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• W2168382825 abstract "The peripherin gene, encoding a neuron-specific intermediate filament protein, is transcriptionally induced when PC12 cells begin to terminally differentiate into neurons in response to nerve growth factor. Previously we identified two regulatory sequences of the peripherin gene: a proximal negative element (centered at −173), which prevents peripherin expression in undifferentiated PC12 cells, and a distal positive region (−2660 to −2308) necessary for full induction of peripherin in differentiated PC12 cells (Thompson, M., Lee, E. Lawe, D., Gizang-Ginsberg, E., and Ziff, E.(1992) Mol. Cell. Biol. 12, 2501-2513). Here we define a distal positive element (DPE, −2445 to −2337) within the distal positive region. Methylation interference footprinting of the DPE identified DNA-protein contact points at a novel inverted repeat sequence (AACCACTGGTT) and an Ets-like recognition sequence (CAGGAG). Functional analysis using site-directed mutagenesis demonstrates that both sites are necessary for the activity of the DPE. In addition, ternary complex formation at the DPE is dependent on both sites. Antibody competition assays confirm that an Ets family member participates in the DNA-protein complex. We have indirect evidence that the inverted repeat binding protein and the Ets-related protein interact directly with each other. Finally, we demonstrate that the DPE is constitutively active and that neuron-specific regulation of peripherin expression may be due to interaction with distal and proximal negative regulatory elements. The peripherin gene, encoding a neuron-specific intermediate filament protein, is transcriptionally induced when PC12 cells begin to terminally differentiate into neurons in response to nerve growth factor. Previously we identified two regulatory sequences of the peripherin gene: a proximal negative element (centered at −173), which prevents peripherin expression in undifferentiated PC12 cells, and a distal positive region (−2660 to −2308) necessary for full induction of peripherin in differentiated PC12 cells (Thompson, M., Lee, E. Lawe, D., Gizang-Ginsberg, E., and Ziff, E.(1992) Mol. Cell. Biol. 12, 2501-2513). Here we define a distal positive element (DPE, −2445 to −2337) within the distal positive region. Methylation interference footprinting of the DPE identified DNA-protein contact points at a novel inverted repeat sequence (AACCACTGGTT) and an Ets-like recognition sequence (CAGGAG). Functional analysis using site-directed mutagenesis demonstrates that both sites are necessary for the activity of the DPE. In addition, ternary complex formation at the DPE is dependent on both sites. Antibody competition assays confirm that an Ets family member participates in the DNA-protein complex. We have indirect evidence that the inverted repeat binding protein and the Ets-related protein interact directly with each other. Finally, we demonstrate that the DPE is constitutively active and that neuron-specific regulation of peripherin expression may be due to interaction with distal and proximal negative regulatory elements. The neuron-specific intermediate filament proteins are a major component of the neuronal cytoskeleton. They include NF-L, NF-M, and NF-H, as well as peripherin, α-internexin, and nestin (reviewed in (2.Liem R.K. Curr. Opin. Cell Biol. 1990; 2: 86-90Crossref PubMed Scopus (24) Google Scholar, 3.Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1124) Google Scholar, 4.Steinert P.M. Liem R.K.H. Cell. 1990; 60: 521-523Abstract Full Text PDF PubMed Scopus (141) Google Scholar)). They are expressed in different regions of the nervous system: NF-L, NF-M, and NF-H are found in all neurons, whereas α-internexin is expressed in the central nervous system(5.Ching G.Y. Liem R.K.H. J. Biol. Chem. 1991; 266: 19459-19468Abstract Full Text PDF PubMed Google Scholar), nestin in neuroepithelial stem cells(6.Lendahl U. Zimmerman L.B. Mckay R.D.G. Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2779) Google Scholar), and peripherin in the peripheral nervous system and a subset of central nervous system neurons(7.Brody B.A. Ley C.A. Parysek L.M. J. Neurosci. 