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• W2000493062 abstract "The promoter-distal half of the spacer separating the tandem Xenopus laevis rRNA genes consists of “0” and “1” repetitive elements that have been considered unimportant in polymerase I transcriptional activation. Utilizing oocyte microinjection, we now demonstrate that the 0/1 region, as well as its component 0 and 1 repeats, substantially stimulate transcription from a ribosomal promoter in cis and inhibit transcription when located in trans. Both the cis and trans responses increase linearly with increasing numbers of 0 or 1 repeats until saturation is approached. The 0/1 block and its component elements stimulate transcription in both orientations, over distances, and when placed downstream of the initiation site, properties for which the 60/81-base pair (bp) repeats have been defined as polymerase I enhancers. In their natural promoter-distal rDNA location, the 0/1 repeats can stimulate transcription from the rRNA gene promoter, above the level afforded by the intervening 60/81-bp repeats and spacer promoter. In addition, as with the 60/81-bp repeats, the 0/1 repeats bind a factor in common with the rDNA promoter. Thus, the entire X. laevis rDNA intergenic spacer (the 0 repeats, 1 repeats, spacer promoter repeats, and 60/81-bp repeats) acts together to enhance ribosomal transcription. The promoter-distal half of the spacer separating the tandem Xenopus laevis rRNA genes consists of “0” and “1” repetitive elements that have been considered unimportant in polymerase I transcriptional activation. Utilizing oocyte microinjection, we now demonstrate that the 0/1 region, as well as its component 0 and 1 repeats, substantially stimulate transcription from a ribosomal promoter in cis and inhibit transcription when located in trans. Both the cis and trans responses increase linearly with increasing numbers of 0 or 1 repeats until saturation is approached. The 0/1 block and its component elements stimulate transcription in both orientations, over distances, and when placed downstream of the initiation site, properties for which the 60/81-base pair (bp) repeats have been defined as polymerase I enhancers. In their natural promoter-distal rDNA location, the 0/1 repeats can stimulate transcription from the rRNA gene promoter, above the level afforded by the intervening 60/81-bp repeats and spacer promoter. In addition, as with the 60/81-bp repeats, the 0/1 repeats bind a factor in common with the rDNA promoter. Thus, the entire X. laevis rDNA intergenic spacer (the 0 repeats, 1 repeats, spacer promoter repeats, and 60/81-bp repeats) acts together to enhance ribosomal transcription. INTRODUCTIONEukaryotic ribosomal RNA (rRNA) constitutes 75% of total cellular RNA and is synthesized by RNA polymerase I (pol I) 1The abbreviations used are: pol Ipolymerase IIGSintergenic spacerkbkilobase(s)bpbase pair(s)DTTdithiothreitolSPspacer promoter. as a precursor to the 18, 5.8, and 28 S RNAs of the ribosome (1Sollner-Webb B. Tower J. Annu. Rev. Biochem. 1986; 55: 801-830Crossref PubMed Google Scholar). The typical eukaryotic cell contains from several hundred (in vertebrates) to several thousand (in plants) rRNA genes organized in head-to-tail tandem arrays located at one or a few chromosomal sites (2Long E.O. Dawid I.B. Annu. Rev. Biochem. 1980; 49: 727-764Crossref PubMed Scopus (1064) Google Scholar). Each rDNA repeating unit consists of the transcribed pre-rRNA region and an intergenic spacer (IGS), whose length varies considerably (e.g. from ∼2.5 kb in Saccharomyces cerevisiae (3Skryabin K.G. Eldarov M.A. Larionov V.L. Bayev A.A. Klootwijk J. de Regt V.C. Veldman G.M. Planta R.J. Georgiev O.I. Hadjiolov A. Nucleic Acids Res. 1984; 12: 2955-2968Crossref PubMed Scopus (85) Google Scholar) to ∼30 kb in humans (4Gonzalez I.L. Sylvester J.E. Genomics. 1995; 27: 320-328Crossref PubMed Scopus (144) Google Scholar)). Within a given species, and even within the individual rDNA repeats of a single organism, the length of the IGS may be polymorphic (e.g. ∼3 kb to ∼9 kb in Xenopus laevis (5Wellauer P.K. Reeder R.H. Carroll D. Brown D.D. Deutch A. Higashinakagawa T. Dawid I.B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2823-2827Crossref PubMed Scopus (110) Google Scholar)). In all cases examined, this length polymorphism is due to differences in the numbers of repetitive sequence elements (6Erickson J.M. Schmickel R.D. Am. J. Hum. Genet. 