Identification of Adjacent Binding Sites for the YY1 and E4BP4 Transcription Factors in the Ovine PrP (Prion) Gene Promoter

Abstract

The PrP gene encodes the cellular isoform of the prion protein (PrPc) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrPc, exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions –287 to –263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype. The PrP gene encodes the cellular isoform of the prion protein (PrPc) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrPc, exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions –287 to –263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype. The ovine PrP gene (PRNP in human, prn-p in mice) encodes the prion protein (PrPC), 4The abbreviations used are: PrPc, cellular prion protein; TSE, transmissible spongiform encephalopathy; YY1, Yin Yang 1 transcription factor; bZIP, basic leucine zipper transcription factor; EMSA, electrophoretic mobility shift assay; NE, nuclear extract; PAR, proline-acidic amino acid-rich transcription factors. which is a crucial component of prion diseases, also known as transmissible spongiform encephalopathies (TSEs). This group of diseases include scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease in humans (1Weissmann C. Aguzzi A. Dormont D. Hunter N. British Medical Bulletin Series of Expert Reviews. Oxford University Press, Oxford2003: 321Google Scholar). The hallmark of TSEs is the aggregation of a pathological isoform PrPSc of the cellular PrPC protein (2Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar). PrPC is highly expressed in neurons of the brain and also found in many other tissues, especially the lymphoreticular system. Prn-p knock-out mice, which do not express endogenous PrPC, exhibit resistance to TSE disease (3Manson J.C. Clarke A.R. Hooper M.L. Aitchison L. McConnell I. Hope J. Mol. Neurobiol. 1994; 8: 121-127Crossref PubMed Scopus (496) Google Scholar). Transgenic mice with differing copy numbers of the prn-p gene show a clear relationship between expression level and disease progression, characterized by changes in incubation periods (4Manson J.C. Clarke A.R. McBride P.A. McConnell I. Hope J. Neurodegeneration. 1994; 3: 331-340PubMed Google Scholar). It is assumed that the TSE agent can only replicate in cells expressing PrPC, so that up- or down-regulation of the PrP promoter can have consequences for the distribution of the agent in the host and, therefore, the risk of transmission; for example, from consumption of specific animal parts or blood transfusion in humans (5Johnson R.T. Lancet Neurol. 2005; 10: 635-642Abstract Full Text Full Text PDF Scopus (174) Google Scholar). Beside its key role in disease, the physiological function of PrPC remains elusive with signal transduction, synaptic transmission, neuroprotection, and immunoregulation among the proposed properties (6Hu W. Rosenberg R.N. Stüve O. Acta Neurol. Scand. 2007; 116: 75-82Crossref PubMed Scopus (21) Google Scholar). The subtle phenotypes observed in PrPC knock-out mice are as diverse as disturbance of circadian rhythm (7Zomosa-Signoret V. Arnaud J.D. Fontes P. Alvarez-Martinez M.T. Liautard J.P. Vet. Res. 2008; 39: 9Crossref PubMed Scopus (63) Google Scholar) and abnormalities in tooth development (8Schneider K. Korkmaz Y. Addicks K. Lang H. Raab W.H. J. Endod. 2007; 33: 110-113Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A detailed understanding of the regulation of the ovine PrP gene expression will help to clarify its physiological and disease-related physiological role. Surprisingly, given its importance in the development of TSEs, very little is known about the DNA motifs involved in the regulation of the PrP gene. Regulatory sequences have been mapped to a region ∼90 base pairs upstream of the rat and bovine genes that appears to depend for activity on elements in intron 1 (9Saeki K. Matsumoto Y. Matsumoto Y. Onodera T. Biochem. Biophys. Res. Commun. 1996; 219: 47-52Crossref PubMed Scopus (32) Google Scholar, 10Inoue S. Tanaka M. Horiuchi M. Ishiguro N. Shinagawa M. J. Vet. Med. Sci. 1997; 59: 175-183Crossref PubMed Scopus (53) Google Scholar) and exon 1 (11Haigh C.L. Wright J.A. Brown D.R. J. Mol. Biol. 2007; 368: 915-927Crossref PubMed Scopus (16) Google Scholar). This upstream region is rich in consensus binding sites for SP1 that are highly conserved between species (12Premzl M. Gamulin V. BMC Genomics. 