1989; 9: 2391-2401Crossref PubMed Google Scholar, 8.Gorham J.D. Baker H. Kegler D. Ziff E.B. Dev. Brain Res. 1990; 57: 235-248Crossref PubMed Scopus (83) Google Scholar, 9.Leonard D.G.B. Gorham J.D. Cole P. Greene L.A. Ziff E.B. J. Cell Biol. 1988; 106: 181-193Crossref PubMed Scopus (165) Google Scholar, 10.Parysek L.M. Goldman R.D. J. Neurosci. 1988; 8: 555-563Crossref PubMed Google Scholar). During development, the expression of these genes is closely associated with the terminally differentiated neuronal phenotype(5.Ching G.Y. Liem R.K.H. J. Biol. Chem. 1991; 266: 19459-19468Abstract Full Text PDF PubMed Google Scholar, 6.Lendahl U. Zimmerman L.B. Mckay R.D.G. Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2779) Google Scholar, 8.Gorham J.D. Baker H. Kegler D. Ziff E.B. Dev. Brain Res. 1990; 57: 235-248Crossref PubMed Scopus (83) Google Scholar, 11.Escurat M. Djabali K. Gumpel M. Gros F. Portier M-M. J. Neurosci. 1990; 10: 764-784Crossref PubMed Google Scholar, 12.Troy C.M. Brown K. Greene L.A. Shelanski M.L. Neuroscience. 1991; 36: 217-237Crossref Scopus (138) Google Scholar). Identification of regulatory proteins that control the expression of these neuronal structural genes may lead to understanding of the cellular mechanism underlying neuronal differentiation. We have studied the regulation of the peripherin gene, which encodes a neuron-specific type III intermediate filament protein(9.Leonard D.G.B. Gorham J.D. Cole P. Greene L.A. Ziff E.B. J. Cell Biol. 1988; 106: 181-193Crossref PubMed Scopus (165) Google Scholar, 10.Parysek L.M. Goldman R.D. J. Neurosci. 1988; 8: 555-563Crossref PubMed Google Scholar, 13.Portier M-M. Bracht P. Croizat B. Gros J. Dev. Neurosci. 1984; 6: 215-226Crossref Scopus (48) Google Scholar, 14.Thompson M.A. Ziff E.B. Neuron. 1989; 2: 1043-1053Abstract Full Text PDF PubMed Scopus (115) Google Scholar). The peripherin gene is a late response gene expressed approximately 12 h after initiation of nerve growth factor (NGF) ( 1The abbreviations used are: NGFnerve growth factorNREnegative regulatory elementbpbase pair(s)DPEdistal positive elementEMSAelectrophoretic mobility shift assayPCRpolymerase chain reactionCATchloramphenicol acetyltransferaseTKthymidine kinaseRSVRous sarcoma virusDNRdistal negative regionSRFserum response factor.) treatment of PC12 cells, corresponding to the time when PC12 cells begin to exhibit a neuronal phenotype(9.Leonard D.G.B. Gorham J.D. Cole P. Greene L.A. Ziff E.B. J. Cell Biol. 1988; 106: 181-193Crossref PubMed Scopus (165) Google Scholar, 15.Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4859) Google Scholar, 16.Greene L.A. Tischler A.S. Adv. Cell. Neurobiol. 1982; 3: 373-414Crossref Google Scholar, 17.Leonard D.G.B. Ziff E.B. Greene L.A. Mol. Cell. Biol. 1987; 7: 3156-3167Crossref PubMed Scopus (222) Google Scholar). In vivo, peripherin expression is limited to sympathetic, parasympathetic, and sensory ganglia of the peripheral nervous system as well as a small subset of neurons in the central nervous system(7.Brody B.A. Ley C.A. Parysek L.M. J. Neurosci. 1989; 9: 2391-2401Crossref PubMed Google Scholar, 9.Leonard D.G.B. Gorham J.D. Cole P. Greene L.A. Ziff E.B. J. Cell Biol. 1988; 106: 181-193Crossref PubMed Scopus (165) Google Scholar, 10.Parysek L.M. Goldman R.D. J. Neurosci. 1988; 8: 555-563Crossref PubMed Google Scholar). Peripherin is first expressed at day 11.5 of rat embryogenesis in the newly formed sympathetic ganglia(8.Gorham J.D. Baker H. Kegler D. Ziff E.B. Dev. Brain Res. 1990; 57: 235-248Crossref PubMed Scopus (83) Google Scholar). Belecky-Adams et al.(18.Belecky-Adams T. Wight D.C. Kopchick J.J. Parysek L.M. J. Neurosci. 