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Switching of DNA Secondary Structure in Proenkephalin Transcriptional Regulation

1997; Elsevier BV; Volume: 272; Issue: 52 Linguagem: Inglês

10.1074/jbc.272.52.33145

1083-351X

Craig Spiro, Cynthia T. McMurray,

Neuroscience and Neuropharmacology Research

Proper transcriptional regulation of the proenkephalin gene requires a switch between distinct factor binding sites that cannot exist at the same time. Each of the sites is formed from a nearly palindromic region that contains two functionally defined cAMP response elements. The region can switch between cruciform and linear duplex. Formation of the cruciform creates an alternative binding site for mediators of second messenger-directed transcription and abolishes the site present in the native duplex form. Use of the cruciform site has been shown to correlate with activated transcription. Analysis of DNA structure, protein binding, and gene expression from plasmids with mutant enhancers shows, however, that both sites are required for regulation of transcription. The two distinct structures form within the same enhancer. Shifting the balance between the two alters transcriptional response. Proper transcriptional regulation of the proenkephalin gene requires a switch between distinct factor binding sites that cannot exist at the same time. Each of the sites is formed from a nearly palindromic region that contains two functionally defined cAMP response elements. The region can switch between cruciform and linear duplex. Formation of the cruciform creates an alternative binding site for mediators of second messenger-directed transcription and abolishes the site present in the native duplex form. Use of the cruciform site has been shown to correlate with activated transcription. Analysis of DNA structure, protein binding, and gene expression from plasmids with mutant enhancers shows, however, that both sites are required for regulation of transcription. The two distinct structures form within the same enhancer. Shifting the balance between the two alters transcriptional response. Transcription of the proenkephalin gene, which encodes the precursor to endogenous opiate-receptor ligands, is regulated by signals from growth factors, neurotransmitters, hormones, and cell depolarization. The pathways converge on an evolutionarily conserved, nearly palindromic region that contains two functionally defined cAMP response elements (CREs) 1The abbreviations used are: CRE, cAMP response element; CREB, cAMP response element binding protein; CAT, chloramphenicol acetyltransferase. (1Comb M. Birnberg N. Seasholtz A. Herbert E. Goodman H.M. Nature. 1986; 323: 353-356Crossref PubMed Scopus (577) Google Scholar,2Comb M. Mermod N. Hyman S.E. Pearlberg J. Ross M.E. Goodman H.M. EMBO J. 1988; 7: 3793-3805Crossref PubMed Scopus (309) Google Scholar). Only CRE-2 can independently bind transcription factors. However, point mutations in either CRE have quantitatively similar effects on transcription (2Comb M. Mermod N. Hyman S.E. Pearlberg J. Ross M.E. Goodman H.M. EMBO J. 1988; 7: 3793-3805Crossref PubMed Scopus (309) Google Scholar), and the response to CRE-binding proteins depends on the presence of CRE-1 as well as CRE-2 (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar). Because the 23-base pair region containing the CREs is nearly palindromic, it can convert to a cruciform structure in which a transcription factor-binding site is formed from the sequences of both CREs on the same strand (3–5; Fig.1 a). The switch to the alternative site eliminates the native CRE-2, substituting a site with the same sequence but with two mismatched GT base pairs (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar, 4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar, 5McMurray C.T. Juranic N. Chandrasekaran S. Macura S. Li Y. Jones R.L. Wilson W.D. Biochemistry. 1994; 33: 11960-11970Crossref PubMed Scopus (12) Google Scholar). The structural change, therefore, creates a single site comprising both CRE-1 and CRE-2 (Fig.1 a). Use of the site made from both CREs correlates with stimulated transcription, according to several types of analysis. In vivo, the site made from CRE-1 and CRE-2, but not the native duplex CRE-2 site, is occupied specifically during transcription (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Dimethyl sulfate treatment of cells actively transcribing proenkephalin specifically modifies adenines that form non-Watson-Crick AC base pairs in the cruciform structure, indicating unusual charge distribution at those bases only during active transcription (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Electron spectroscopic images show that a CREB dimer occupies twice as much DNA in the proenkephalin gene