Circadian Transcription

The E-Box is a widely used DNA control element. Despite its brevity and broad distribution the E-Box is a remarkably versatile sequence that affects many different genetic programs, including proliferation, differentiation, tissue-specific responses, and cell death. The circadian clock is one of the latest pathways shown to employ this element. In this context, E-Boxes are likely to play a key role in establishing the robust waves of gene expression characteristic of circadian transcription. The regulatory flexibility of the E-Box hinges on the sequence ambiguity allowed at its core, the strong influence of the surrounding sequences, and the recruitment of spatially and temporally regulated E-Box-binding factors. Therefore, understanding how a particular E-Box can accomplish a specific task entails the identification and systematic analysis of these cis- and trans-acting E-Box modifiers. In the present study we compared the E-Box-containing minimal promoters of vasopressin andcyclin B1, two genes that can respond to the transcriptional oscillators driving the circadian clock and cell cycle, respectively. Results of this comparison will help elucidate the manner in which discreet DNA modules associate to either augment or restrain the activation of potential circadian E-Boxes in response to competing regulatory signals. The E-Box is a widely used DNA control element. Despite its brevity and broad distribution the E-Box is a remarkably versatile sequence that affects many different genetic programs, including proliferation, differentiation, tissue-specific responses, and cell death. The circadian clock is one of the latest pathways shown to employ this element. In this context, E-Boxes are likely to play a key role in establishing the robust waves of gene expression characteristic of circadian transcription. The regulatory flexibility of the E-Box hinges on the sequence ambiguity allowed at its core, the strong influence of the surrounding sequences, and the recruitment of spatially and temporally regulated E-Box-binding factors. Therefore, understanding how a particular E-Box can accomplish a specific task entails the identification and systematic analysis of these cis- and trans-acting E-Box modifiers. In the present study we compared the E-Box-containing minimal promoters of vasopressin andcyclin B1, two genes that can respond to the transcriptional oscillators driving the circadian clock and cell cycle, respectively. Results of this comparison will help elucidate the manner in which discreet DNA modules associate to either augment or restrain the activation of potential circadian E-Boxes in response to competing regulatory signals. enhancer upstream stimulatory factor BMAL1·CLOCK complex vasopressin CYC, cyclin B1 Renilla luciferase Firefly luciferase electrophoretic mobility shift assay thymidine kinase cyclic adenosine monophosphate responsive element Homographs are words that share the same spelling but differ in sound, origin, or meaning. By extension, an enhancer (E)1-Box can be defined as a “transcriptional homograph”: a short DNA element (word) that, depending upon the features of the sequence and cellular environment (context), can dispense very different transcriptional outputs (messages). By recruiting the proto-oncogene c-myc, for example, an E-Box can drive cells to become growth factor independent, to speed through G1 of the cell cycle, to avoid differentiation, or, paradoxically, to undergo apoptotic death (1Littlewood T. Evan G. Adv. Dent. Res. 1990; 4: 69-79Crossref PubMed Scopus (33) Google Scholar, 2Fuhrmann G. Rosenberger G. Grusch M. Klein N. Hofmann J. Krupitza G. Mutat. Res. 1999; 437: 205-217Crossref PubMed Scopus (47) Google Scholar). The ability of an E-Box to support myogenic factor-mediated muscle differentiation, even in many non-muscle cell types (3Funk W. Ouellette M. Wright W. Mol. Biol. Med. 1991; 8: 185-195PubMed Google Scholar), is another example of the regulatory plasticity provided by this multifunctional site. In fact, since its identification in 1985 as a control element in the immunoglobulin heavy chain gene promoter (4Church G. Ephrussi A. Gilbert W. Tonegawa S. Nature. 