2007; 8: 1Crossref PubMed Scopus (48) Google Scholar). Two polymorphisms in the bovine PrP gene promoter have been shown to alter the binding of transcription factors RP58 and SP1, thereby changing the expression of reporter genes (13Sander P. Hamann H. Drögemüller C. Kashkevich K. Schiebel K. Leeb T. J. Biol. Chem. 2005; 280: 37408-37414Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). There are also four highly conserved sequences, designated motifs 1–4, located within the region –220 to –290 (14Westaway D. Cooper C. Turner S. Dacosta M. Carlson G.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6418-6422Crossref PubMed Scopus (91) Google Scholar). Of particular interest is the presence of a T to C polymorphism within motif 1 (–278 to –290) of the ovine PrP promoter (15O’Neill G.T. Donnelly K. Marshall E. Cairns D. Goldmann W. Hunter N. J. Anim. Breed Genet. 2003; 120: 114-123Crossref Scopus (13) Google Scholar, 16O’Neill Cairns D. Goldmann W. Toovey L. Hunter N. J. Anim. Breed. Genet. 2005; 122: 86-94Crossref PubMed Scopus (7) Google Scholar). Additionally, motif 2 (–266 to –278) contains a species-specific sequence variation where the ruminant PrP promoter contains a C (TTACGTAA), whereas the non-ruminant promoter containsaTatthe same position (TTATGTAA). The aim of this study was to identify factors that bind to the ovine PrP gene promoter and to determine their role in transcriptional regulation. Our data reveal binding of the transcription factors YY1 to motif 1 and of E4BP4 to motif 2. Whereas YY1 acted as an inhibitor but only bound to one PrP haplotype, E4BP4 acted as an activator of transcription binding with different affinities to all PrP haplotypes. DNA Probes-Complimentary pairs of oligonucleotides (Eurofins MWG, Germany) containing the conserved motif sequences were annealed forming short double-stranded DNA fragments (probes) that were radiolabeled with [γ-32P]ATP (Amersham Biosciences) using T4 polynucleotide kinase (Promega, Southampton, UK). The following probes (only top strand sequence given) were used: M1T, 5′-TAATCTTTCATTTTCTCCAT-3′; M1C, 5′-TAATCTTCCATTTTCTCCAT-3′; M2C, 5′-TTCTCCATTACGTAACGAGA-3′; M2T, 5′-TTCTCCATTATGTAACGAGA-3′; M1CM2C, 5′-TAATCTTCCATTTTCTCCATTACGTAACGAGA-3′; NC-1, 5′-ATTCGATCGGGGCGGGGCGAGC-3′. Nuclear Extracts and Electrophoretic Mobility Shift Assay probes were in binding assays with nuclear from ovine brain and murine cells or from tissues from mice and from using the nuclear were using the shift of 2 of binding NE, and to a of for To the 1 of double-stranded DNA was and for a of double-stranded DNA probes were The were in a in for 1 and to were as for the of the the was to the The was for The used were the for YY1 of and and the for E4BP4 of and factor binding sites were analyzed with the using the E. H. I. V. T. M. I. Res. PubMed Scopus Google Scholar). as in (15O’Neill G.T. Donnelly K. Marshall E. Cairns D. Goldmann W. Hunter N. J. Anim. Breed Genet. 2003; 120: 114-123Crossref Scopus (13) Google Scholar) contains a promoter to of the PrP sequence has the of in motif 1 and as in Cairns D. Goldmann W. Toovey L. Hunter N. J. Anim. Breed. Genet. 2005; 122: 86-94Crossref PubMed Scopus (7) Google Scholar). with the and cells was used to to on to This was used in of in to to The promoter was from all with oligonucleotides and were with and was by in to C in position to The four different promoter fragments were with and and and sites in The promoter sequences of the four were by of both and from brain were as 1994; PubMed Scopus Google they a of and Two ovine from the and were from a by of A of Scholar). was in from brain and and incubation with for the was and the cells were The was in and the was cells were in containing of the in were to and were to cells were by cells were in in 2 1 with the of bovine cells were and and cells were all cells were in a of cells on were with using in a containing of were for in the which was with 2 of were analyzed were were and test was for cells were with and with of was for with and was by for and were in of cells using the Assay to the 1 and motif 2 are highly conserved sequences 1) located base pairs upstream of the ovine PrP promoter transcription reporter gene of ovine promoter as used in factor binding and and conserved motifs as in (14Westaway D. Cooper C. Turner S. Dacosta M. Carlson G.