Transcriptional State of the Mouse Mammary Tumor Virus Promoter Can Affect Topological Domain Size in Vivo
1999; Elsevier BV; Volume: 274; Issue: 40 Linguagem: Inglês
10.1074/jbc.274.40.28590
ISSN1083-351X
AutoresPhillip R. Kramer, G. Fragoso, William D. Pennie, Han Htun, Gordon L. Hager, Richard R. Sinden,
Tópico(s)Immunotherapy and Immune Responses
ResumoUnrestrained DNA supercoiling and the number of topological domains were measured within a 1.8 megabase pair chromosomal region consisting of about 200 tandem repeats of a mouse mammary tumor virus promoter-driven ha-v-rasgene. When uninduced, unrestrained negative supercoiling was organized into 32-kilobase pair (kb) topological domains. Upon induction, DNA supercoiling throughout the region was completely relaxed. Supercoiling was detected, however, when elongation was blocked before or following induction. The formation of transcription initiation complexes upon addition of dexamethasone decreased the domain size to 16 kb. During transcription the domain size was 9 kb, the length of one repeat. These results suggest that topological domain boundaries can be “functional” in nature, being established by the formation of activated and elongating transcription complexes. Unrestrained DNA supercoiling and the number of topological domains were measured within a 1.8 megabase pair chromosomal region consisting of about 200 tandem repeats of a mouse mammary tumor virus promoter-driven ha-v-rasgene. When uninduced, unrestrained negative supercoiling was organized into 32-kilobase pair (kb) topological domains. Upon induction, DNA supercoiling throughout the region was completely relaxed. Supercoiling was detected, however, when elongation was blocked before or following induction. The formation of transcription initiation complexes upon addition of dexamethasone decreased the domain size to 16 kb. During transcription the domain size was 9 kb, the length of one repeat. These results suggest that topological domain boundaries can be “functional” in nature, being established by the formation of activated and elongating transcription complexes. Topological domains, often referred to as chromosomal loops, have been detected in many organisms including Escherichia coli(1Sinden R.R. Pettijohn D.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 224-228Crossref PubMed Scopus (199) Google Scholar), Drosophila (2Jupe E.R. Sinden R.R. Cartwright I.L. Biochemistry. 1995; 34: 2628-2633Crossref PubMed Scopus (33) Google Scholar), and humans (3Jackson D.A. Dickinson P. Cook P.R. EMBO J. 1990; 9: 567-571Crossref PubMed Scopus (204) Google Scholar) (for review see Ref. 4Freeman L.A. Garrard W.T. Crit. Rev. Eukaryotic Gene Expression. 1992; 2: 165-209PubMed Google Scholar). The formation of a topological domain requires restraint of the DNA helix such that rotation of one strand of the DNA double helix around the other is prevented. Consequently, DNA within a topological domain can contain a linking number deficit that is manifest as unrestrained superhelical energy (for review see Ref. 5Sinden R.R. DNA Structure and Function. Academic Press, San Diego1994Google Scholar). Domain boundaries may result from the attachment of the DNA at specialized sites (i.e. MARs 1The abbreviations used are:MARmatrix attachment regionSARscaffold attachment regionMMTVmouse mammary tumor virusMe2SOdimethyl sulfoxideCHEFclamped homogeneous electric fieldBPVbovine papilloma virusLTRlong terminal repeatkbkilobase pair(s)bpbase pair(s)Xlcross-linksPIPES1,4-piperazinediethanesulfonic acid or SARs) (6Cockerill P.N. Garrard W.T. Cell. 1986; 44: 273-282Abstract Full Text PDF PubMed Scopus (744) Google Scholar, 7Gasser S.M. Laemmli U.K. Cell. 1986; 46: 521-530Abstract Full Text PDF PubMed Scopus (433) Google Scholar) onto the nuclear matrix. Recently a bipartite sequence element has been identified within matrix attachment regions that may be important in chromosome organization or function via these sites (8van Drunen C.N. Sewalt R.G.A.B. Oosterling R.W. Weisbeek P.J. Smeekens S.C.M. van Driel R. Nucleic Acids Res. 1999; 27: 2924-2930Crossref PubMed