as in the prodynorphin gene, consistent with binding to a cruciform in the proenkephalin gene (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The prodynorphin CRE is identical in sequence to proenkephalin CRE-2 but is in a region unable to form alternative structure (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Finally, point mutations that do not alter transcription factor binding but that could modify hairpin stability modulate response to receptor signaling (2Comb M. Mermod N. Hyman S.E. Pearlberg J. Ross M.E. Goodman H.M. EMBO J. 1988; 7: 3793-3805Crossref PubMed Scopus (309) Google Scholar, 4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar). Within the proenkephalin gene, therefore, CRE-1 and CRE-2 form a single binding site that is essential for transcriptional response to extracellular signals. The native CRE-2 is a relatively low-affinity binding site for CREB (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar, 7Williams J.S. Dixon J.E. Andrisani O.M. DNA Cell Biol. 1993; 12: 183-190Crossref PubMed Scopus (19) Google Scholar), but GT base pairs in the stem of the hairpin made from CRE-1 and CRE-2 enhance CREB binding (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar). The higher affinity site on the hairpin arm could correlate with high activity transcription because it is better able to recruit enhancer-binding proteins. To understand the mechanism of proenkephalin enhancer action and to test the hypothesis that enhanced protein affinity for the altered site allows more efficient transcription, we have analyzed gene expression, protein binding, and DNA structure of reporter plasmids with point mutations in the region of the CREs. Binding to the CREs is necessary for transcriptional response, but our analysis demonstrates that efficiency of binding of CREB alone does not correlate with transcription. CREB preference for the alternative site cannot by itself explain the enhanced activity of the hairpin versuslinear duplex binding site. The relative availability of the alternative sites does, however, affect transcription. Mutants in which strong binding to the linear duplex CRE-2 is favored, relative to the native gene, are less responsive to cAMP. Mutants with greater preference for hairpin rather than linear duplex CRE-2 site are more responsive to cAMP. Thus, proper regulation of transcription mediated by the native enhancer requires switching between both sites. Construction of mutant proenkephalin enhancer region-CAT reporter plasmids by overlap polymerase chain reaction (11Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (7053) Google Scholar) has been described previously (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The mutations are described under “Results” (see Fig. 1 b). SK-N-MC neuroblastoma cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. Proenkephalin-CAT reporter plasmids were introduced by DEAE-dextran-mediated transformation (250 μg/ml DEAE dextran + DNA for 2–2.5 h followed by 10% dimethyl sulfoxide for 2 min). Forskolin (20 μm final) or dimethyl sulfoxide carrier (“control”) was added 15 h after 10% dimethyl sulfoxide treatment. Cells (10 cm dish) were harvested 24 h after forskolin treatment into 75 μl of enzyme extract; 15 μl was used for the CAT assay (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 12Collins-Hicok J. Lin L. Spiro C. Laybourn P.J. Tschumper R. Rapacz B. McMurray C.T. Mol. Cell. Biol. 1994; 14: 2837-2848Crossref PubMed Scopus (49) Google Scholar). Phosphor storage screen was exposed to dried chromatograms and then scanned by PhosphorImager 425 (Molecular Dynamics). Acetylated and unacetylated spots were quantified from digital images by IP Lab Gel (Signal Analytics), using supplied macros (“profile plot,” “auto peak find,” and “analyze peaks”). Percent acetylation and standard deviation of replicate plates were calculated in Excel (Microsoft), and data were plotted using KaleidaGraph (Abelbeck). Bacterially expressed CREB protein was expressed and purified as described previously (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Protein-DNA complexes formed on the same plasmids used for expression studies (overnight at 4 °C) were analyzed by DNase I digestion and primer extension as described previously (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). After separation of fragments on 8% sequencing gels, the gels were dried for exposure of phosphor storage screen. ImageQuant software was used to convert the data from the phosphor storage screen to images of gels and to numbers used to quantify band strength. Scale of scanned images was adjusted to reduce variation in signal between lanes. Comparison of bands was as described previously (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar): equivalent