1993; 13: 5056-5065Crossref PubMed Google Scholar) demonstrated that a transgene containing 5.8 kilobases of peripherin 5′-flanking sequence achieved correct temporal and nervous system-specific expression in transgenic mice, although intragenic sequences contribute to correct expression in certain neuronal subtypes. These in vivo data imply that the regulatory elements located in the 5′-flanking region are capable of directing neuron-specific expression of the peripherin gene. nerve growth factor negative regulatory element base pair(s) distal positive element electrophoretic mobility shift assay polymerase chain reaction chloramphenicol acetyltransferase thymidine kinase Rous sarcoma virus distal negative region serum response factor. In order to identify the regulatory factors necessary for the cell-specific expression of peripherin, we have previously dissected its 5′-flanking sequence and localized a negative regulatory element (NRE) centered at −173 whose deletion results in elevated basal expression of the gene(1.Thompson M. Lee E. Lawe D. Gizang-Ginsberg E. Ziff E. Mol. Cell. Biol. 1992; 12: 2501-2513Crossref PubMed Scopus (37) Google Scholar). In addition, there are two positive regulatory regions required for full induction in PC12 cells treated with NGF: a distal positive region approximately 2450 bp upstream of the transcription start site and a proximal constitutive region within 111 bp of the transcription start site(1.Thompson M. Lee E. Lawe D. Gizang-Ginsberg E. Ziff E. Mol. Cell. Biol. 1992; 12: 2501-2513Crossref PubMed Scopus (37) Google Scholar). We proposed a two-step model of transcriptional activation of peripherin by NGF in which dissociation of a repressor from the protein complex at the NRE, coupled with a positive signal from the distal positive element, results in complete activation of the gene. We also showed that a multiprotein complex containing a member of the CTF/NF-1 family as the DNA-binding core protein interacts with the NRE(19.Adams A.D. Choate D.M. Thompson M. J. Biol. Chem. 1995; 270: 6975-6983Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The composition of this complex changes after NGF treatment, coincident with derepression of the peripherin gene. We have now finely mapped the distal positive element (DPE) necessary for the full expression of peripherin in NGF-treated PC12 cells. The isolated DPE is constitutively active in PC12 cells and retains significant activity in non-neuronal cells as well. We show that a distal negative element 5′ to the DPE (between −3710 and −2660) may restrict the activity of the DPE in non-neuronal cells. Here we present a detailed analysis of the minimal cis-acting sequences within the DPE that are necessary for its activity. Methylation interference footprinting of the DPE demonstrates two protein binding sites, a unique inverted repeat sequence as well as a sequence 38 bp upstream that resembles an Ets protein binding site. The ets protooncogene family is a novel class of eukaryotic sequence-specific DNA-binding proteins ((20.Le Prince D. Gegonne A. Coll J. DeTaisne C. Schneeberger A. Lagrou C. Stehelin D. Nature. 1983; 306: 395-397Crossref PubMed Scopus (331) Google Scholar), reviewed in (21.Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar, 22.Macleod K. Leprince D. Stehelin D. Trends Biochem. Sci. 1992; 17: 251-256Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 23.Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar)). The Ets proteins are related to each other based on sequence similarity in the ETS domain, which is the DNA binding domain. The Ets family proteins recognize purine-rich sequences characterized by an invariant GGA core sequence(23.Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar, 24.Nye J.A. Petersen J.M. Gunther C.V. Jonsen M.D. Graves B.J. Genes & Dev. 1992; 6: 975-990Crossref PubMed Scopus (311) Google Scholar, 25.Woods D.B. Ghysdael J. Owen M.J. Nucleic Acids Res. 1992; 20: 699-704Crossref PubMed Scopus (95) Google Scholar). Several members of the Ets family function as transcriptional activators, and a subset of these Ets family proteins are thought to require cooperation with other DNA-binding proteins for their activity ((26.Wasylyk C. Kerckaert J-P. Wasylyk B. Genes & Dev. 1992; 6: 965-974Crossref PubMed Scopus (115) Google Scholar), reviewed in (21.Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar) and (23.Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar)). In this report we present evidence that an Ets-related protein interacts with a novel inverted repeat binding protein to form a transcriptionally active complex at the DPE. Stock cultures of PC12 cells were maintained as described previously(27.Gizang-Ginsberg E. Ziff E.B. Genes & Dev. 1990; 4: 477-491Crossref PubMed Scopus (224) Google Scholar). PC12 cells grown for nuclear extract preparation were plated on plastic culture dishes coated with collagen (Celtrix Laboratories, Palo Alto, CA) at a density of 3 × 106 cells/150-mm dish. NGF was added 24 h after plating (50 ng/ml; 2.5 S, Bioproducts for Science, Indianapolis, IN). NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% defined and supplemented calf serum (HyClone, Logan, UT). Nuclear extracts were prepared from PC12 cells and NIH 3T3 cells according to the method of Dignam et al.(28.Dignam J.D. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 14: 1475-1489Crossref Scopus (9155) Google Scholar). Differentiated PC12 cells were treated with NGF (50 ng/ml) for 5 days prior to harvesting, unless otherwise indicated. The conditions for the DNA-protein binding reactions were essentially as described previously (1.Thompson M. Lee E. Lawe D. Gizang-Ginsberg E. Ziff E. Mol. Cell. Biol. 1992; 12: 2501-2513Crossref PubMed Scopus (37) Google Scholar). Competition experiments included 150-fold molar excess of unlabeled double-stranded oligonucleotides unless otherwise indicated. DNA-protein complexes were resolved on 5 or 4% polyacrylamide gels (30:0.8, acrylamide/bisacrylamide) under conditions described previously(29.Gilman M.Z. Wilson R.N. Weinberg R.A. Mol. Cell. Biol. 1986; 6: 4305-4316Crossref PubMed Scopus (301) Google Scholar). Supershift assays were performed by preincubating nuclear extract with 2 μl of polyclonal antibodies for 1.5 h at 4°C prior to the addition of the DNA probe. The anti-ets-1 antibody was raised against a peptide representing a portion of the DNA binding domain that is highly conserved among Ets family members (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-HMGI(Y) and pre-immune serum were generous gifts from Dimitris Thanos (Columbia University). The anti-human interleukin-8 was obtained from Endogen (Boston, MA). The −3710-CAT, −2660-CAT, and −2308-CAT reporter constructs were constructed as described previously (1.Thompson M. Lee E. Lawe D. Gizang-Ginsberg E. Ziff E. Mol. Cell. Biol. 1992; 12: 2501-2513Crossref PubMed Scopus (37) Google Scholar). A series of reporter constructs containing 5′ and 3′ deletions within the distal positive region were generated by PCR technology. Successively smaller segments of the distal positive region (−2660 to −2308) were synthesized by PCR using primers with HindIII tails. Reactions were run with Taq polymerase (Perkin-Elmer) for 25 amplification cycles in a DNA thermal cycler (Perkin-Elmer) as described by Saiki et al.(30.Saiki R.D. Gelfand D.H. Stoffel S. Scharf J.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13489) Google Scholar). Reaction products were cut with HindIII, gel-purified, and subcloned into the HindIII site at the 5′ boundary of the −2308-CAT construct. This introduces 10 bp of polylinker sequence between the PCR fragment and the peripherin sequence in the −2308-CAT plasmid. Comparison of the activity of the −2660-CAT plasmid produced using a −2660 to −2308 PCR fragment with the activity of the uninterrupted −2660-CAT plasmid demonstrated no significant effect of this insertion (data not shown). The orientation and sequence of the inserted fragment were confirmed by dideoxynucleotide sequencing(31.Sanger F. Coulson A.R. Barrell B.G. Smith A.J. Roe B. J. Mol. Biol. 1980; 143: 161-178Crossref PubMed Scopus (2194) Google Scholar). Plasmids with mutations in the Ets-like site, the inverted repeat site, or both sites were generated by site-directed mutagenesis according to the method of Kunkel et al.(32.