1985; 37: 311-325PubMed Google Scholar, 7Gerlach W.L. Bedbrook J.R. Nucleic Acids Res. 1979; 7: 1858-1869Crossref Scopus (1269) Google Scholar, 8Long E.O. Dawid I.B. Nucleic Acids Res. 1979; 7: 205-215Crossref PubMed Scopus (57) Google Scholar, 9Sylvester J.E. Whiteman D.A. Podolsky R. Pozsgay J.M. Respess J. Schmickel R.D. Hum. Genet. 1986; 73: 193-198Crossref PubMed Scopus (92) Google Scholar, 10Wellauer P.K. Dawid I.B. Brown D.D. Reeder R.H. J. Mol. Biol. 1976; 105: 461-486Crossref PubMed Scopus (157) Google Scholar). Indeed, all metazoan organisms for which sequence information is available have one or more types of reiterated sequence elements that constitute a substantial portion of their promoter-proximal IGSs.The IGS of X. laevis rDNA is composed almost entirely of four types of repeated elements (Fig. 1) (11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 12Moss T. Boseley P.G. Birnstiel M. Nucleic Acids Res. 1980; 8: 467-485Crossref PubMed Scopus (84) Google Scholar). The promoter-proximal portion of the IGS consists of blocks of 60/81-bp repeats (6-12 copies of 60- or 81-bp elements) that alternate with a pol I spacer promoter (SP). This unit is generally repeated two to three times/spacer, but can be repeated up to eight times. The SP is ∼90% identical to the gene promoter (−140 to −1) and the 60/81-bp repeats have ∼80% identity to 50 bp of the gene promoter (−121 to −72) (11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 13Sollner-Webb B. Reeder R.H. Cell. 1979; 18: 485-499Abstract Full Text PDF PubMed Scopus (352) Google Scholar). The promoter-distal portion of the X. laevis IGS consists of “0” repeats, 34-bp elements reiterated ∼2-10 times/spacer, and “1” repeats, 100-bp elements reiterated ∼six to nine times/spacer (11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 12Moss T. Boseley P.G. Birnstiel M. Nucleic Acids Res. 1980; 8: 467-485Crossref PubMed Scopus (84) Google Scholar, 14Botchan P. Reeder R.H. Dawid I.B. Cell. 1977; 11: 599-607Abstract Full Text PDF PubMed Scopus (90) Google Scholar).The promoter-proximal portion of the X. laevis IGS can substantially affect transcription from the rRNA gene promoter (15Busby S.J. Reeder R.H. Cell. 1983; 34: 989-996Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 16Moss T. Nature. 1983; 302: 223-228Crossref PubMed Scopus (156) Google Scholar). The 60/81-bp repeats are pol I transcriptional enhancers because they function in both orientations and over considerable distances to stimulate transcription from an rDNA promoter located in cis, relative to one on a separate DNA molecule (17Labhart P. Reeder R.H. Cell. 1984; 37: 285-289Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 18Labhart P. Reeder R.H. Nucleic Acids Res. 1985; 13: 8999-9009Crossref PubMed Scopus (17) Google Scholar, 19Reeder R.H. Roan J.G. Dunaway M. Cell. 1983; 35: 449-456Abstract Full Text PDF PubMed Scopus (97) Google Scholar). The 60/81-bp repeats can also stimulate a promoter in cis in the absence of a competitor template (20Pape L.K. Windle J.J. Mougey E.B. Sollner-Webb B. Mol. Cell. Biol. 1989; 9: 5093-5140Crossref PubMed Scopus (30) Google Scholar), and their cis-stimulatory and trans-competitive effects can each be ∼10-fold (17Labhart P. Reeder R.H. Cell. 1984; 37: 285-289Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 20Pape L.K. Windle J.J. Mougey E.B. Sollner-Webb B. Mol. Cell. Biol. 1989; 9: 5093-5140Crossref PubMed Scopus (30) Google Scholar). Although the SP does not stimulate transcription from the gene promoter by itself, it potentiates the enhancement observed from the 60/81-bp repeats, in a process that is not yet understood (21De Winter R.F.J. Moss T. Cell. 1986; 44: 313-318Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 22De Winter R.F.J. Moss T. J. Mol. Biol. 1987; 196: 813-827Crossref PubMed Scopus (34) Google Scholar).In contrast to the transcriptional effects of the promoter-proximal repetitive elements, previous studies have concluded that the promoter-distal region of the X. laevis rDNA IGS containing the 0 repeats and the 1 repeats had no appreciable effect on transcription (19Reeder R.H. Roan J.G. Dunaway M. Cell. 