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6418-6422Crossref PubMed Scopus (91) Google variants of and SP1, binding sites for transcription factors and four containing the PrP gene promoter or with in motif 1 binding or motif 2 binding in of the were used to activity and from are on the of the the presence or of the binding sequences for YY1 and were the motif 1 sequence and the motif 1 sequence for their to with ovine the with to a nuclear protein shown by the shift of the to a This binding was by the of and of with the on the of the that binding to the motif 1 was the to show a shift with the ovine a signal to the was with ovine nuclear This differential binding was also observed with both the and the probes this was using the or not A of transcription factor binding revealed the between the consensus motif of transcription factor Yin Yang 1 (YY1) and the motif 1 sequence Y. Biophys. 1997; PubMed Scopus Google Scholar). To determine YY1 was of the motif 1 an in assays with the with The of the in the of the and with The was with murine with the same not The was with ovine NE, and of the were The of the as was it was of the This that it was the transcription factor YY1 that was bound to the motif 1 The was with the and an with on the that the with the was to have been by the and not a component of the for the was from the that in the was observed the ruminant motif 2 was with the to the motif 1 probes was also analyzed with from ovine and murine brain and specific and and were the of which to be to the YY1 observed with the ovine The was using the as a agent as was that the YY1 transcription factor also to the in the ovine brain A on other has been reported with the same E. K. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a has also been Rosenberg Sci. PubMed Scopus Google Scholar). The two were not by the YY1 or the of the DNA the binding of other as yet unidentified are not to as is with the whereas to both the and probes not appears to be in nuclear from murine and not that this factor may be ruminant cattle, motif 2 sequence from the non-ruminant human, and motif 2 sequence by a base of C to T that the ruminant motif 2 was bound with by in the ovine as as in the murine 2 and the non-ruminant motif 2 was bound with by may be the same on of their electrophoretic and was by the of a of and binding was not by the of a of and that the are The same were observed with both the and probes this was using the ovine and not A for motif 2 binding was the E4BP4 transcription which the motif of PubMed Scopus Google Scholar). To determine the protein bound to motif 2 was E4BP4 an in a with ovine nuclear The assays were by the not or the of the radiolabeled two different of incubation were for or not of the was observed with ovine of the To that the for a were the was with murine in with the to the ovine a with the murine and was observed the was the shift and the were both by the of but not with and A was observed for the with the murine NE, that the E4BP4 transcription factor is to bind to both the ruminant and non-ruminant of motif 2 in a was for the with the A in electrophoretic mobility to E4BP4 was observed with from ovine and murine brain tissues and the 2 and A slight in electrophoretic was observed between the from the murine brain and 2 and and also between the and and The of these remains to be they may represent the binding of and to the same of between 1 and 2 the ovine PrP promoter, motifs 1 and 2 are and their may the pattern of transcription factor binding. The of both factors to bind the same was using a of probes that the different of motifs 1 and 2 and their on the from the motif the binding for both factors is for the of the motif 1 and the ruminant motif 2 Two were observed with the and NE, which were with the of a or a were by the of a of but were not by the of a of with one of the motif probes or a as to the of the of the motif 1 or motif 2 but not of both and only the was the of that this the binding