1985; 313: 798-801Crossref PubMed Scopus (199) Google Scholar), the E-box has been found to influence the expression of a large number of genes that share no additional obvious relationships. The list includes genes such as actin (5Biben C. Kirschbaum B. Garner I. Buckingham M. Mol. Cell. Biol. 1994; 14: 3504-3513Crossref PubMed Scopus (25) Google Scholar), ornithine decarboxylase (6Walhout A. Gubbels J. Bernards R. van der Vliet P. Timmers H. Nucleic Acids Res. 1997; 25: 1493-1501Crossref PubMed Scopus (71) Google Scholar),prothymosin (7Gaubatz S. Meichle A. Eilers M. Mol. Cell. Biol. 1994; 14: 3853-3862Crossref PubMed Google Scholar), vasopressin (8Coulson J. Fiskerstrand C. Woll P. Quinn J. Biochem. J. 1999; 344: 961-970Crossref PubMed Google Scholar, 9Jin X. Shearman L. Weaver D. Zylka M. de Vries G. Reppert S. Cell. 1999; 96: 57-68Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar),TGF-β (10Scholtz B. Kingsley-Kallesen M. Rizzino A. J. Biol. Chem. 1996; 271: 32375-32380Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), BRCA2 (11Davis P. Miron A. Andersen L. Iglehart J. Marks J. Oncogene. 1999; 18: 6000-6012Crossref PubMed Scopus (50) Google Scholar), cyclin B1 (12Farina A. Gaetano C. Crescenzi M. Puccini F. Manni I. Sacchi A. Piaggio G. Oncogene. 1996; 13: 1287-1296PubMed Google Scholar), glycophorin-B (13Camara-Clayette V. Rahuel C. Bertrand O. Cartron J. Biochem. Biophys. Res. Commun. 1999; 265: 170-176Crossref PubMed Scopus (12) Google Scholar), and myosin(14Navankasattusas S. Sawadogo M. van Bilsen M. Dang C. Chien K. Mol. Cell. Biol. 1994; 14: 7331-7339Crossref PubMed Scopus (67) Google Scholar), among many others. The perplexing diversity of E-Box-dependent processes has fueled an intense search for trans-acting factors regulating a given E-Box. Predictably, such efforts have resulted in an ever-expanding list of transcription factors that can recognize E-Box consensus sequences. Virtually every such factor uses a basic helix-loop-helix (bHLH) (15Murre C. McCaw P. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (2051) Google Scholar) motif to interact specifically with promoters and other proteins. A partial catalog of E-Box-binding factors includes Myc (5Biben C. Kirschbaum B. Garner I. Buckingham M. Mol. Cell. Biol. 1994; 14: 3504-3513Crossref PubMed Scopus (25) Google Scholar), Arnt (16Antonsson C. Arulampalam V. Whitelaw M. Pettersson M. Poellinger L. J. Biol. Chem. 1995; 270: 13968-13972Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), Max (17Zhang H. Fan S. Prochownik E. J. Biol. Chem. 1997; 272: 17416-17424Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), MyoD (18Huang J. Blackwell T. Kedes L. Weintraub H. Mol. Cell. Biol. 1996; 16: 3893-3900Crossref PubMed Scopus (39) Google Scholar), Mad (19Hurlin P. Queva C. Koskinen P. Steingrimsson E. Ayer D. Copeland N. Jenkins N. Eisenman R. EMBO J. 1995; 14: 5646-5659Crossref PubMed Scopus (256) Google Scholar), upstream stimulatory factor (USF) (20Potter J. Cheneval D. Dang C. Resar L. Mezey E. Yang V. J. Biol. Chem. 1991; 266: 15457-15463Abstract Full Text PDF PubMed Google Scholar), Mxi1 (21Lee T. Ziff E. J. Biol. Chem. 1999; 274: 595-606Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), E47 (22Ellenberger T. Fass D. Arnaud M. Harrison S. Genes Dev. 1994; 8: 970-980Crossref PubMed Scopus (360) Google Scholar), TFE3 (23Artandi S. Cooper C. Shrivastava A. Calame K. Mol. Cell. Biol. 1994; 14: 7704-7716Crossref PubMed Scopus (42) Google Scholar), TAL1 (24Hsu H. Huang L. Tsan J. Funk W. Wright W., Hu, J. Kingston R. Baer R. Mol. Cell. Biol. 1994; 14: 1256-1265Crossref PubMed Scopus (148) Google Scholar), and the more recently described BMAL1 (25Hogenesch J., Gu, Y. Jain S. Bradfield C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5474-5479Crossref PubMed Scopus (641) Google Scholar) and CLOCK (26Darlington T. Wager-Smith K. Ceriani K. Staknis D. Gekakis N. Steeves T. Weitz C. Takahashi J. Kay S. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (706) Google Scholar, 27Gekakis N. Staknis D. H. F. L. D. Takahashi J. Weitz C. Science. 1998; 280: PubMed Scopus Google Scholar) proteins. the sequence surrounding an E-Box play a role in different promoters different of an that has been the of The of as a of these efforts a complex in which the and number of E-Boxes L. S. Eilers M. Genes Dev. 1996; PubMed Scopus Google Scholar, S. M. R. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), F. D. P. W. D. C. EMBO J. PubMed Scopus Google Scholar, H. W. Bradfield C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A. P. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar, K. Weintraub H. 