1983; 35: 449-456Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 22De Winter R.F.J. Moss T. J. Mol. Biol. 1987; 196: 813-827Crossref PubMed Scopus (34) Google Scholar, 23Reeder R.H. Cell. 1984; 38: 349-351Abstract Full Text PDF PubMed Scopus (149) Google Scholar). However, the assays used in these experiments were considerably less sensitive than those used to demonstrate the effects of the promoter proximal elements, and they might not have detected the effects of the 60/81-bp repeats either. This report re-examines the transcriptional role of the 0/1 repetitive elements.Following the X. laevis paradigm, the promoter-proximal repetitive elements of the rDNA IGS in mouse (24Kuhn A. Deppert U. Grummt I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7527-7531Crossref PubMed Scopus (56) Google Scholar, 25Pikaard C.S. Pape L.K. Henderson S.L. Ryan K. Paalman M. Lopata M.A. Reeder R.H. Sollner-Webb B. Mol. Cell. Biol. 1990; 10: 4816-4825Crossref PubMed Scopus (70) Google Scholar), Arabidopsis (26Doelling J.H. Gaudino R.J. Pikaard C.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7528-7532Crossref PubMed Scopus (84) Google Scholar), and Acanthamoeba (27Yang Q. Zwick M.G. Paule M.R. Nucleic Acids Res. 1994; 22: 4798-4805Crossref PubMed Scopus (17) Google Scholar), the promoter-proximal spacer promoter repeats in Drosophila (28Grimaldi G. Di Nocera P.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5502-5506Crossref PubMed Scopus (62) Google Scholar, 29Grimaldi G. Fiorentini P. Di Nocera P.P. Mol. Cell. Biol. 1990; 10: 4667-4677Crossref PubMed Scopus (31) Google Scholar) and mouse (30Paalman M.H. Henderson S.H. Sollner-Webb B. Mol. Cell. Biol. 1995; 15: 4648-4656Crossref PubMed Scopus (30) Google Scholar), and the promoter-proximal spacer promoter and/or repetitive elements in rat (31Cassidy B.G. Yang-Yen H.-F. Rothblum L.I. Mol. Cell. Biol. 1986; 6: 2766-2773Crossref PubMed Scopus (34) Google Scholar) have been found to stimulate transcription from the respective cis-located gene promoters, much as in frog. Thus, there are many examples of promoter-proximal rDNA repeats acting to stimulate pol I transcription.Using oocyte microinjection assays under conditions that can separately detect cis stimulation and trans competition, we have directly examined the effects of the promoter-distal half of the X. laevis IGS. We show that the 0 repeats, the 1 repeats, and the combined 0/1 repeats can significantly influence transcription from the rRNA gene promoter. In cis, these repeats serve as potent enhancers of both spacer promoter and gene promoter transcription, acting independent of orientation and over distances, both upstream and downstream of the initiation site. When located in trans, these promoter-distal repetitive elements instead act as inhibitors of transcription. By footprint competition, the 0 and 1 repeats were found to specifically interact with a factor that binds to the rDNA promoter; gel shift analysis indicates that the pol I transcription factor UBF can bind to these sequences. Thus, all of the criteria that establish the 60/81-bp repeats as pol I transcriptional enhancers also apply to the promoter-distal 0/1 transcriptional enhancers, indicating that virtually the entire X. laevis IGS consists of repetitive elements that enhance ribosomal transcription.DISCUSSIONThe X. laevis 0 and 1 spacer repeats are enhancers of pol I transcription. The X. laevis rDNA intergenic spacer consists almost entirely of four kinds of repeated elements. The promoter-proximal portion contains the 60/81-bp enhancer repeats and spacer promoters, both of which serve to stimulate pol I transcription, while the promoter-distal portion contains two other types of repeats, the 0 and 1 repeats (11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 23Reeder R.H. Cell. 1984; 38: 349-351Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 47Moss T. Mitchelson K. De Winter R. Oxford Survey on Eukaryotic Genes. Vol 2. Oxford University Press, Oxford, England1985: 207Google Scholar). We report here that the 0/1 repeats, as well as the individual 0 and 1 repeats, are enhancers of pol I transcription and exhibit all of the known properties of the 60/81-bp enhancers (17Labhart P. Reeder R.H. Cell. 1984; 37: 285-289Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 