Scopus (61) Google Scholar). Alternatively, topological domain boundaries may be functional in nature resulting from the attachment of functional proteins such as RNA or DNA polymerase complexes to the nuclear membrane (9Jackson D.A. Dolle A. Robertson G. Cook P.R. Cell Biol. Int. Rep. 1992; 16: 687-696Crossref PubMed Scopus (48) Google Scholar). For example, in Salmonella typhimurium membrane attachment of TetA leads to the formation of a topological domain boundary defined by the transcriptional complex where the RNA is being translated (10Chen D. Bowater R. Dorman C.J. Lilley D.M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8784-8788Crossref PubMed Scopus (65) Google Scholar). In addition, in yeast, telomeric sequences can act as functional anchor points for chromosome organization to provide blocks to the transmission of supercoils across the block (11Mirabella A. Gartenberg M.R. EMBO J. 1997; 16: 523-533Crossref PubMed Scopus (22) Google Scholar). Other DNA·enzyme complexes in bacteria, such as that created by UvrAB can also act as a boundary to supercoiling (12Koo H.S. Claassen L. Grossman L. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1212-1216Crossref PubMed Scopus (68) Google Scholar). matrix attachment region scaffold attachment region mouse mammary tumor virus dimethyl sulfoxide clamped homogeneous electric field bovine papilloma virus long terminal repeat kilobase pair(s) base pair(s) cross-links 1,4-piperazinediethanesulfonic acid DNA in living bacterial cells is organized with a linking number deficit leading to a state of unrestrained negative supercoiling (1Sinden R.R. Pettijohn D.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 224-228Crossref PubMed Scopus (199) Google Scholar,13Sinden R.R. Carlson J.O. Pettijohn D.E. Cell. 1980; 21: 773-783Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Although, DNA is negatively supercoiled in bacterial cells, not all supercoiling is unrestrained. About half the supercoils in DNA inEscherichia coli are restrained, possibly by the wrapping of DNA around histone-like proteins (14Pettijohn D.E. Pfenninger O. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1331-1335Crossref PubMed Scopus (89) Google Scholar, 15Broyles S. Pettijohn D.E. J. Mol. Biol. 1986; 187: 47-60Crossref PubMed Scopus (234) Google Scholar, 16Drlica K. Rouviere-Yaniv J. Microbiol. Rev. 1987; 51: 301-319Crossref PubMed Google Scholar, 17Drlica K. Trends Genet. 1990; 6: 433-437Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 18Drlica K. Mol. Microbiol. 1992; 6: 425-433Crossref PubMed Scopus (289) Google Scholar). Consequently, in vivo measurements of levels of supercoiling are about half that measured for the purified chromosome (19Bliska J.R. Cozzarelli N.R. J. Mol. Biol. 1987; 194: 205-218Crossref PubMed Scopus (220) Google Scholar, 20McClellan J.A. Boublikova P. Palecek E. Lilley D.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8373-8377Crossref PubMed Scopus (149) Google Scholar, 21Rahmouni A.R. Wells R.D. Science. 1989; 246: 358-363Crossref PubMed Scopus (173) Google Scholar, 22Zheng G. Kochel T. Hoepfner R.W. Timmons S.E. Sinden R.R. J. Mol. Biol. 1991; 221: 107-129Crossref PubMed Scopus (75) Google Scholar). Initial studies of supercoiling in eukaryotic cells failed to detect unrestrained supercoiling averaged over the entire chromosome (13Sinden R.R. Carlson J.O. Pettijohn D.E. Cell. 1980; 21: 773-783Abstract Full Text PDF PubMed Scopus (287) Google Scholar), presumably due to the restraint of supercoils through the organization of DNA into nucleosomes. However, analyses of individual genes inDrosophila, mouse, and human cells have revealed the presence of unrestrained supercoiling associated with gene regions (2Jupe E.R. Sinden R.R. Cartwright I.L. Biochemistry. 1995; 34: 2628-2633Crossref PubMed Scopus (33) Google Scholar,23Ljungman M. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6055-6059Crossref PubMed Scopus (95) Google Scholar, 24Jupe E.R. Sinden R.R. Cartwright I.L. EMBO J. 1993; 12: 1067-1075Crossref PubMed Scopus (67) Google Scholar, 25Ljungman M. Hanawalt P.C. Nucleic Acids Res. 1995; 23: 1782-1789Crossref PubMed Scopus (54) Google Scholar, 26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar), whereas DNA outside the functional hsp70 domain at locus 87ADrosophila was completely relaxed (2Jupe E.R. Sinden R.R. Cartwright I.L. Biochemistry. 1995; 34: 2628-2633Crossref PubMed Scopus (33) Google Scholar). However, a relationship between transcription or transcriptional activation and unrestrained supercoiling remains to be clearly established. For example, the level of supercoiling within a transcriptionally active hygromycin resistance gene introduced into different regions of the human genome can vary from highly supercoiled to relaxed (26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar). The mouse mammary tumor virus promoter provides a model system in which the nucleosomal organization and nucleosomal reorganization upon transcription activation are extremely well characterized (27Hager G.L. Archer T.K. Fragoso G. Bresnick E.H. Tsukagoshi Y. John S. Smith C.L. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 63-71Crossref PubMed Scopus (49) Google Scholar). Transcription rapidly follows the addition of the glucocorticoid hormone dexamethasone, allowing analysis of the chromatin under conditions of gene repression or activation. When stably introduced into the genome, the mouse mammary tumor virus (MMTV) promoter acquires six positioned nucleosome families (28Richard-Foy H. Hager G.L. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (450) Google Scholar), positioned over the binding sites for the glucocorticoid receptor and associated factors involved in promoter activation. Steroid hormone induction of the MMTV promoter with dexamethasone renders the DNA sequences associated with the B nucleosome family accessible to restriction endonuclease digestion (28Richard-Foy H. Hager G.L. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (450) Google Scholar). The hormone-dependent increased nucleosome accessibility is believed to be mechanistically responsible for the loading of transcription factors and the subsequent induction of transcriptional activation (29Archer T.K. Cordingley M.G. Wolford R.G. Hager G.L. Mol. Cell. Biol. 1991; 11: 688-698Crossref PubMed Scopus (294) Google Scholar, 30Archer T.K. LeFebvre P. Wolford R.G. Hager G.L. Science. 1992; 255: 1573-1576Crossref PubMed Scopus (350) Google Scholar). The increase in enzyme accessibility of nucleosome B in a stably integrated MMTV promoter is 15–20% in one mouse cell line, suggesting that transcriptional activation of the promoter occurs at similar levels (31Fragoso G. John S. Roberts M.S. Hager G.L. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar). The well characterized MMTV promoter and the availability of a cell line containing multiple tandem copies of this promoter afford an excellent system in which to study the effects of gene activation and transcriptional elongation on the level of DNA supercoiling and the number of topological domains. We have studied the change in supercoiling and topological domain size in a DNA region containing approximately 200 copies of the MMTV promoter 5′ of theha-v-ras gene to understand the role of chromatin organization and supercoiling on gene expression. The transcriptional state of the gene influences the topological domain size and the level of unrestrained supercoiling. The average domain size within the tandem array of MMTV promoter-driven genes changes upon transcriptional activation and elongation suggesting that transcription complexes are functional topological domain boundaries. Cell line 3134 contains approximately 200 copies of the 9-kb fragment (pM18D) of the plasmid pM18 (28Richard-Foy H. Hager G.L. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (450) Google Scholar) in a tandem array in a single chromosomal location. Cells were maintained as a monolayer in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. To incorporate BrdUrd into the DNA, cells were grown in Dulbecco's modified Eagle's medium + 10% fetal bovine serum containing 10 mm bromo-2-deoxyuridine. At this concentration, 45% of the thymidines were substituted with bromo-2-deoxyuridine in hamster cells (32Kaufman E.R. Davidson R.