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PubMed Scopus Google Scholar), and the E-Box element is a the that the circadian even the features to an E-Box circadian control are to be characteristic by circadian E-Boxes is ability to specifically a BMAL1·CLOCK complex X. Shearman L. Weaver D. Zylka M. de Vries G. Reppert S. Cell. 1999; 96: 57-68Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, J., Gu, Y. Jain S. Bradfield C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5474-5479Crossref PubMed Scopus (641) Google Scholar, T. Wager-Smith K. Ceriani K. Staknis D. Gekakis N. Steeves T. Weitz C. Takahashi J. Kay S. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (706) Google Scholar, 27Gekakis N. Staknis D. H. F. L. D. Takahashi J. Weitz C. Science. 1998; 280: PubMed Scopus Google Scholar). fact, shift to the features that the for a a circadian E-Box and the to the E-Box in the of genes have provided a understanding of the sequence upon the a circadian E-Box G. A. T. D. W. L. Kay S. A. J. 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A. 1998; 95: 5474-5479Crossref PubMed Scopus (641) Google Scholar). is how this consensus the circadian of a E-Box in its we the of these by the of the sequence the in by and cyclin B1 gene Despite the of a E-Box in promoters and at very these two genes are to different of transcriptional The promoter has been shown to be circadian by the in the X. Shearman L. Weaver D. Zylka M. de Vries G. Reppert S. Cell. 1999; 96: 57-68Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, T. Wager-Smith K. Ceriani K. Staknis D. Gekakis N. Steeves T. Weitz C. Takahashi J. Kay S. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (706) Google Scholar). in the transcription of the gene is by and W. E. Sci. Google Scholar). the other the promoter to cell A. A. W. R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and transcriptional J. M. M. J. L. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). has been that cyclin B1 expression be by G. R. R. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). how different the DNA can to the circadian of a E-Box. A by a in number and in The by a to cyclin B1 promoter in of the in The CLOCK and BMAL1 expression provided by N. Gekakis and C. Weitz expression for and a M. Sawadogo of of the by or DNA the The at the of the in the and used to the of the promoter to the and used as to the promoter and The to in the promoter E-Box sequence in to to to to to to to to to to to the of the the of the promoter upstream of the and by a the and E-Boxes of the promoter the in the of the E-Box. promoter and by a in the which at to the of the with a the or upstream of the respectively. of and the same the of a luciferase cells at a of cells in a and with a of and of a expression or or in of the of to the which in by the by a thymidine kinase Renilla luciferase DNA for the to of expression for and by analysis the and of BMAL1 either to the of this or the of the that we used and of BMAL1 in in which the of expression an for of the promoter through its E-Box. Firefly luciferase and Renilla luciferase the and the a of at two in analysis by a for as described W. R. Mol. Res. PubMed Scopus Google Scholar) with cells in and with the and the that the with and DNA to in by a with and A and and on an of either or to the assay the of the of the for at with of the or a control in a of The to the assay as described of an to the to of and for at in a the of the the and to a for the the features that a E-Box we compared the Y. J. H. Res. Mol. Res. 1990; 8: PubMed Scopus Google Scholar) and (12Farina A. Gaetano C. Crescenzi M. Puccini F. Manni I. Sacchi A. Piaggio G. Oncogene. 1996; 13: 1287-1296PubMed Google Scholar) upstream regulatory a E-Box at to the transcription we the of these two E-Box-containing minimal promoters and by of an or gene cells E-Boxes can many transcription processes the is as of gene as as with X. Shearman L. Weaver D. Zylka M. de Vries G. Reppert S. Cell. 1999; 96: 57-68Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, W. R. Mol. Res. PubMed Scopus Google Scholar), the by an that upon the of transcription factors In the to the of the circadian the for the we used in this study to the features of a circadian E-Box. The of in the the that this promoter is the of the cell A. A. W. R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). cells this in an can support of cyclin B1 gene transcription. The analysis of that the minimal promoter by a factor of the promoter to of the E-Box-dependent transcription factor to as X. Sawadogo M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), the