19Reeder R.H. Roan J.G. Dunaway M. Cell. 1983; 35: 449-456Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 20Pape L.K. Windle J.J. Mougey E.B. Sollner-Webb B. Mol. Cell. Biol. 1989; 9: 5093-5140Crossref PubMed Scopus (30) Google Scholar). The 0, 1, and 0/1 repeats stimulate transcription from a promoter when located in cis (Fig. 3, A and B), when in the reverse orientation relative to the promoter (Fig. 3B), when moved hundreds of bp upstream from the promoter (Fig. 4A), and when located within the transcribed region downstream from the promoter (Fig. 4B). They also stimulate transcription from a promoter in cis relative to that from a coinjected promoter in trans (Fig. 7A). Thus, virtually the entire multikilobase X. laevis IGS is made up of pol I enhancer repeats.The extent of cis stimulation afforded a pol I promoter by the 0 and 1 repeats increases with an increasing number of repeats until the saturation level is approached (Fig. 5). This saturation point varies between individual frogs, a phenomenon also observed with the 60/81-bp enhancers (20Pape L.K. Windle J.J. Mougey E.B. Sollner-Webb B. Mol. Cell. Biol. 1989; 9: 5093-5140Crossref PubMed Scopus (30) Google Scholar, 42Pikaard C.S. Reeder R.H. Mol. Cell. Biol. 1988; 8: 4282-4288Crossref PubMed Scopus (29) Google Scholar). Notably, the level of cis stimulation afforded by the 0 and 1 repeats is comparable to and in most frogs somewhat greater than that afforded by similarly positioned 60/81-bp enhancer repeats, on a per length basis.In oocytes from individuals in which the maximal level of enhanced transcription is very high relative to the level from the promoter alone, we also observe that the 0/1 repeats in their natural promoter-distal location in the IGS stimulate transcription from the downstream gene promoter, above the level directed by the intervening promoter-proximal 60/81-bp enhancers and spacer promoter (Fig. 7B). In the mature oocytes of other frogs, the level of gene promoter transcription appears maximal with a single 60/81-bp block and no additional stimulation from the more distal spacer promoter and 0/1 repeats is observed (data not shown). This presumably explains why previous studies concluded that the 0 and 1 repetitive elements do not exert a significant transcriptional effect (19Reeder R.H. Roan J.G. Dunaway M. Cell. 1983; 35: 449-456Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 22De Winter R.F.J. Moss T. J. Mol. Biol. 1987; 196: 813-827Crossref PubMed Scopus (34) Google Scholar, 23Reeder R.H. Cell. 1984; 38: 349-351Abstract Full Text PDF PubMed Scopus (149) Google Scholar). These conclusions were based entirely on the type of experiment of Fig. 7B, where detection of stimulation by the distal 0/1 repeats is frog-dependent; in the oocytes examined, the maximal level of transcription may have been reached in the absence of the 0/1 region. These early studies did not specifically examine the promoter-distal repeats in experiments like those of Fig. 3-7.How Might the 0 and 1 Repeats Function?In trans competition assays, the subcloned 0, 1, and 0/1 enhancer repeats inhibit transcription from a coinjected ribosomal promoter on a separate plasmid molecule (Fig. 6), as do the 60/81-bp enhancer repeats (17Labhart P. Reeder R.H. Cell. 1984; 37: 285-289Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 20Pape L.K. Windle J.J. Mougey E.B. Sollner-Webb B. Mol. Cell. Biol. 1989; 9: 5093-5140Crossref PubMed Scopus (30) Google Scholar), suggesting that both types of enhancers may bind a pol I transcription factor (see also 23Reeder R.H. Cell. 1984; 38: 349-351Abstract Full Text PDF PubMed Scopus (149) Google Scholar and 48Paule M.R. Transcription: Mechanisms and Regulation. Raven Press, Ltd., New York1994: 83Google Scholar). This hypothesis was strengthened by the finding that formation of a footprint on the rDNA promoter that is diagnostic for a stable transcription complex is specifically inhibited by the 0, 1, and 0/1 repeats (Fig. 8), as well as by the 60/81-bp repeats, but not by various nonspecific DNAs. Additionally, the 1 repeats exhibit a strong sequence identity with the upstream domain of the rRNA gene promoter (Fig. 10A; a 63-bp segment of >75% identity), suggesting that a common factor may bind to the promoter and to the enhancers. The 60/81-bp repeats show a comparable extent of sequence homology with the central region of the rRNA gene promoter (a 43-bp segment of ∼80% identity (−114 to −72); 11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 12Moss T. Boseley P.G. Birnstiel M. Nucleic Acids Res. 1980; 8: 467-485Crossref PubMed Scopus (84) Google Scholar, 13Sollner-Webb B. Reeder R.H. Cell. 1979; 18: 485-499Abstract Full Text PDF PubMed Scopus (352) Google Scholar); and an artificially polymerized version of the central region of the rRNA gene promoter can function as a pol I enhancer (49Pikaard C.