regions of each lane were selected and quantified (ImageQuant); data were exported to KaleidaGraph; curves were normalized to correct for uneven loading; and relative intensities of individual bands were estimated by peak-to-valley measurement. Oligonucleotide probes are described under “Results” (Fig. 5). Crude extract from bacteria expressing recombinant CREB protein (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) was incubated for 15 min at room temperature in the presence of 1.5 μg of sonicated salmon sperm DNA and 0.1 pmol of 32P-end-labeled oligonucleotide probe. Complexes were analyzed on 5%, 0.5 × Tris glycine gel as described previously (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar, 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The ability of specific mutations to either enhance or reduce transcription contributed to an understanding of the effect of secondary structure in proenkephalin gene expression (4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar). Certain mutations eliminate protein binding, which can explain their deleterious effect on transcription (e.g. enk −88C,−89A; Refs. 3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar and 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and Figs. 1 b and3 a). In contrast, other mutations do not abolish binding yet have a significant effect on transcription (e.g. enk subCRE1; Refs. 3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar, 4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar, and 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar; and Figs. 1 b and3 a). To understand the relationships among binding efficiency, structure, and gene expression, we have produced and analyzed mutant proenkephalin-CAT reporter plasmids (Fig.1 b). Binding and structural analyses were carried out on intact plasmids, identical to those used for transcriptional analysis. The plasmids analyzed included the previously described mutants lacking a functional CRE-2 (enk −88C,−89A) or a functional CRE-1 (enk subCRE1) as well as mutants with single or two-base changes designed to alter the cruciform. Because CRE-2 is essential for binding to both the linear duplex and the hairpin site (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), modification of CRE-2 was avoided in this latter group of mutant plasmids. These mutations have been chosen for their ability to alter stability of the cruciform. The mutants designated enk −98T and enk −105G,−106A have a less stable cruciform structure because of reduced hydrogen bonding in the stem (Fig. 1 b; Refs. 4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar, 5McMurray C.T. Juranic N. Chandrasekaran S. Macura S. Li Y. Jones R.L. Wilson W.D. Biochemistry. 1994; 33: 11960-11970Crossref PubMed Scopus (12) Google Scholar, 17Gacy A.M. McMurray C.T. Biochemistry. 1994; 33: 11951-11959Crossref PubMed Scopus (23) Google Scholar, and 18.Gacy, A. M. (1996) The Role of DNA Hairpin Structure in the Regulation of Transcription and Replication. Ph.D. thesis, Mayo Graduate School.Google Scholar). The mutants designated enk −91A and enk −102A and the double mutant enk −91A,−102A have either one or both of the mismatched base pairs in the stem corrected and, consequently, a more stable stem-loop structure (Fig. 1 b). Alterations in the loop have been shown previously to modify hairpin stability (18.Gacy, A. M. (1996) The Role of DNA Hairpin Structure in the Regulation of Transcription and Replication. Ph.D. thesis, Mayo Graduate School.Google Scholar). Substitution of an A or T for the G at position −95 creates a more stable hairpin, as determined by thermal melting analysis (17Gacy A.M. McMurray C.T. Biochemistry. 1994; 33: 11951-11959Crossref PubMed Scopus (23) Google Scholar, 18.Gacy, A. M. (1996) The Role of DNA Hairpin Structure in the Regulation of Transcription and Replication. Ph.D. thesis, Mayo Graduate School.Google Scholar). Reporter plasmids with the desired mutations were constructed for use in the analyses (Fig.1 b; and see “Experimental Procedures”). Transcription was analyzed by transient transfection of proenkephalin-CAT reporter plasmids into the SK-N-MC human neuroblastoma cell line, in which the endogenous proenkephalin gene is expressed. CAT activity of mutant plasmids was compared with that of the native plasmid (Fig.2). Mutations that eliminate either CRE-1 (enk subCRE-1) or CRE-2 (enk −88C,−89A) reduce transcriptional efficiency (Fig. 2), as has been shown previously (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The strongest inhibition of transcriptional response is caused by the mutation that alters the conserved CGTCA in CRE-2 (Fig. 2, enk −88C,−89A), and, thereby, eliminates binding to the enhancer region (Fig. 3 a; Ref. 