the and with to that the complex can these two of luciferase the and promoters and for the shown in the to the of in as have the response of the and promoters for this the of the is or The response of the and E-Box-containing to the of different for factors. this we compared the ability of two in the and to factors in an electrophoretic mobility shift assay to robust and specific with as the of E-Box-binding factors in A with that the of the or E-Box and a the of and are Despite the in the by the two we that the E-Box-dependent complex a of the E-Box for the factor in the the specific of the two and the present in large with this a E-Box sequence as as a E-Box at either the E-box or E-Box the complex shown the and E-Boxes can factors in with different and respond very to the and E-Box-containing responsive to in the of the promoter that of an E-Box for an factor as be to a of by a to the of in the an in be to support robust of a circadian E-Box as or the but of in a E-Box the E-Box and in the of the the and in the same as the and a robust of even the to In to a response to that of the to is either or by DNA the The to search for these of E-Box we on the by different of the promoter the sequences. found that which a a of to but a The the the sequence used to the sequence the E-Box the of a in the the other of by we a in as as a in The in and a to a the which has been shown to a complex A. P. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar). Despite this the a of these the of a enhancer element upstream of the E-Box a upstream of the E-Box. with this of the in the minimal promoter a on the ability of the sequence to respond to a upstream element to be to the response of the E-Box to the of this we an additional of the E-Box and the of the minimal in this through to the of In the response of these to that and in this minimal promoter play an and specific role in E-Box-dependent which includes a to through the transcription factor search of the K. K. H. E. T. Nucleic Acids Res. 1995; PubMed Scopus Google Scholar). among these are and we the influence of on the response of the E-Box to been that a at and an at are of an consensus for the complex in (25Hogenesch J., Gu, Y. Jain S. Bradfield C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5474-5479Crossref PubMed Scopus (641) Google Scholar). to the E-Box is by an at and a at the of the for the additional in which the and at and to the of the The of with at these with the role of these The to at resulted in a in the response to the other the of the to at through its of the of the to at the E-Box is by a at which is with the response of the to is the that the sequence is to the of expression of a E-Box and that the sequence is likely to a enhancer the upstream enhancer in is to robust the we the to as The of this promoter is a that is the one in this of the robust that we with the The of expression by can for this is that additional are to or a be provided by one or more of the consensus the E-Box and the of transcription C. G. Quinn J. 1999; PubMed Scopus (25) Google Scholar). this either or in and the for the E-Box for its found that of of the in the promoter and its ability to respond to is to that of these a response to that the E-Boxes more to a is to a to specific to in or In a of we found that of the element no on the ability of this to respond to of the either by or in with the a that but a enhancer role is that the of the the E-Box and the upstream that the of the E-Box of a strong response to in the this we a the and of the E-Box in the and this sequence a on which upon the of the upstream enhancer to upstream and of the E-Box to be the response The E-Box is one of the DNA widely used for transcription. The of its in circadian gene expression to the for the of an E-Box in A that have this have on the E-Box the circadian regulatory sequence of the promoter M. M. P. Mol. Cell. Biol. PubMed Scopus (71) Google Scholar, L. T. H. J. Kay S. P. J. Biol. PubMed Scopus Google Scholar, T. L. P. Kay S. J. Biol. PubMed Google Scholar). present that the E-Box element to for the activation by the circadian through the the of In the present study we compared the vasopressin and cyclin B1 E-Box-containing two control the of which is as a circadian Despite the of the cyclin B1 promoter to support B1 a robust in its as a of cell A. A. W. R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), of which are to the E-Box at (12Farina A. Gaetano C. Crescenzi M. Puccini F. Manni I. Sacchi A. Piaggio G. Oncogene. 