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 464-468Crossref PubMed Scopus (21) Google Scholar).Fig. 10The 0 and 1 repeats have sequence identity to regions of the gene promoter and to elements in the IGSs of other Xenopus species. Panel A, the program BESTFIT of the GCG package was used to identify a >75% identity between residues −167 to −105 of the X. laevis rDNA promoter (13Sollner-Webb B. Reeder R.H. Cell. 1979; 18: 485-499Abstract Full Text PDF PubMed Scopus (352) Google Scholar) and the 1 repeats (12Moss T. Boseley P.G. Birnstiel M. Nucleic Acids Res. 1980; 8: 467-485Crossref PubMed Scopus (84) Google Scholar). Panel B, the X. borealis rDNA repeat of pXbr101A (62Labhart P. Reeder R.H. Nucleic Acids Res. 1987; 15: 3623-3624Crossref PubMed Scopus (12) Google Scholar) is schematically represented. Regions of identity between the 0 and 1 repetitive elements and repetitive elements in the spacers of X. borealis (62Labhart P. Reeder R.H. Nucleic Acids Res. 1987; 15: 3623-3624Crossref PubMed Scopus (12) Google Scholar) and X. clivii (57Bach R. Allet B. Crippa M. Nucleic Acids Res. 1981; 9: 5311-5330Crossref PubMed Scopus (56) Google Scholar) were identified as in A. BESTFIT analysis showed the X. borealis region designated 0 to have 100% identity within the 0 repeats of X. laevis, and the X. borealis region 2 to have a >70% identity within the X. laevis 1 repeats. Identities between the E region of X. borealis and the 60/81-bp repeats of X. laevis, as well as between the spacer and gene promoters of X. borealis and X. laevis have been noted (47Moss T. Mitchelson K. De Winter R. Oxford Survey on Eukaryotic Genes. Vol 2. Oxford University Press, Oxford, England1985: 207Google Scholar). Region ga contains the simple repeat GA, and all other labels are as in Fig. 1. The identity between the X. laevis 0 repeats and the X. clivii 1 repeats has been noted (47Moss T. Mitchelson K. De Winter R. Oxford Survey on Eukaryotic Genes. Vol 2. Oxford University Press, Oxford, England1985: 207Google Scholar).View Large Image Figure ViewerDownload (PPT)The pol I transcription factor UBF that acts at the rDNA promoter (see 38McStay B. Hu C.H. Pikaard C.S. Reeder R.H. EMBO J. 1991; 10: 2297-2303Crossref PubMed Scopus (69) Google Scholar, 45Bell S.P. Learned R.M. Jantzen H.-M. Tjian R. Science. 1988; 241: 1192-1197Crossref PubMed Scopus (258) Google Scholar, and 50, and references therein) is the only protein reported to bind to the 60/81-bp repeats and has been proposed to be important in mediating enhancement by the 60/81-bp repeats (25Pikaard C.S. Pape L.K. Henderson S.L. Ryan K. Paalman M. Lopata M.A. Reeder R.H. Sollner-Webb B. Mol. Cell. Biol. 1990; 10: 4816-4825Crossref PubMed Scopus (70) Google Scholar, 40Putnam C.D. Pikaard C.S. Mol. Cell. Biol. 1992; 12: 4970-4980Crossref PubMed Scopus (30) Google Scholar, 46Pikaard C.S. McStay B. Schultz M.C. Bell S.P. Reeder R.H. Genes Dev. 1989; 3: 1779-1788Crossref PubMed Scopus (78) Google Scholar). Since analogous assays show that UBF can bind similarly to the 0 and the 1 repeats (Fig. 9), UBF is also a candidate for mediating the action of the 0 and 1 enhancers. However, competition studies using footprinting, gel shift, and UV cross-linking do not reveal the extent of binding specificity for UBF to the promoter or to any of the enhancers, as is typical for pol II transcription factors (51Hu C.H. McStay B. Jeong S.W. Reeder R.H. Mol. Cell. Biol. 1994; 14: 2871-2882Crossref PubMed Scopus (47) Google Scholar; Fig. 9) 3E. B. Mougey, unpublished observation. and mutations of the 60/81-bp sequence that inhibit enhancer function can have minimal effect on UBF binding (42Pikaard C.S. Reeder R.H. Mol. Cell. Biol. 1988; 8: 4282-4288Crossref PubMed Scopus (29) Google Scholar). Furthermore, UBF appears to recognize structural features of DNA rather than a certain nucleotide sequence (51Hu C.H. McStay B. Jeong S.W. Reeder R.H. Mol. Cell. Biol. 1994; 14: 2871-2882Crossref PubMed Scopus (47) Google Scholar, 52Bell S.P. Pikaard C.S. Reeder R.H. Tjian R. Cell. 1989; 59: 489-497Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 53Copenhaver G.P. Putnam C.D. Denton M.L. Pikaard C.S. Nucleic Acids Res. 1994; 22: 2651-2657Crossref PubMed Scopus (90) Google Scholar, 54Jantzen H.