of the YY1 transcription factor was an shift of the binding of both to the promoter sequence the same that the binding of these factors is by with this sequence motif that both motifs can four in which the was the of the PrP shown in ovine motif 2 was or in with or motif 1 both in the and to other as found in the ovine PrP cells were and activity was for The activity for motif 1 motif 2) was to A was observed in two of the other of motifs 1 and 2. A highly to was with motif 1 motif that factor YY1 a to was for motif 1 with motif 2) which appears to reveal the activator role There was between motif 1 with motif 2) and which can be as an of of the transcription factors binding to motif 1 and 2. The of short sequence motifs in the and PrP gene was first by (14Westaway D. Cooper C. Turner S. Dacosta M. Carlson G.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6418-6422Crossref PubMed Scopus (91) Google Scholar) but has been the that motif 1 and motif 2 are transcription factor binding was given to the that the for these factors may be associated with ovine and of motif 1 (15O’Neill G.T. Donnelly K. Marshall E. Cairns D. Goldmann W. Hunter N. J. Anim. Breed Genet. 2003; 120: 114-123Crossref Scopus (13) Google Scholar) and a species-specific sequence variation in motif 2. The YY1 to the 1 with nuclear from have shown that the motif 1 sequence as for and PrP not a the used in the mobility shift a base from T to C in motif which has been found as a polymorphism in (15O’Neill G.T. Donnelly K. Marshall E. Cairns D. Goldmann W. Hunter N. J. Anim. Breed Genet. 2003; 120: 114-123Crossref Scopus (13) Google Scholar, 16O’Neill Cairns D. Goldmann W. Toovey L. Hunter N. J. Anim. Breed. Genet. 2005; 122: 86-94Crossref PubMed Scopus (7) Google appears to have a on protein binding. The sequence and highly specific binding with nuclear from ovine and murine protein binding to motif 1 was by of a ovine PrP gene promoter from using T. C. L. A. T. K. N. and W. The with the to specific of the of the which us to that the factor bound to the motif 1 C is the transcription factor YY1 is a transcription factor with a of its binding sequence is located on a of mammalian gene in which it as an activator of transcription or a Y. Biophys. 1997; PubMed Scopus Google Scholar). The binding motif for YY1 contains an so that the motif 1 sequence of the PrP gene not the YY1 consensus binding whereas the motif 1 sequence a consensus YY1 binding motif E. T. Res. PubMed Scopus Google Scholar, W. D. M. Res. PubMed Scopus Google Scholar). a has been within the where a polymorphism a YY1 binding within this gene The presence of this polymorphism a in the expression of the and the that this may have a on with N. A. C. J. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the ovine PrP promoter the binding of YY1 to in and this may in to expression of YY1 is to gene transcription by with other as on of and it is that YY1 this by the DNA E. T. PubMed Scopus Google Scholar). The ovine PrP gene promoter contains a binding in to the YY1 motif only on the this SP1 a with YY1 but the SP1 and different expression regulation remains to be YY1 sites are found to or other binding and of transcription is by of other factors J. A. P.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This may be the for the ovine PrP promoter, in which the YY1 (motif 1) is to the E4BP4 (motif data from the nuclear that binding factors other YY1 may in a role. The two protein designated and proteins of that were observed with the brain nuclear will for binding with YY1 in a to the E4BP4 with other factors. Our proposed YY1 and E4BP4 therefore, be other are the of in brain from and that motif 1 can bind and this may motif 1 is conserved in all PrP promoters. assays L. 2003; PubMed Scopus Google Scholar) be used to a of the role that YY1, and these other factors in PrP gene expression in The E4BP4 to species-specific C to T sequence variation of motif 2 has been where the ruminant sequence is and the non-ruminant sequence is (14Westaway D. Cooper C. Turner S. Dacosta M. Carlson G.