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Modification of CRE-1 (enk subCRE-1) does not alter CRE-2, but it does destroy the palindromic character of the region, precluding formation of a cruciform (Refs. 3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar and 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar; and Fig. 1). Two changes (enk −98T and enk −105G,−106A) cause reduced transcriptional response very much like that caused by the mutations that abolish CRE-1 in the mutant enk subCRE-1 (Fig. 2). Both modifications (in enk −98T and in enk −105G,−106) destabilize hairpins. Enk −98T has a single-base change in CRE-1, which introduces another mismatched base pair into each stem (Fig. 1 b), and enk −105G,−106A has two mutations which substitute mismatched base pairs at the base and reduce the stem to 8-base pairs (Fig. 1 b). Neither of these plasmids has a mutation in or close to CRE-2 in the linear duplex form (Fig. 1 b, left side), and one of them (enk −105G,−106A) modifies neither CRE at all, yet they both show an effect similar to the complete elimination of CRE-1 (Fig. 2). Mutations that correct mismatched base pairs in the stem, by substituting an A for the G at either −91 or −102 or at both, stabilize the cruciform (Ref. 4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar; Fig. 1 b). Either or both of these changes enhance transcriptional response (Fig. 2). We do note, however, that each of these mismatch-correcting mutations complicates direct comparison with the native. The mutation at −102 creates within CRE-1 the conserved CGTCA of CREs, so that transcriptional response could be due in part to the creation of a different binding site. The mutation at −91 modifies CRE-2 so that the binding site is not identical to the CRE-2 in the native. Because of these ambiguities, we do not consider these mutant plasmids in our structural analysis (see below). The final two mutant plasmids were altered at base −95, which is between the two CREs in the linear duplex and forms the middle base in the loop (Fig. 1 b). Changes at the middle position of the loop (−95) all create more stable hairpins than native, as determined by thermal melting analysis of synthetic enhancers (17Gacy A.M. McMurray C.T. Biochemistry. 1994; 33: 11951-11959Crossref PubMed Scopus (23) Google Scholar, 18.Gacy, A. M. (1996) The Role of DNA Hairpin Structure in the Regulation of Transcription and Replication. Ph.D. thesis, Mayo Graduate School.Google Scholar). The loop then contributes to hairpin stability. Analysis of the proenkephalin-CAT reporter plasmids shows that alteration to either A or T at position −95 enhances transcription, with a greater response than native to addition of forskolin (Fig. 2). In summary, mutants with predicted stabilization of the hairpin show enhanced transcriptional response, while destabilization correlates with reduced response. In short synthetic enhancers, insertion of GT base pairs into a duplex site increases CREB protein binding, indicating that GT base pairs enhance binding when the structure is otherwise held constant (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar). The cruciform site, which has mismatched GT base pairs (Fig. 1 a; Ref. 5McMurray C.T. Juranic N. Chandrasekaran S. Macura S. Li Y. Jones R.L. Wilson W.D. Biochemistry. 1994; 33: 11960-11970Crossref PubMed Scopus (12) Google Scholar), can, therefore, have an advantage over the linear duplex site in recruiting transcription factors such as CREB, which mediates proenkephalin expression in vivo via the CREs (8Konradi C. Kobierski L.A. Nguyen T.V. Heckers S. Hyman S.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7005-7009Crossref PubMed Scopus (145) Google Scholar, 9Borsook D. Konradi C. Falkowski O. Comb M. Hyman S.E. Mol. Endocrinol. 1994; 8: 240-248PubMed Google Scholar, 10Konradi C. Cole R.L. Heckers S. Hyman S.E. J. Neurosci. 1994; 14: 5623-5634Crossref PubMed Google Scholar). Conversely, stabilizing the structure, even at the expense of eliminating the mismatched base pairs, can also enhance receptor response (Fig. 2, enk −91A and/or enk −102A). CREB binds to the enhancer in the plasmid with the substituted CRE-1 even at the lowest concentration of protein (Figs. 1 b and3 a, enk subCRE1; and Ref. 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The enk subCRE1 enhancer, however, cannot support efficient transcription (Fig. 2; Ref. 