-M. Admon A. Bell S.P. Tjian R. Nature. 1990; 344: 830-836Crossref PubMed Scopus (509) Google Scholar, 55Leblanc B. Read C. Moss T. EMBO J. 1993; 12: 513-525Crossref PubMed Scopus (50) Google Scholar). From such considerations (see also 49Pikaard C.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 464-468Crossref PubMed Scopus (21) Google Scholar and 56Putnam C.D. Copenhaver G.P. Denton M.L. Pikaard C.S. Mol. Cell. Biol. 1994; 14: 6476-6488Crossref PubMed Scopus (71) Google Scholar), it appears that UBF alone may not mediate transcriptional enhancement, and it remains to be proven whether UBF is indeed directly relevant to the in vivo action of pol I enhancers, both the 60/81-bp repeats and the 0 and 1 repeats.Are Promoter-distal Repetitive Enhancers More General?The promoter-distal intergenic spacer of Xenopus borealis rDNA contains extensive repetitive regions that have a high degree of sequence identity to the 0 and 1 repeats of X. laevis. There is >70% identity over a 70-bp region of a sequence that is repeated nine times in the X. borealis IGS region 2 with the X. laevis 1 repeat (Fig. 10B, bottom). There is also 100% identity between a 13-bp sequence that is repeated three times in the more promoter-distal X. borealis IGS and sequences of the X. laevis 0 repeat (Fig. 10B, top). Furthermore, there is 70% identity between a 28-bp sequence that is repeated four times in the promoter-distal X. clivii IGS (57Bach R. Allet B. Crippa M. Nucleic Acids Res. 1981; 9: 5311-5330Crossref PubMed Scopus (56) Google Scholar) and sequences of the X. laevis 0 repeat (Fig. 10B, top). These repetitive elements in the spacers of other Xenopus species that have sequence identities to the 0 and 1 repeats may also be promoter-distal enhancers of pol I transcription. Furthermore, in mouse, Drosophila, Arabidopsis, and Acanthamoeba where promoter-proximal repetitive enhancers have been demonstrated, there are also more promoter-distal repetitive elements (26Doelling J.H. Gaudino R.J. Pikaard C.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7528-7532Crossref PubMed Scopus (84) Google Scholar, 27Yang Q. Zwick M.G. Paule M.R. Nucleic Acids Res. 1994; 22: 4798-4805Crossref PubMed Scopus (17) Google Scholar, 58Arnheim N. Seperack P. Banerji J. Lang R.B. Miesfeld R. Marcu K.B. Cell. 1980; 22: 179-185Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 59Kominami R. Urano Y. Mishima Y. Muramatsu M. Nucleic Acids Res. 1981; 9: 3219-3233Crossref PubMed Scopus (49) Google Scholar, 60Simeone A. La Volpe A. Boncinelli E. Nucleic Acids Res. 1985; 13: 1089-1101Crossref PubMed Scopus (46) Google Scholar, 61Tautz D. Tautz C. Webb D. Dover G.A. J. Mol. Biol. 1987; 195: 525-542Crossref PubMed Scopus (99) Google Scholar); these might also function as promoter-distal enhancers of pol I transcription. INTRODUCTIONEukaryotic ribosomal RNA (rRNA) constitutes 75% of total cellular RNA and is synthesized by RNA polymerase I (pol I) 1The abbreviations used are: pol Ipolymerase IIGSintergenic spacerkbkilobase(s)bpbase pair(s)DTTdithiothreitolSPspacer promoter. as a precursor to the 18, 5.8, and 28 S RNAs of the ribosome (1Sollner-Webb B. Tower J. Annu. Rev. Biochem. 1986; 55: 801-830Crossref PubMed Google Scholar). The typical eukaryotic cell contains from several hundred (in vertebrates) to several thousand (in plants) rRNA genes organized in head-to-tail tandem arrays located at one or a few chromosomal sites (2Long E.O. Dawid I.B. Annu. Rev. Biochem. 1980; 49: 727-764Crossref PubMed Scopus (1064) Google Scholar). Each rDNA repeating unit consists of the transcribed pre-rRNA region and an intergenic spacer (IGS), whose length varies considerably (e.g. from ∼2.5 kb in Saccharomyces cerevisiae (3Skryabin K.G. Eldarov M.A. Larionov V.L. Bayev A.A. Klootwijk J. de Regt V.C. Veldman G.M. Planta R.J. Georgiev O.I. Hadjiolov A. Nucleic Acids Res. 1984; 12: 2955-2968Crossref PubMed Scopus (85) Google Scholar) to ∼30 kb in humans (4Gonzalez I.L. Sylvester J.E. Genomics. 