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6418-6422Crossref PubMed Scopus (91) Google Scholar). The ruminant motif 2 a with a factor in the ovine and murine whereas the non-ruminant a but with The ruminant motif 2 sequence contains a consensus binding for the protein E4BP4 known as nuclear which is a transcription factor of two PubMed Scopus Google Scholar). E4BP4 with the proline-acidic amino acid-rich of transcription factors PubMed Scopus Google Scholar). It has been proposed that between E4BP4 and factors for the same binding as a of S. T. T. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). Mobility shift assays of the with an and with murine and nuclear in the binding of E4BP4 to this nuclear to a or which may be to sequence changes in the of ovine the has not been with ovine it be that factor bound to motif 2 in the ovine nuclear the murine very mobility to the found in ovine nuclear the consensus binding for E4BP4 is the sequence (TTACGTAA), and this is very in transcription factor binding as will bind to one strand and top strand Sci. PubMed Scopus Google Scholar). The differential of the ovine nuclear protein for the sequence therefore, for the of a C the not alter the of the protein in the ovine nuclear not in and it therefore, be that the may have a on protein binding as the non-ruminant (TTATGTAA). this was not the it is to that the binding is as for E4BP4 to the strand which is not by but to the non-ruminant sequence This also that of motif 2 in the ovine promoter DNA is to gene regulation by The E4BP4 transcription factor has been shown to a role in the regulation of circadian PubMed Scopus Google Scholar, S. S. T. Y. H. PubMed Scopus Google Scholar). This is of interest in of the that may exhibit a role in circadian as shown for prn-p knock-out mice I. T. P. M. T. M. McBride P.A. J.C. 1996; PubMed Scopus Google Scholar) and suggested by the of a prion disease in humans in which from P. L. M. L. P. P. E. Med. PubMed Scopus Google Scholar). prn-p has been shown to be in a circadian 91: PubMed Scopus Google Scholar). The that which has in the regulation of circadian with the PrP gene promoter may for the understanding of the function of and the of TSE and E4BP4 has been shown to have in the of and in signal Y. Tanaka T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google both of which are in the of prion diseases E. M. PubMed Scopus (21) Google Scholar, A. Brown D.R. J. M. J. 1997; PubMed Google Scholar). The reporter gene with in cells that E4BP4 as activator in the context of the PrP promoter, and this activity can be by this E4BP4 and YY1 to have an to bind to their motifs, and E4BP4 or YY1 so that on all cells the is to the E4BP4 and YY1 binding this is in the in tissues remains to be Our data a role for E4BP4 in the regulation of the PrP they do not a role for other factors in these to this in data that the cellular level PrP transcription is by transcription factors that have been shown to be involved in (YY1) and regulation this cellular that the to and of TSE disease in a it is to a role this regulation by A. D. D. G.A. 2008; PubMed Scopus Google Scholar) that PrP was one of the genes in the variation of PrP expression be as yet involved in TSE infectivity it was shown in and cellular that is a between disease and PrP expression I. J.C. Turner Hooper Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar, M. J. C. S. A. T. M. J. 2008; PubMed Scopus (16) Google Scholar). YY1 was reported to as an activator of the 1) gene promoter K. C. U. A. A. S. J. PubMed Scopus Google it also transcription of protein which is involved in the and of protein N. S. V. A. T. Biochem. J. 1997; PubMed Scopus Google Scholar). by YY1 of PrP expression be as a in protein to the it have on the development of this regulation is and which that be with differing of in these diseases on PrP promoter genotype. in scrapie but also in TSEs as P. Med. 2005; Scopus Google Scholar) may reveal the importance of these PrP promoter two motifs in the PrP promoter were previously on the of their between Our data for the first that protein binding can be to these four different protein were of which identified two as being the transcription factors YY1 and The ovine motif 1 polymorphism and the PrP promoter sequence variation among between the TSE and the TSE host sheep, have an for PrP expression for The and of University of for help and on the and for with the