) Google Scholar), and even serum starvation (11Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (160) Google Scholar). Taken together, these data indicate that Stra13 expression is closely associated with activation and stress in many cell types. Recently, Sun et al. (6Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (151) Google Scholar) have used gene targeting to generate Stra13 −/− mice. Surprisingly, homozygous Stra13 mutant mice are born and survive to adulthood. However, aging Stra13 mutant mice develop an autoimmune disorder. This effect has been traced to impaired CD4+ T-cell activation, with reduced interleukin-2 production, reduced clonal expansion, impaired T-cell differentiation, and reduced clearance of activated lymphocytes (6Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (151) Google Scholar). Despite indications that Stra13 may regulate activation and stress in a number of cell types, Stra13-binding DNA elements have yet to be identified. Consequently, the effect of this transcription factor on its theoretical DNA target(s) is also unknown. The putative Stra13 DNA-binding/dimerization domain is somewhat related to bHLH domains of enhancer of split family (E(spl))/Hes transcriptional repressor proteins. However, Stra13 contains a proline residue in a distinct location within the basic domain. Therefore it is not clear whether Stra13 even binds directly to DNA. In addition, a fusion construct between the GAL4 DNA-binding domain and Stra13 can repress transcription from GAL upstream activator sequence sites through the recruitment of a histone deacetylase and perhaps through direct effects on the basal transcription factor TFIIB (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar, 11Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (160) Google Scholar). However, it is not clear whether Stra13 functions as a DNA-binding repressor, a co-repressor, or perhaps even a transcription activating protein that is behaving inappropriately when fused to GAL4 (12Ruden D.M. Ma J. Li Y. Wood K. Ptashne M. Nature. 1991; 350: 250-252Crossref PubMed Scopus (130) Google Scholar). Indeed, the conformation of a GAL4-Stra13 fusion protein on DNA would be dramatically different from the conformation of Stra13 bound through its own putative DNA-binding domain. Consequently, the biochemical mechanism by which Stra13 functions remains unknown. Here we report identification of the Stra13 DNA target site, the well described CACGTG E-box element, with preference for this site preceded by T and/or followed by an A residue. In addition we have determined that Stra13 can function as a homodimer to repress transcription from these sites. These data identify Stra13 as a transcriptional repressor protein that functions to regulate E-box elements through cross-interference with E-box-binding transcriptional activators that bind such sites and through direct transcriptional repression from the class B E-box and related sites. COS-7 cells were maintained in Iscove's medium supplemented with 10% fetal bovine serum, whereas HC11 cells were maintained in RPMI 1640 supplemented with epidermal growth factor (10 ng/ml), insulin (5 μg/ml), and 10% fetal bovine serum (13Wartmann M. Cella N. Hofer P. Groner B. Liu X. Hennighausen L. Hynes N.E. J. Biol. Chem. 1996; 271: 31863-31868Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14Ball R.K. Friis R.R. Schoenenberger C.A. Doppler W. Groner B. EMBO J. 1988; 7: 2089-2095Crossref PubMed Scopus (419) Google Scholar). For transfections, HC11 or COS-7 cells were seeded at a dilution of 1:20 (60-mm plates) or 1:30 (6-well plates) in their respective media. Transfections were carried out using the Superfect (Qiagen) transfection reagent according to manufacturer's specifications. Typically, for 60-mm cell cultures 4–6 and 10–12 μg of total DNA were used for COS-7 and HC11 cells, respectively. Cells were lysed 36–48 h post-transfection using ice-cold 1× Triton X-100 lysis buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 mm NaF). Soluble and insoluble fractions were separated by centrifugation at 13,200 rpm for 15 min at 4 °C. Soluble protein was then used to perform luciferase/β-galactosidase assays and/or immunoprecipitation/Western blotting experiments. Murine Stra13 was cloned from mammary gland cDNA through a combination of reverse transcriptase PCR and high stringency hybridization based on the identification of an expressed sequence tag sequence, which at the time represented a fragment from a novel bHLH domain containing cDNA. 2B. St-Pierre and S. E. Egan, unpublished data. TheStra13 constructs described in the following section were subcloned into pcDNA3 for expression in transfected COS-7 and HC11 cells (Invitrogen). FLAG-Stra13 andStra13-Myc were created using PCR to add an in-frame epitope tag at the N- and C-terminal ends, respectively, of theStra13 open reading frame. The FLAG epitope tag is DYKDDDDK, which is recognized by the M2 monoclonal antibody (Sigma), whereas the Myc epitope tag is EQKLISEED, and is recognized by the 9E10 monoclonal and A-14 rabbit polyclonal antibodies (Santa Cruz Biotechnology). The Stra13 C-terminal deletion mutants were created using pBSK-FLAG-Stra13 as a substrate for exonuclease III (Exo-Size deletion kit, New England Biolabs). For this purpose, a double-stranded oligonucleotide was inserted downstream of theStra13 stop codon. It contained a unique site a unique site and stop The of deletion was determined by on their deletion mutants were for The Stra13 basic domain mutant was created by through a site Stra13 cDNA respectively, to and of the Stra13 open reading frame. The mutant cDNA fragment in which the basic domain is from the Stra13 sequence was used to the Stra13 sequence through fusion of the fragment with the Stra13 using an was created by a double-stranded oligonucleotide for an in-frame in et al. D. K. H. C. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google in the site of For and oligonucleotide 1 was inserted at a unique site of the and The sequence of to is as The sequence of from to is as E-box of and were created by double-stranded oligonucleotide of fragment of the site of The were to the sequence of for the at the of the was created by the kinase into the site of The site was as described M. E. Genes Dev. PubMed Scopus Google Scholar). For electrophoretic mobility shift assays μg of oligonucleotide were in a of of mm 10 mm mm μg of was with for 1 h at with separated from by through and into of for a of were in the using and were using 1 μg of DNA in the according to manufacturer's specifications. were by of protein from a with of double-stranded oligonucleotide in a of (10 mm pH mm glycerol, 1 mm with a total of of μg of BSA, and μg of DNA. were at for For 1 μg of monoclonal antibody was to were by on which were and to with an screen at °C. with T and A and M2 were used for In data not 1 CACGTG was found to bind Stra13 with if not affinity with with in the CACGTG not bind Stra13 Stra13 of not 1 μg of antibody was to protein and at 4 for 1 h or to was to the which was then for an min at 4 °C. were using 1× Triton X-100 lysis buffer or and by in buffer containing mm The protein were separated on and then were in (10 mm pH 150 mm NaCl, and and then with the rabbit polyclonal antibody in 1% for 1 The were then 15 in 1% followed by with at a dilution of for 1 h in 1% the were with 1% 15 followed by using blotting according to the manufacturer's For experiments in and COS-7 cells or HC11 cells were transfected with or of with of pcDNA3 or mutant In the for transfected was according to the from a or The and of the for were The in the of construct was a of For of Stra13 protein were determined through immunoprecipitation/Western on transfected with 4 μg or 10 μg of Stra13 expression was a of at the of 10 of protein was with of and the of the was determined for using a the from a of protein was with of buffer μg/ml 10 mm 1 and mm was to for min at and was using a active repression of the early by Stra13 in COS-7 expression was in COS-7 cells transfected with or with of expression was in COS-7 cells transfected with or with of expression was in COS-7 cells transfected with with of basic domain mutant expression for or expression was in COS-7 cells transfected with with as in E. of or mutant Stra13 protein were as determined through immunoprecipitation/Western of transfected with 4 μg of expression a of the Stra13 open reading with C-terminal deletion HC11 or COS-7 cells were seeded on and h later with phosphate-buffered in ice-cold and in 10 Cells were then in 1% for 1 h and for 1 h with at a 1:20 dilution in 1% were then with 10 and then using In a screen to genes expressed in the mammary we identified a cDNA that was identified in a number of tissues and cell This Stra13, the of a novel family of genes bHLH proteins with an basic domain sequence (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar, M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell Neurosci. 1997; 9: 460-475Crossref PubMed Scopus (109) Google Scholar, M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Crossref PubMed Scopus (145) Google Scholar, T. M. M. H. K. T. Oncogene. 1997; PubMed Scopus Google Scholar). bHLH proteins and/or in to bind DNA target elements (1Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1392) Google Scholar). In Stra13 has been to and with other bHLH including and (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar, T. M. M. H. K. T. Oncogene. 1997; PubMed Scopus Google Scholar). whether Stra13 in we transfected COS-7 cells with different of Stra13 1 In from cells with Myc Stra13 and FLAG Stra13, contained In from cells transfected with Stra13, antibodies not These data reveal that Stra13 in transfected COS-7 domains are for of bHLH transcription factors, we if an fragment of Stra13 containing the bHLH domain was for antibodies Stra13 from of cells with and not from of cells transfected with cDNA 1 we whether the basic of the bHLH domain was for by it or by it with an domain D. K. H. C. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). Basic domain and Stra13 proteins were both to with wild Stra13 1 we determined the of wild Stra13, and the Stra13 in transfected COS-7 cells and HC11 mammary In Stra13 protein was found in the 1 bHLH proteins DNA elements that are related to the E-box However, as Stra13 contains a proline residue at an in its basic it is not clear whether this protein binds directly to DNA. used a site to for a Stra13 homodimer site in M. E. Genes Dev. PubMed Scopus Google Scholar). the bHLH domain of Stra13 is related to enhancer of split family bHLH domains and these proteins were to bind elements K. N. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, N. M. E. Mol. PubMed Scopus Google Scholar), we stringency which in Stra13 bind to elements This in the of and This affinity Stra13 complex was used as a to Stra13 from in the early of site of Stra13 of bound and PCR we high affinity Stra13-binding sites to a we would on the The DNA was by and M. E. Genes Dev. PubMed Scopus Google Scholar). This for of site in of that contained the Interestingly, the Stra13 site identified in screen was which is a class B E-box C. Kato Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In addition, the CACGTG sequence was preceded by a T residue and followed by an A these are not for high affinity to Stra13 in a for the of on Stra13 to the E-box identified in we assays to for to sites high stringency in the of and D.M. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). This bound a protein or proteins in the in However, when the in was with a cDNA and this was with a novel complex was that be through of monoclonal The antibody not for of Stra13 on related sites. For example, we determined whether Stra13 bound to and to for the of within the E-box and for the of T and A that were in Interestingly, Stra13 a complex with as determined by the of this complex in the of antibody in the This site has a in the E-box with residues. In Stra13 not with which has the E-box T and A we by whether E-box elements Stra13 of not not and not complex B and data not Interestingly, elements including and not Stra13 from B and data not In addition, the mutant not and mutant E-box elements that from the CACGTG by not with for to Stra13 B and data not Stra13 binds with high affinity to CACGTG elements. It can also bind to mutant when these elements are by and with affinity CACGTG elements not for Stra13 in In GAL4-Stra13 fusion proteins were to repress transcription from upstream activator sequence elements that bind GAL4 (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar). whether Stra13 or represses transcription from its DNA target site, of the were inserted upstream of the and kinase in the and respectively. These constructs were transfected into COS-7 cells that not Stra13 protein and also into a mammary that The addition of E-box elements dramatically the by these in both of the expression and effect of E-box-binding proteins. of FLAG-Stra13 expression of in a Interestingly, the bHLH domain construct was as active as Stra13 in transcription in these suggesting that repression was as a result of Stra13 proteins with E-box activator proteins for to CACGTG elements A and that repression by Stra13 with E-box transcription factors for target CACGTG we whether and mutants function as in this basic domain mutants were to repress transcription from the CACGTG elements at wild Stra13 transcription by B This effect was associated with of DNA as both proteins were 1 and found in the 1 whether the basic domain mutant proteins function to wild Stra13 from E-box we Stra13 with or The repressor function of wild Stra13 was by of of these mutant proteins whether Stra13 repress transcription in the of a complex of of the upstream of the in the not gene expression in cells transcription of this in a not repress the 4 whether E-box elements were for transcriptional repression we inserted Stra13 mutant and into the Interestingly, the mutant site was 4 with the that elements bound Stra13 in In the mutant elements and that not bind were not to Stra13 in from E-box elements an basic domain in Stra13, it was not when or mutants were with 4 In addition, this effect represented active repression in to the repression on repression of the in the C-terminal of Stra13 not in Stra13 for active repression of the in this we and a of C-terminal mutants The C-terminal of the transcriptional repression domain was to between and C-terminal mutants including were whereas and C-terminal mutants this 4 The of to repress transcription was not by a of expression of this mutant protein 4 Stra13 is a bHLH protein associated with cell activation and stress in many Indeed, this protein is induced in activated neuronal cells, and a number of cell has that Stra13 is for T-cell activation and of clearance (6Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (151) Google Scholar). In addition, Stra13 has been implicated in repression of Dev. Cell. 2: Full Text Full Text PDF PubMed Scopus Google Scholar). Despite the of Stra13 in these biological the biochemical mechanism by which it functions remains unknown. For example, it yet to be determined whether Stra13, which a proline residue at an unprecedented site within its basic binds directly to DNA. have used a screen to that Stra13 binds to a DNA element, the CACGTG class B E-box element, with preference for sites preceded by T and followed by A. of this et al. L. J. S. B. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) have that Stra13 binds CACGTG elements in can repress transcription from containing these sites. sites regulate expression of many genes associated with proliferation, differentiation, and cell have also determined that Stra13 can repress transcription of that class B E-box elements. The domain of Stra13 for transcriptional repression from CACGTG elements to in the of This result is with data from et al. (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar) used GAL4-Stra13 to repress transcription from upstream activator sequence sites and to a repression domain between and In addition, Sun and Taneja (11Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (160) Google Scholar) have determined that and bind to Stra13 between and Interestingly, the Stra13 deletion mutant that to repress transcription bound suggesting that Stra13 may transcriptional repression not These are important that Stra13 binds to and proteins M. Taneja R. Oncogene. 2001; 20: PubMed Scopus Google Scholar) and represses expression of c-Myc (11Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (160) Google Scholar), of transcription activating proteins that bind to CACGTG E-box elements. Stra13 also represses expression of its own through a mechanism histone deacetylase (11Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (160) Google Scholar). Interestingly, the Stra13 contains CACGTG elements and upstream of the M. Nakamasu K. Noshiro M. Y. O. M. S. Kawamoto T. Y. Kato Y. J. Biochem. 2001; PubMed Scopus Google Scholar). work has identified gene as a target of transcriptional repression of Dev. Cell. 2: Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, the that to repression contains sites for proteins E-box elements identified in In addition, the bHLH domain of Stra13 was to repress expression of Stra13 represses expression and through direct of will be to the mechanism by which Stra13 represses expression of Stra13 and and to identify E-box elements in the that are to by Stra13 and the protein, Indeed, the E-box elements identified in this are to represent in a of activator and repressor proteins cell activation, proliferation, and and of the and for and

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