1995; 27: 320-328Crossref PubMed Scopus (144) Google Scholar)). Within a given species, and even within the individual rDNA repeats of a single organism, the length of the IGS may be polymorphic (e.g. ∼3 kb to ∼9 kb in Xenopus laevis (5Wellauer P.K. Reeder R.H. Carroll D. Brown D.D. Deutch A. Higashinakagawa T. Dawid I.B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2823-2827Crossref PubMed Scopus (110) Google Scholar)). In all cases examined, this length polymorphism is due to differences in the numbers of repetitive sequence elements (6Erickson J.M. Schmickel R.D. Am. J. Hum. Genet. 1985; 37: 311-325PubMed Google Scholar, 7Gerlach W.L. Bedbrook J.R. Nucleic Acids Res. 1979; 7: 1858-1869Crossref Scopus (1269) Google Scholar, 8Long E.O. Dawid I.B. Nucleic Acids Res. 1979; 7: 205-215Crossref PubMed Scopus (57) Google Scholar, 9Sylvester J.E. Whiteman D.A. Podolsky R. Pozsgay J.M. Respess J. Schmickel R.D. Hum. Genet. 1986; 73: 193-198Crossref PubMed Scopus (92) Google Scholar, 10Wellauer P.K. Dawid I.B. Brown D.D. Reeder R.H. J. Mol. Biol. 1976; 105: 461-486Crossref PubMed Scopus (157) Google Scholar). Indeed, all metazoan organisms for which sequence information is available have one or more types of reiterated sequence elements that constitute a substantial portion of their promoter-proximal IGSs.The IGS of X. laevis rDNA is composed almost entirely of four types of repeated elements (Fig. 1) (11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 12Moss T. Boseley P.G. Birnstiel M. Nucleic Acids Res. 1980; 8: 467-485Crossref PubMed Scopus (84) Google Scholar). The promoter-proximal portion of the IGS consists of blocks of 60/81-bp repeats (6-12 copies of 60- or 81-bp elements) that alternate with a pol I spacer promoter (SP). This unit is generally repeated two to three times/spacer, but can be repeated up to eight times. The SP is ∼90% identical to the gene promoter (−140 to −1) and the 60/81-bp repeats have ∼80% identity to 50 bp of the gene promoter (−121 to −72) (11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 13Sollner-Webb B. Reeder R.H. Cell. 1979; 18: 485-499Abstract Full Text PDF PubMed Scopus (352) Google Scholar). The promoter-distal portion of the X. laevis IGS consists of “0” repeats, 34-bp elements reiterated ∼2-10 times/spacer, and “1” repeats, 100-bp elements reiterated ∼six to nine times/spacer (11Boseley P. Moss T. Mächler M. Portmann R. Birnstiel M. Cell. 1979; 17: 19-31Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 12Moss T. Boseley P.G. Birnstiel M. Nucleic Acids Res. 1980; 8: 467-485Crossref PubMed Scopus (84) Google Scholar, 14Botchan P. Reeder R.H. Dawid I.B. Cell. 1977; 11: 599-607Abstract Full Text PDF PubMed Scopus (90) Google Scholar).The promoter-proximal portion of the X. laevis IGS can substantially affect transcription from the rRNA gene promoter (15Busby S.J. Reeder R.H. Cell. 1983; 34: 989-996Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 16Moss T. Nature. 1983; 302: 223-228Crossref PubMed Scopus (156) Google Scholar). The 60/81-bp repeats are pol I transcriptional enhancers because they function in both orientations and over considerable distances to stimulate transcription from an rDNA promoter located in cis, relative to one on a separate DNA molecule (17Labhart P. Reeder R.H. Cell. 1984; 37: 285-289Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 18Labhart P. Reeder R.H. Nucleic Acids Res. 1985; 13: 8999-9009Crossref PubMed Scopus (17) Google Scholar, 19Reeder R.H. Roan J.G. Dunaway M. Cell. 1983; 35: 449-456Abstract Full Text PDF PubMed Scopus (97) Google Scholar). The 60/81-bp repeats can also stimulate a promoter in cis in the absence of a competitor template (20Pape L.K. Windle J.J. Mougey E.B. Sollner-Webb B. Mol. Cell. Biol. 1989; 9: 5093-5140Crossref PubMed Scopus (30) Google Scholar), and their cis-stimulatory and trans-competitive effects can each be ∼10-fold (17Labhart P. Reeder R.H. Cell. 1984; 37: 285-289Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 20Pape L.K. Windle J.J. Mougey E.B. Sollner-Webb B. Mol. Cell. Biol. 1989; 9: 5093-5140Crossref PubMed Scopus (30) Google