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and the response to CREB itself is abrogated in plasmids lacking a functional CRE-1 (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar). In plasmid enk subCRE1 protection is limited to the region around CRE-2; it does not extend into CRE-1 (Fig. 3 a). The plasmid designed to abolish CRE-2 (enk −88C,−89A) shows little protection even at the highest concentration of protein (Fig. 3 a). Thus CRE-2 alone is sufficient for binding and is necessary for CREB binding to either CRE. Binding, however, does not correlate with efficient transcription. Other mutants, which differ less from the native, are likely to be more informative as to the importance of individual bases in the action of the CREs. Thus, we have analyzed for binding those plasmids with least disruption of the CREs. Mutations in four plasmids (enk −98T, enk −105G,−106A, enk −95T, and enk −95A) do not modify CRE-2 and do not create possible new CREB-binding sites in CRE-1. Two of these four plasmids show diminished response (Fig. 2, enk −98T and enk −105G,−106A), while two of them are more responsive transcriptionally (Fig. 2, enk −95T and enk −95A). Only one of this group (enk −98T) has a change in either of the CREs (Fig. 1 b). The plasmids were incubated with CREB protein over a 30-fold range and then analyzed for protection from DNase I digestion. Binding was detected at all of the enhancers, but the depth and shape of the protected area along the top strand differed among these mutant plasmids and in comparison to the native (Fig.3 b). There was no greater protection at low levels of CREB in those mutants that were more active transcriptionally (enk −95T and enk −95A) as compared with those that were less active (enk −98T and enk −105G,−106A). On the contrary, at the lowest level of protein (second lane from left in each set), enk −105G,−106A showed greater protection around CRE-2 than the native, enk −95T or enk −95A (lower rectangles along the gels in Fig. 3 b). The protected area in mutant plasmid enk subCRE1 (Fig.3 a) is a useful reference for protection of CRE-2, since enk subCRE1 lacks CRE-1 and cannot form a stem-loop structure (Fig. 1 b). Thus the area from −78 through −95 can be protected along the top strand by binding to CRE-2 (Fig.3 a). The footprint in plasmid enk −98T is similar to that in plasmid enk subCRE1. That is, there is protection of CRE-2 but little within CRE-1 (upper rectangle along enk −98T in Fig. 3 b), even at the highest levels of protein. In mutant plasmid enk −105G,−106A, which has a diminished response to forskolin as compared with native, there is a footprint over CRE-1 (along upper rectangles in Fig. 3 b). In summary, more efficient binding of CREB protein alone does not correlate with stronger transcriptional response. Switching between structures cannot be detected in the absence of protein (Fig.3 b, 0 protein lane). Because CREB can bind to the site in the cruciform enhancer and protect from DNase I digestion, it is a probe for the cruciform. CREB binds to the linear duplex site as well, but the pattern of protection differs between the two sites (Fig.4). Binding to the linear duplex site should protect bases only in CRE-2 on both strands (Fig. 4, left side); binding to the cruciform should protect bases within both CRE-1 and CRE-2 but only on the top strand (Fig. 4, right side). Switching creates one site as it eliminates the other (Fig.1 a, Fig. 4, top). We have designed mutations to alter the stability of the cruciform, but the mutations have little to no influence on duplex stability. A single binding reaction might, therefore, contain some molecules with linear duplex enhancer and others with cruciform enhancer. Because protection in CRE-1 results only from binding to the cruciform site, increasing protection there indicates switching to the cruciform (Fig. 4, predicted patterns of protection). The pattern of protection in the mutant enk subCRE1 (Fig.3 a) can serve as a reference. The removal of the palindromic character of the region makes formation of the cruciform site impossible, but CRE-2 is intact (Fig. 1 b). Protection from DNase I is limited to CRE-2 (Fig. 3 a). In contrast, the native and the mutants with single or two base changes show some protection in CRE-1, indicating that there is switching to the alternative site (Fig. 3 b). Two of the plasmids (enk −98T and enk −105G,−106A) contain cruciform destabilizing mutations; two (enk −95T and enk −95A) contain loop mutations that stabilize cruciform. All four mutant plasmids and the native share an identical CRE-2 with the enk subCRE1 mutant (Fig. 1 b). Consequently, they show protection of CRE-2 but differ in protection at CRE-1 (Fig. 3). Differences in relative amount of protection in CRE-1 and CRE-2 can indicate changes in the balance of availability of the sites. In the native plasmid, there is protection throughout CRE-1 (native, Fig. 3 b). At the lowest concentration of protein, however, CRE-1 is not protected, but CRE-2 is (Fig. 3 b). This is consistent with the prediction that CRE-2 would be protected on the top strand in both linear duplex and cruciform (that is, all molecules) (Fig. 4, left