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). Briefly, a 138-bp HindIII DNA fragment containing the peripherin sequence (−2446 to −2308) was subcloned into M13MP19 and used as a template for mutagenesis. In vitro mutagenesis was then performed with the following oligonucleotides: oligonucleotide I, 5′-CAATGGAGAAGGTCATGAATACTCAGTCACCTTCC-3′ (mutant inverted repeat site); and oligonucleotide II, 5′-CCCTTTAACACAGACAGTAATACTTTGCTG-3′ (mutant Ets-like site). The underlined nucleotides are the mutated ones. Clones containing a single mutation (I or II) or both mutations (I and II) were confirmed by dideoxynucleotide DNA sequencing, and the −2445 to −2308 HindIII insert was subcloned back into −2308-CAT to generate the mutant −2445-CAT constructs. Orientation of the mutant insert was confirmed by dideoxynucleotide DNA sequencing. Wild type −2445-CAT and the mutant −2445-CAT mutant constructs were used as templates for PCR to generate wild type and mutant 109-bp fragments (−2445 to −2337) with HindIII ends for use as probes and competitors in EMSAs. The wild type 109-bp fragment (−2445 to −2337) with HindIII ends was subcloned into the pBLCAT2 vector (33.Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar) to generate a hybrid peripherin-TK-CAT reporter construct. PBLCAT2 contains nucleotides −105 to +51 of the thymidine kinase (TK) promoter. The orientation and sequence of the inserted fragment was confirmed by dideoxynucleotide sequencing before use. Transient transfection of equimolar amounts of supercoiled peripherin-CAT plasmids into PC12 cells by electroporation was performed as described previously(34.Flug F. Copp R.P. Casanova J. Horowitz Z.D. Janocko L. Plotnick M. Samuels H. J. Biol. Chem. 1987; 262: 6373-6382Abstract Full Text PDF PubMed Google Scholar). After electroporation, cells in each electroporation cuvette were distributed to two collagen-coated 10-cm dishes. Cells in one plate of each pair were stimulated with NGF for a total of 44-46 h of exposure. Cells were harvested 44-48 h after the time of transfection, and CAT enzymatic assays were performed as described by Gorman et al.(35.Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5294) Google Scholar). CAT activity was normalized to β-galactosidase activity expressed by an internal standard plasmid containing the Rous sarcoma virus (RSV) promoter linked to the β-galactosidase gene. B-galactosidase activity was determined as described by An et al.(36.An G. Hidaka K. Siminovitch L. Mol. Cell. Biol. 1982; 2: 1628-1632Crossref PubMed Google Scholar). Transient transfection assays using 3T3 cells were performed by the calcium phosphate method as described by Velcich and Ziff(37.Velcich A. Ziff E.B. Cell. 1985; 40: 705-715Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The wild type 109-bp PCR fragment with HindIII ends spanning −2445 to −2337 (see above) was cloned into pBluescript II SK- (Stratagene, La Jolla, CA). The cloned plasmid was linearized with either HindIII or BamHI, treated with calf intestine alkaline phosphatase (Boehringer Mannheim), end-labeled with [32P]ATP by polynucleotide kinase (New England Biolabs, Boston, MA), and recut with the second restriction enzyme (BamHI or HindIII) to generate labeled coding or noncoding DNA strands. The labeled fragments were purified by 10% polyacrylamide gel electrophoresis and partially methylated by dimethyl sulfate as described by Baldwin et al.(38.Baldwin Jr., A.S. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates and Wiley Interscience, Brooklyn, NY1987Google Scholar). Binding reaction mixtures contained 30 μg of nuclear extract protein and 5 × 105 cpm of partially methylated probe. After EMSA, DNA was eluted in 0.5 M ammonium acetate, 1 mM EDTA from gel pieces containing bound or free DNA, as described by Thompson et al.(1.Thompson M. Lee E. Lawe D. Gizang-Ginsberg E. Ziff E. Mol. Cell. Biol. 1992; 12: 2501-2513Crossref PubMed Scopus (37) Google Scholar). The rest of the procedure was performed as described by Baldwin et al.(38.Baldwin Jr., A.S. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates and Wiley Interscience, Brooklyn, NY1987Google Scholar). Maxam and Gilbert G-specific and purine-specific reactions were run for orientation (39.Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Oligonucleotides used in DNA mobility shift assays were flanked by HindIII restriction sites, synthesized on a Milligen Biosearch Cyclone Plus DNA synthesizer, and gelpurified. Previously, a region between −2660 and −2308 of the peripherin 5′-flanking DNA was shown to be necessary for significant activity of the peripherin gene in response to NGF(1.Thompson M. Lee E. Lawe D. Gizang-Ginsberg E. Ziff E. Mol. Cell. Biol. 1992; 12: 2501-2513Crossref PubMed Scopus (37) Google Scholar). In order to define the minimal sequence necessary for activity, we used PCR technology to construct a series of peripherin-CAT reporter constructs with progressive deletions from −2660 to −2308 (Fig. 1B). The peripherin-CAT reporter constructs were transiently transfected into PC12 cells, which were subsequently treated with or without NGF. Fig. 1A summarizes their activities in PC12 cells in response to NGF. The −2660-CAT construct responds 5-fold to NGF and has 10-fold more activity than the −2308-CAT construct. There is a 7-fold drop in the activity of the −2370-CAT construct relative to the −2396-CAT construct in NGF-treated cells, defining a 26-bp sequence necessary for positive activity in response to NGF. The observation that a construct missing −2397 to −2308 (−2397 3′-CAT) responds less than 2-fold to NGF treatment confirms the importance of the region 3′ of −2396 containing the 26-bp element. The increase in activity upon deletion from −2660 to −2445 suggests the presence of an additional negative element in this regulatory region. Upon inspection, we noted that the sequence of the 26-bp region (−2396 to −2370) necessary for activity of the peripherin promoter in response to NGF contains an inverted repeat sequence, AACCACTGGTT (Fig. 2B). This is suggestive of a protein recognition element, since DNA-binding proteins are often dimers that recognize a site with dyad symmetry(40.Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2205) Google Scholar, 41.Ziff E.B. Trends Genet. 1990; 6: 69Abstract Full Text PDF PubMed Scopus (188) Google Scholar) . We attempted to perform DNA mobility shift assays (EMSAs) using a 32P-labeled oligonucleotide corresponding to this 26-bp sequence and nuclear extracts from differentiated PC12 cells. No specific DNA-protein complex was observed (data not shown). However, when nuclear extracts from differentiated PC12 cells (treated for either 12 h or 5 days with NGF) were incubated with a 109-bp fragment (-2445 to -2337) containing the 26-bp sequence and flanking DNA, a strong and specific retarded DNA-protein complex was observed (Fig. 2A, lane 1). This 109-bp sequence (-2445 to -2337) is referred to as the DPE. In EMSAs, a 100-fold molar excess of unlabeled competitor fragments containing either the 26-bp sequence or the region upstream of the 26-bp sequence (-2445 to -2397) successfully competes for binding of proteins to the radiolabeled probe (Fig. 2A, lanes 3, 4, and 5). However, competitor fragments containing either the region 3′ to the 26-bp sequence or an extraneous oligonucleotide (E-box) were significantly less effective as competitors (Fig. 2A, lanes 6 and 7). This indicates that binding is dependent on both the 26-bp element and a sequence immediately upstream. Inspection of this 5′ sequence reveals two regions with GGA motifs that form the invariant core of DNA binding sites recognized by Ets family proteins(23.Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar, 24.Nye J.A. Petersen J.M. Gunther C.V. Jonsen M.D. Graves B.J. Genes & Dev. 1992; 6: 975-990Crossref PubMed Scopus (311) Google Scholar, 25.Woods D.B. Ghysdael J. Owen M.J. Nucleic Acids Res. 1992; 20: 699-704Crossref PubMed Scopus (95) Google Scholar) . One site is centered around nucleotide -2430, and the second site is immediately upstream from the 26-bp element, around -2402. To determine where DNA-protein contacts occur within the DPE, we performed methylation interference footprinting with the 109-bp DPE probe. Fig. 3 demonstrates that two sites are contacted by proteins. First, probe molecules methylated at two Gs in the inverted repeat site and one G residue immediately 5′ are underrepresented in the retarded DNA-protein complex. A second region of protein-DNA contacts involves the GGA motif around nucleotide −2430. It is noteworthy that at this site footprinting implicates contact nucleotides in and adjacent to the GGA motif rather than the immediately 3′ GGAA, which is actually a more complete Ets protein core motif (see Fig. 3 and (23.Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar, 24.Nye J.A. Petersen J.M. Gunther C.V. Jonsen M.D. Graves B.J. Genes & Dev. 1992; 6: 975-990Crossref PubMed Scopus (311) Google Scholar, 25.Woods D.B. Ghysdael J. Owen M.J. Nucleic Acids Res. 1992; 20: 699-704Crossref PubMed Scopus (95) Google Scholar)). In order to confirm that formation of the specific DNA-protein complex depends just upon the inverted repeat site and the Ets-like site, EMSAs were performed using the same radiolabeled probe as in Fig. 2B (DPE; −2445 to −2337) and nuclear extracts from PC12 cells treated with NGF for 5 days. Competitor fragments identical in length to the DPE probe but containing mutated nucleotides in the Ets-like site, the inverted repeat site, or both sites were generated by PCR using mutant templates created by site-directed mutagenesis. Mutations introduced into the Ets-like site or the inverted repeat site were as listed for the mutant oligonucleotides (E-, I-) in Table 1. Each nucleotide shown by methylation interference footprinting to contact protein was mutated; in addition, the 3′ GGAA was mutated in the E- oligonucleotide and additional palindromic nucleotides were mutated in the I- oligonucleotide. As expected, the unlabeled DPE competitor efficiently competed away proteins binding to the radiolabeled DPE probe; however, the 109-bp fragment with mutations in both footprinted sites (E-I-) is an ineffective competitor (Fig. 3B). Both fragments mutated in only one site were able to compete for the protein complex. However, the 109-bp fragment mutated in the Ets-like site (E-I+) alone was a much more effective competitor than the 109-bp fragment mutated in the inverted repeat site alone (E+I-). This competition data confirms the dependence of specific complex formation on the two sites identified by methylation interference footprinting.Tabled 1 The sequence surrounding the footprinted GGA motif at nucleotide −2430 is similar to the consensus sites for Ets-1 and Ets-2 (22.Macleod K. Leprince D. Stehelin D. Trends Biochem. Sci. 1992; 17: 251-256Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 23.Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar, 24.Nye J.A. Petersen J.M. Gunther C.V. Jonsen M.D. Graves B.J. Genes & Dev. 1992; 6: 975-990Crossref PubMed Scopus (311) Google Scholar, 25.Woods D.B. Ghysdael J. Owen M.J. Nucleic Acids Res. 1992; 20: 699-704Crossref PubMed Scopus (95) Google Scholar) in 6 out of 10 bp, five of which are contiguous and include the GGA core. To test the hypothesis that an Ets family member participates in the formation of the specific DNA-protein complex at the DPE, antibody competition assays were performed using a polyclonal anti-ETS domain antibody. This antibody was raised against a peptide in the highly conserved DNA binding domain (ETS domain) of Ets-1 and cross-reacts with most Ets proteins (Santa Cruz Biotechnology). The −2445 to −2337 radiolabeled DPE probe was incubated with nuclear extracts from differentiated PC12 cells in the presence or absence of antibodies. The left panel of Fig. 4 shows that formation of the DNA-protein complex is disrupted by incubation of extract with anti-ETS domain antibodies prior to addition of the DNA probe (Fig. 4A, lane 2). Two antibodies of unrelated specificity (anti-HMG and anti-interleukin 8), as well as preimmune serum, have no effect on DNA-protein complex formation (Fig. 4B, lanes 2-4). A further control experiment in the right panel of Fig. 4 shows that the anti-ETS domain antibody has no effect on DNA-protein complex formation with an Sp1 probe, as expected. These results support the hypothesis that an Ets family protein binds to the DPE at a novel Ets-binding site. To determine the relative dependence of the formation of the specific DNA-protein complex on" @default.
• W2168382825 created "2016-06-24" @default.
• W2168382825 creator A5075001959 @default.
• W2168382825 creator A5088204152 @default.
• W2168382825 date "1996-03-01" @default.
• W2168382825 modified "2023-10-18" @default.
• W2168382825 title "Activity of the Distal Positive Element of the Peripherin Gene Is Dependent on Proteins Binding to an Ets-like Recognition Site and a Novel Inverted Repeat Site" @default.
• W2168382825 cites W1426173733 @default.
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