Scholar). Although the SP does not stimulate transcription from the gene promoter by itself, it potentiates the enhancement observed from the 60/81-bp repeats, in a process that is not yet understood (21De Winter R.F.J. Moss T. Cell. 1986; 44: 313-318Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 22De Winter R.F.J. Moss T. J. Mol. Biol. 1987; 196: 813-827Crossref PubMed Scopus (34) Google Scholar).In contrast to the transcriptional effects of the promoter-proximal repetitive elements, previous studies have concluded that the promoter-distal region of the X. laevis rDNA IGS containing the 0 repeats and the 1 repeats had no appreciable effect on transcription (19Reeder R.H. Roan J.G. Dunaway M. Cell. 1983; 35: 449-456Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 22De Winter R.F.J. Moss T. J. Mol. Biol. 1987; 196: 813-827Crossref PubMed Scopus (34) Google Scholar, 23Reeder R.H. Cell. 1984; 38: 349-351Abstract Full Text PDF PubMed Scopus (149) Google Scholar). However, the assays used in these experiments were considerably less sensitive than those used to demonstrate the effects of the promoter proximal elements, and they might not have detected the effects of the 60/81-bp repeats either. This report re-examines the transcriptional role of the 0/1 repetitive elements.Following the X. laevis paradigm, the promoter-proximal repetitive elements of the rDNA IGS in mouse (24Kuhn A. Deppert U. Grummt I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7527-7531Crossref PubMed Scopus (56) Google Scholar, 25Pikaard C.S. Pape L.K. Henderson S.L. Ryan K. Paalman M. Lopata M.A. Reeder R.H. Sollner-Webb B. Mol. Cell. Biol. 1990; 10: 4816-4825Crossref PubMed Scopus (70) Google Scholar), Arabidopsis (26Doelling J.H. Gaudino R.J. Pikaard C.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7528-7532Crossref PubMed Scopus (84) Google Scholar), and Acanthamoeba (27Yang Q. Zwick M.G. Paule M.R. Nucleic Acids Res. 1994; 22: 4798-4805Crossref PubMed Scopus (17) Google Scholar), the promoter-proximal spacer promoter repeats in Drosophila (28Grimaldi G. Di Nocera P.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5502-5506Crossref PubMed Scopus (62) Google Scholar, 29Grimaldi G. Fiorentini P. Di Nocera P.P. Mol. Cell. Biol. 1990; 10: 4667-4677Crossref PubMed Scopus (31) Google Scholar) and mouse (30Paalman M.H. Henderson S.H. Sollner-Webb B. Mol. Cell. Biol. 1995; 15: 4648-4656Crossref PubMed Scopus (30) Google Scholar), and the promoter-proximal spacer promoter and/or repetitive elements in rat (31Cassidy B.G. Yang-Yen H.-F. Rothblum L.I. Mol. Cell. Biol. 1986; 6: 2766-2773Crossref PubMed Scopus (34) Google Scholar) have been found to stimulate transcription from the respective cis-located gene promoters, much as in frog. Thus, there are many examples of promoter-proximal rDNA repeats acting to stimulate pol I transcription.Using oocyte microinjection assays under conditions that can separately detect cis stimulation and trans competition, we have directly examined the effects of the promoter-distal half of the X. laevis IGS. We show that the 0 repeats, the 1 repeats, and the combined 0/1 repeats can significantly influence transcription from the rRNA gene promoter. In cis, these repeats serve as potent enhancers of both spacer promoter and gene promoter transcription, acting independent of orientation and over distances, both upstream and downstream of the initiation site. When located in trans, these promoter-distal repetitive elements instead act as inhibitors of transcription. By footprint competition, the 0 and 1 repeats were found to specifically interact with a factor that binds to the rDNA promoter; gel shift analysis indicates that the pol I transcription factor UBF can bind to these sequences. Thus, all of the criteria that establish the 60/81-bp repeats as pol I transcriptional enhancers also apply to the promoter-distal 0/1 transcriptional enhancers, indicating that virtually the entire X. laevis IGS consists of repetitive elements that enhance ribosomal transcription." @default.
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• W2000493062 title "Virtually the Entire Xenopus laevis rDNA Multikilobase Intergenic Spacer Serves to Stimulate Polymerase I Transcription" @default.
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