side). CRE-1, in contrast, would be protected only in those molecules in which CREB protein stabilized the alternative (cruciform) binding site (Fig. 4, right side). Direct comparison of the native with each of the mutants shows that mutations affect protection of CRE-2 and CRE-1. Plasmid enk −98T shows little protection in CRE-1 as compared with the other plasmids (Fig.3 b), but at 1 μl (lowest amount of added protein) protection of CRE-2 is similar to the native (Fig. 3 b; see also Table I). Thus, this mutation, which adds another mismatched base pair near the hairpin loop (Fig.1 b), shifts the balance of protection toward the linear duplex site as compared with native. The plasmid enk −105G,−106A is modified in neither CRE, so that both linear duplex and hairpin binding sites are identical to the native (Fig. 1 b). Base changes upstream from CRE-1 (Fig. 1 b, left side) destabilize hairpin structure by shortening the stem (Fig. 1 b, right side). There is in enk −105G,−106A protection in CRE-1, so that a cruciform site is present. At the lowest concentration of protein there is greater protection throughout CRE-2, as compared with the native.Table IStrength of bands in DNase I protection assayBasenativeenk -98Tenk -105G–106Aenk -95Tenk -95ABase017.530017.530017.530017.530017.530−127275286 (1.04)268 (0.97)280 (1.02)372338 (0.91)361 (0.97)347 (0.94)159169 (1.06)157 (0.99)188 (1.18)295284 (0.96)263 (0.89)283 (0.96)309329 (1.06)303 (0.98)272 (0.88)−127−119691743 (1.08)690 (1.00)732 (1.06)946933 (0.99)965 (1.02)952 (1.01)744744 (1.00)759 (1.02)728 (0.98)13991280 (0.91)1399 (1.00)1369 (0.98)15481558 (1.01)1533 (0.99)1538 (0.99)−119−116154157 (1.02)157 (1.02)117 (0.76)219252 (1.15)212 (0.97)167 (0.76)175184 (1.05)163 (0.93)175 (1.00)338317 (0.94)297 (0.88)194 (0.57)385384 (1.00)347 (0.90)290 (0.75)−116−113196199 (1.02)204 (1.04)117 (0.60)261302 (1.16)274 (1.05)144 (0.55)291284 (0.98)207 (0.71)220 (0.76)414391 (0.94)327 (0.79)219 (0.53)428467 (1.09)342 (0.80)304 (0.71)−113−104149149 (1.00)203 (1.36)29 (0.19)189214 (1.13)316 (1.67)161 (0.85)155151 (0.97)78 (0.50)87 (0.56)198175 (0.88)135 (0.68)40 (0.20)238246 (1.03)171 (0.72)63 (0.26)−104−103362407 (1.12)489 (1.35)103 (0.28)456505 (1.11)720 (1.58)405 (0.89)845711 (0.84)220 (0.26)58 (0.07)532476 (0.89)362 (0.68)151 (0.28)683706 (1.03)533 (0.78)96 (0.14)−103−101373426 (1.14)414 (1.11)353 (0.95)489516 (1.06)694 (1.42)561 (1.15)347296 (0.85)257 (0.74)358 (1.03)556492 (0.88)428 (0.77)429 (0.77)627611 (0.97)633 (1.01)695 (1.03)−101−99251245 (0.98)173 (0.69)82 (0.33)190191 (1.01)188 (0.99)177 (0.93)583545 (0.93)630 (1.08)617 (1.06)367333 (0.91)305 (0.83)262 (0.71)412394 (0.96)293 (0.71)172 (0.42)−99−95432411 (0.95)52 (0.12)52 (0.12)305289 (0.95)67 (0.22)67 (0.22)438257 (0.59)39 (0.09)85 (0.19)578394 (0.68)121 (0.21)96 (0.17)492396 (0.80)103 (0.21)103 (0.21)−95−91558421 (0.75)61 (0.11)40 (0.07)651508 (0.78)78 (0.12)32 (0.05)557333 (0.60)56 (0.10)29 (0.05)14171072 (0.76)156 (0.11)72 (0.05)17501321 (0.75)179 (0.10)48 (0.03)−91−90308220 (0.71)37 (0.12)20 (0.07)369298 (0.81)62 (0.17)39 (0.11)338177 (0.52)27 (0.08)43 (0.13)452452 (1.00)50 (0.11)72 (0.16)500381 (0.76)80 (0.16)59 (0.12)−90−88213179 (0.84)68 (0.32)86 (0.40)376395 (1.05)109 (0.29)100 (0.27)286227 (0.79)74 (0.26)198 (0.69)641463 (0.72)224 (0.35)184 (0.29)768637 (0.83)247 (0.32)244 (0.32)−88−87367192 (0.52)33 (0.09)12 (0.03)343224 (0.65)41 (0.12)53 (0.15)262103 (0.39)18 (0.07)64 (0.24)120102 (0.85)35 (0.29)19 (0.16)375292 (0.79)79 (0.21)66 (0.18)−87−86130120 (0.92)25 (0.19)34 (0.26)214174 (0.81)62 (0.29)60 (0.28)156133 (0.85)39 (0.25)86 (0.55)184134 (0.73)39 (0.21)77 (0.42)266234 (0.88)89 (0.33)36 (0.14)−86−81587446 (0.76)117 (0.20)103 (0.18)683492 (0.72)157 (0.23)183 (0.27)505255 (0.50)91 (0.18)189 (0.37)660457 (0.69)198 (0.30)197 (0.30)807667 (0.83)235 (0.29)146 (0.18)−81−78584449 (0.77)222 (0.38)249 (0.42)745694 (0.93)313 (0.42)278 (0.37)628383 (0.61)226 (0.36)311 (0.50)824655 (0.79)404 (0.49)496 (0.60)992826 (0.83)440 (0.44)437 (0.44)−78 Open table in a new tab Both mutations at −95 (in plasmids enk −95T and enk −95A) enhance the transcriptional response (Fig. 2) and within synthetic enhancers can increase the stability of hairpin structures (17Gacy A.M. McMurray C.T. Biochemistry. 