1996; 13: 1287-1296PubMed Google Scholar, J. M. M. J. L. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). even the genes a E-Box in and and with a of are the of different regulatory that a analysis of these minimal promoters help DNA that either or a E-Box by the circadian The basic in this study is that the E-Box is in an environment that is to at in cells Therefore, can be to play a key role in the response to by the two The of such is likely to the that DNA the two a response to the of in the of a promoter a regulatory this a one every E-Box-containing promoter be at responsive to the circadian circadian on the other a more robust of analysis of the the of by the to the E-Box that can either the of the for the of expression by or transcription factors. the of the compared with the promoter as as the in the response of to to with this is the of to in the these are with the of a regulatory element upstream of the E-Box. is with the with a of which allowed to the of this enhancer to a and upstream of the E-Box The of such a by the that the of the and E-Boxes but the of this to respond to these that we have in the the example of a element to to The for the influence of this sequence upon is is that the to an E-Box help the of factors that interact with the site. In fact, is a with the and of the by E-Box factors L. S. Eilers M. Genes Dev. 1996; PubMed Scopus Google Scholar, A. P. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar, and K. Weintraub H. Science. 1990; PubMed Scopus Google Scholar). BMAL1 to different as a of the that is provided in (25Hogenesch J., Gu, Y. Jain S. Bradfield C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5474-5479Crossref PubMed Scopus (641) Google Scholar). is the study to the of by a complex the of a The comparison of the of these with to the the E-Box that are for in The of a complex and a of been to be (25Hogenesch J., Gu, Y. Jain S. Bradfield C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5474-5479Crossref PubMed Scopus (641) Google Scholar). study this at on the of the E-Box. have on and to be among a of strong circadian of a for the at to the resulted in a in that to the the at by a is to that we compared the and either the or E-Box-containing the the more intense the used that the E-Box can E-Box-binding such as the with the analysis this is with the of A. P. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar) in which a of and among the is to that E-Boxes with for such factors be and for with the more for a and E-Box-binding factor as a for by factors. at in the of the E-Box and be in by E-Boxes to or avoid circadian of will the a promoter by the In fact, gene of in in the and X. Shearman L. Weaver D. Zylka M. de Vries G. Reppert S. Cell. 1999; 96: 57-68Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar), the pathways can a robust In to the strong of the upstream we have found that of the in the response to The of E-Box been the and of circadian regulated gene which E-Box In to the we the influence of the and of the E-Box. of the no on the of of the to a and in promoter in response to that this as a enhancer The of the promoters used in this study with E-Box the transcriptional to the circadian of a E-Box a of a the potential of a E-Box to respond to can through many to The of the can be with a of for factors the of that of expression on the other be in a by a shift in the factors and the complex and by the of specific with promoter and that an element a to and other factors are likely to in different promoters to a of circadian these temporally is that such a for of flexibility in the of in gene to the promoter context, be to and the circadian response of other E-Box-containing promoters the basic is an as analysis of circadian regulated genes M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, C. F. A. G. V. T. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The present study has the of the cellular environment in circadian and of transcription. example, on the of the E-Box for the we that the of to in the of this which a of The role of in circadian in the transcription of E-Box-containing promoters is a two are V. A. D. M. M. A. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. R. Sawadogo M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). for the of the expression for and provided by of a cyclin B1 luciferase provided by J. of and of and BMAL1 and CLOCK expression provided by Gekakis and to of for of the and