1994; 33: 11951-11959Crossref PubMed Scopus (23) Google Scholar, 18.Gacy, A. M. (1996) The Role of DNA Hairpin Structure in the Regulation of Transcription and Replication. Ph.D. thesis, Mayo Graduate School.Google Scholar). Plasmids with each of these mutations show protection within CRE-2 that is similar to the native (compare 1 μl, Fig. 3 b, second lane, and Table I) but show greater protection in CRE-1 (which is protected only when protein is bound to the cruciform site) than the native (Fig.3 b, Table I, 7.5 μl). Thus, within the plasmids, the single change at −95 increases the availability of the hairpin binding site and increases the response to cAMP. This is consistent with previous observations that availability of the cruciform site correlates with enhanced expression (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar, 4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar, 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We considered the possibility that the loop mutants (enk −95T and enk −95A) might show different pattern of protection due to use of a different site. However, for these mutants, as for the native, binding depends on the presence of a functional CRE-2 (Fig.5). For gel shift analysis, probes were synthesized as 74-base oligos containing complementary proenkephalin sequence (−78 to −112) connected by a 4-T loop (Fig. 5 a). One oligonucleotide contained the mutations that abolish CRE-1 to force formation of a linear duplex (CRE-2-only) probe (enk subCRE1, Fig. 1 b, Fig. 5 a). One probe contained an insert of (TA)11T in the portion complementary to the 23-base pair palindrome to force hairpin formation and permit only the alternative binding site (forced hairpin, Fig. 5 a). Binding to four of the probes is similar, but elimination of CRE-2 abolishes binding (Fig. 5 b, enk −95A/−88C,−89A), consistent with what is seen in enk −88C,−89A plasmid (Fig.3 a). Protection in CRE-1 on the top strand indicates presence of the cruciform enhancer (Fig. 4, right side), and change in shape of the top strand footprint is direct evidence that the mutations affect enhancer structure (Fig. 3 b). Protection along the bottom strand provides additional evidence about structural switching. Bottom strand protection (Fig. 6) is a result of CREB's binding to molecules with the linear duplex enhancer (Fig. 4, left side). Protection of the bottom strand is limited to CRE-2 in native as well as in hyporesponsive (enk −98T) or hyperresponsive (enk −95A) mutants (Fig. 6). This is consistent with bottom strand protection resulting only from binding to the linear duplex site (Fig. 4, left side) and with the formation of the cruciform site from sequences on the top strand only (Fig. 4,right side). Stabilizing cruciform (enk −95A) or destabilizing cruciform (enk −98T) changes the shape of the top strand but not of the bottom strand footprint. Thus, in these plasmids, two distinct, double-stranded binding sites can be detected, one in the linear duplex and the other within a cruciform. Changing the relative availability of the sites by mutation modifies transcriptional response. In the brain, transcriptional control of the proenkephalin gene depends on CREB interaction with CRE-1 and CRE-2 (8Konradi C. Kobierski L.A. Nguyen T.V. Heckers S. Hyman S.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7005-7009Crossref PubMed Scopus (145) Google Scholar, 9Borsook D. Konradi C. Falkowski O. Comb M. Hyman S.E. Mol. Endocrinol. 1994; 8: 240-248PubMed Google Scholar, 10Konradi C. Cole R.L. Heckers S. Hyman S.E. J. Neurosci. 1994; 14: 5623-5634Crossref PubMed Google Scholar). The gene for another opioid peptide precursor (prodynorphin) is also regulated by CREB, at least in part via a CRE with a sequence identical to proenkephalin CRE-2 (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 12Collins-Hicok J. Lin L. Spiro C. Laybourn P.J. Tschumper R. Rapacz B. McMurray C.T. Mol. Cell. Biol. 1994; 14: 2837-2848Crossref PubMed Scopus (49) Google Scholar, 20Cole R.L. Konradi C. Douglass J. Hyman S.E. Neuron. 1995; 14: 813-823Abstract Full Text PDF PubMed Scopus (325) Google Scholar, 21Douglass J. McKinzie A.A. Pollock K.M. Mol. Endocrinol. 1994; 8: 333-344PubMed Google Scholar). However, the kinetics of the transcriptional response of these two genes differ in the same tissue and in response to the same stimulus (22Douglass J. Grimes L. Shook J. Lee P.H.K. Hong J. Mol. Brain Res. 1991; 9: 79-86Crossref PubMed Scopus (73) Google Scholar). After systemic administration of the excitatory amino acid kainic acid, prodynorphin mRNA levels in hippocampus rise rapidly, reaching a maximum at 3 h; in contrast, proenkephalin mRNA increases more gradually up to 24 h (22Douglass J. Grimes L. Shook J. Lee P.H.K. Hong J. Mol. Brain Res. 1991; 9: 79-86Crossref PubMed Scopus (73) Google Scholar). The structure of the palindromic enhancer in the proenkephalin gene may account for differences in transcriptional response. We have probed structural changes in mutant and native proenkephalin-CAT plasmids by DNase I digestion of plasmid-CREB complexes and have analyzed the transcriptional response by transient transfection. The four plasmids analyzed for relative availability of the two sites (Fig. 3 b, Table I) were designed to be as close to the native as possible, and these mutants showed protection in the region of the CREs only slightly different from the native. The changes, however, correlated with altered transcriptional response. Increase in the ratio of availability of the hairpin over the linear duplex site (when compared with the native plasmid) correlates with greater than normal response to forskolin (enk −95T and enk −95A, Figs. 2 and 3 b, Table I). Conversely, reducing the hairpin over linear duplex site ratio correlates with lower than normal response to forskolin. The mutant enk −105G,−106A shows very strong protection in CRE-2, but protection is apparent in CRE-1 as well. For the mutant enk −98T there is little protection detected in CRE-1 even at the highest amount of protein (Figs. 2 and 3 b, Table I). Although the hyporesponsive plasmids (enk −98T and enk −105G,−106A) can respond to forskolin, their response is reduced compared with the native (Fig. 2). Both sites, with bound proteins, may enhance transcription in response to second messengers but at different efficiencies. In vivo analysis of the actively transcribed proenkephalin gene has shown that only the cruciform site is detected during active transcription in C6 glioma cells (6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Thus factors other than CREB protein must be available to provide energy and stabilize the hairpin site, so that hairpin formation is greatly favored over linear duplex (Figs. 1 a and 4, right side would be favored). Affinity and gel shift analysis of nuclear extracts show that hairpin probes bind sequence and structure-specific factors not bound by linear duplex probes.2 The loop mutations described above (enk −95T and enk −95A) allow a DNA-directed shift toward availability of the hairpin site, and these mutations greatly enhance the receptor response (Fig. 2). The effect of the loop mutations indicates that both sites must be used for proper regulation of transcription. CRE-1 alone cannot independently bind transcription factors, and even several copies of CRE-1 cannot respond to receptor signals (23Tan Y. Low K.G. Boccia C. Grossman J. Comb M. Mol. Cell. Biol. 1994; 14: 7546-7556Crossref PubMed Scopus (41) Google Scholar), but CRE-1 is an essential part of the high activity binding site created by the alternative structure. Furthermore, response to CREB depends on the presence of CRE-1 (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar). We have previously hypothesized (3Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (49) Google Scholar, 4McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (46) Google Scholar, 5McMurray C.T. Juranic N. Chandrasekaran S. Macura S. Li Y. Jones R.L. Wilson W.D. Biochemistry. 1994; 33: 11960-11970Crossref PubMed Scopus (12) Google Scholar, 6Spiro C. Bazett-Jones D.P. Wu X. McMurray C.T. J. Biol. Chem. 1995; 270: 27702-27710Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) that occupancy and activity of the hairpin site was due at least in part to its higher affinity for the transcription factor CREB. The structure may, however, affect transcription in other ways as well. We have demonstrated that by mutation the availability of the sites and the transcriptional response can be altered. Binding of CREB protein by itself does not account for the properties of the two sites within the palindromic region. The DNA may play an allosteric role: orientation of bound transcription factors would be different at the two sites. Polarity has previously been shown to alter selection of co-factors and, as a consequence, the direction of transcriptional response mediated by nuclear receptors (24Kurokawa R. Soderstrom M. Horlein A. Halachmi S. Brown M. Rosenfeld M.G. Glass C.K. Nature. 1995; 377: 451-454Crossref PubMed Scopus (488) Google Scholar). In addition, the cruciform may create new binding sites. Binding of factors to these sites could stabilize the cruciform and/or contribute to recruiting and stabilizing transcriptional machinery. Footprint analysis with nuclear extracts indicates that the pattern of protection distant from the CRE is modified by mutation or competitors for binding at the CREs. 2C. Spiro, M. Ruan, and C. T. McMurray, unpublished observations. This is consistent with the structure of the binding sites playing a critical role in assembly of the transcriptional complex. The data presented here demonstrate that DNA structure plays a regulatory role in gene expression. Two distinct sites are formed by structural switching within the same 23-base pair region, and both must be used for proper transcriptional regulation of the proenkephalin gene. We thank Dr. A. M. Gacy for critical discussion throughout the study; J. Z. Yao and Dr. X. Wu for purified protein; and Dr. Y. Bao and J. Collins-Hicok for preparing the proenkephalin-CAT mutants.