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4982-4986Crossref PubMed Scopus (68) Google Scholar). 24 h before use, cells were incubated in Dulbecco's modified Eagle's medium lacking phenol red and containing 10% charcoal-stripped serum (Hyclone). For transcriptional activation, cells were treated with dexamethasone at a concentration of 0.5 μm for 1 h, either before or after a 60-min treatment with 50 μg/ml of α-amanitin (Roche Molecular Biochemicals). Genomic DNA from approximately 1.2 × 106 3134 cells, lysed in agarose blocks, was digested overnight at 37 °C with various restriction enzymes. For clamped homogeneous electric field (CHEF) gel analysis, the digested DNAs were separated for 83 h in a 0.8% agarose gel equilibrated with 1X TAE buffer (0.04 m Tris base, 0.04 mglacial acetic acid, 0.001 m EDTA) in a Bio-Rad CHEF Mapper XA pulsed field electrophoresis system with the auto algorithm supplied by the manufacturer for optimal separation of 1 to 3 mega base pair DNAs. Following electrophoresis, location of the Hansenula wingei chromosomal marker was determined by ethidium bromide staining. Genomic DNA was transferred to a nylon membrane (Amersham Hybond-N+) by capillary action under alkali conditions. This blot was hybridized for 48 h under moderate stringency (2× SSC (0.3m NaCl, 0.03 m sodium citrate, pH 7.0); 5× Denhardt's solution (0.1% Ficoll (Mr 400,000), 0.1% polyvinyl pyrrolidone (Mr 400,000), and 0.1% bovine serum albumin); 50% deionized formamide; 1% SDS; 100 μg/ml denatured sheared salmon sperm; 55 °C) with a radioactive RNA probe derived from the 69% transforming portion of bovine papilloma virus (BPV; sense strand position 1234–3224 of BPV-1 DNA (GenBankTM accession number X02346)). The blot was washed at 42 °C in 0.2× SSC, 0.1% SDS following treatment with RNase A (25 μg/ml) and RNase T1 (10 μg/ml) at room temperature in 2× SSC. Subsequently, the blot was placed on a PhosphorImager plate to collect the radioactive signal. For field inversion gel electrophoresis, the genomic DNAs digested in plugs as described above were separated in a 1% agarose gel in 0.5× TBE buffer (0.045 m Tris base, 0.045 m boric acid, 0.001 m EDTA) using the auto algorithm feature optimal for separation of 2- to 50-kilobase pair DNA. Reference marker DNA in this case was HindIII-digested lambda DNA (range of fragments, 8.3 to 48.5 kb). The separated genomic DNA was analyzed by hybridizing to the BPV RNA probe. The S1 analyses were performed as described previously (33Pennie W.D. Hager G.L. Smith C.L. Mol. Cell. Biol. 1995; 15: 2125-2134Crossref PubMed Scopus (42) Google Scholar). Briefly, total cellular RNA was extracted by phenol-chloroform, precipitated with ethanol, and dissolved in H2O. Single-stranded MMTV probes were synthesized by primer extension in the presence of [α-32P]ATP, using an oligonucleotide priming from +85 in the long terminal repeat (LTR) (5′-TCTGGAAAGTGAAGGATAAGTGACGA), and SacI-cut pM18 as a template (end point at −105) (34Ostrowski M.C. Richard-Foy H. Wolford R.G. Berard D.S. Hager G.L. Mol. Cell. Biol. 1983; 3: 2045-2057Crossref PubMed Scopus (54) Google Scholar). The probes were purified by electrophoresis in denaturing gels, recovered by the “crush-and-soak” method, and 105 cpm were hybridized to 10 μg of RNA in 80% formamide, 0.2 m NaCl, 1 mm EDTA, 40 mm PIPES, pH 6.4, for 6–12 h at 37 °C. Hybrids were treated with 100 units of S1 nuclease for 1 h at room temperature, in 50 mm NaCl, 1 mm zinc acetate, 30 mm sodium acetate, pH 4.6, and then extracted with phenol-chloroform and precipitated with ethanol. Digestion products were resolved in denaturing 8% acrylamide sequencing gels. Visualization of the separated fragment was achieved with a PhosphorImager, and quantitation performed with the ImageQuant program (Molecular Dynamics). Preparation of nuclei, restriction enzyme treatment, and analysis by primer extension was performed as described previously (31Fragoso G. John S. Roberts M.S. Hager G.L. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar). Briefly, treated or untreated cells were scraped into cold phosphate-buffered saline and homogenized with a dounce (“A” pestle) in 0.3 m sucrose, 3 mm CaCl2, 2 mm magnesium acetate, 10 mm HEPES, pH 7.8, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1% Triton X-100. The homogenate was diluted 1:1 with digestion buffer (25% glycerol, 5 mm magnesium acetate, 10 mm HEPES, pH 7.8, 0.5 mm dithiothreitol, 0.1 mm EDTA), and centrifuged through a pad containing dilution buffer for 15 min at 1,000 × g and 4 °C. Nuclear pellets were resuspended in 25% glycerol, 5 mm HEPES, pH 7.8, 0.1 mm EDTA, 0.5 mm dithiothreitol. 10 μg of DNA equivalents of nuclei was restricted with 100 units of SacI, for 15 min at 37 °C in 50 mm NaCl, 50 mmTris-Cl, pH 8, 0.5 mm MgCl2, 1 mmβ-mercaptoethanol (35Workman J.L. Langmore J.P. Biochemistry. 1985; 24: 4731-4738Crossref PubMed Scopus (12) Google Scholar) extracted with phenol-chloroform, and precipitated with ethanol. The extracted DNA was cut to completion withDpnII before primer extension to determine the fractional cleavage. To obtain a linear amplification of the signal, primer extensions with Taq polymerase were thermally cycled in 50 mm KCl, 50 mm Tris-Cl, pH 8.8, 200 μm dNTP, 3.5 mm MgCl2, 0.1% Triton X-100, using an oligonucleotide priming from +27 in the LTR (5′-ACAAGAGGTGAATGTTAGGACTGTTGC). Extension products were extracted with phenol-chloroform and precipitated with ethanol before electrophoresis in sequencing gels and analysis in the PhosphorImager. Following growth in the presence of bromo-2-deoxyuridine, the DNA nicking was accomplished by exposure of the cells to 313-nm light using a mercury vapor lamp with a K2CrO4/NaOH filter as described previously (26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar, 36Sinden R.R. Pettijohn D.E. J. Mol. Biol. 1982; 162: 659-677Crossref PubMed Scopus (33) Google Scholar, 37Sinden R.R. Ussery D.W. Methods Enzymol. 1992; 212: 319-335Crossref PubMed Scopus (28) Google Scholar, 38Kramer P.R. Bat O. Sinden R.R. Methods Enzymol. 1999; 304: 639-650Crossref PubMed Scopus (12) Google Scholar, 39Sinden R.R. Bat O. Kramer P.R. Methods Companion Methods Enzymol. 1999; 17: 112-124Crossref Scopus (9) Google Scholar). Subsequent treatment with psoralen and 360-nm light, and DNA purification protocols were described previously (26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar). For Southern analysis, 7 μg of chromosomal DNA were digested to completion at 37 °C with 100 units of SpeI in 60 μl of restriction buffer. After digestion, DNA was purified and treated with glyoxal aldehyde and dimethyl sulfoxide (Me2SO) as described previously (26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar). DNA samples were separated on a 1% agarose gel in 10 mm sodium phosphate (pH 7.0) as described (40McMaster G.K. Carmichael G.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4835-4838Crossref PubMed Scopus (1727) Google Scholar), the gel was denatured, and the DNA was transferred to a nylon membrane (26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar). For hybridization analysis of the MMTV promoter, a 3.0-kbSpeI fragment was isolated from plasmid pM18 (28Richard-Foy H. Hager G.L. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (450) Google Scholar) and labeled with [α-32P]CTP using the random priming method (Roche Molecular Biochemicals). The membrane was hybridized at 65 °C for 12 h, washed twice with 2× SSC (300 mm NaCl, 30 mm sodium citrate, pH 7.0) containing 0.1% SDS at 65 °C for 60 min. Quantitation was completed using a Molecular Dynamics PhosphorImager using ImageQuant software. The fraction of DNA cross-linked (Fx) was calculated by dividing the area of the cross-linked peak by the sum of the area for the cross-linked and noncross-linked peaks. The cross-links per kilobase (Xl/kb) were calculated using the formula Xl/kb = −ln(1−Fx)/S, as described (24Jupe E.R. Sinden R.R. Cartwright I.L. EMBO J. 1993; 12: 1067-1075Crossref PubMed Scopus (67) Google Scholar), where S is the size of the restriction fragment (S = 3.0 kb for the SpeI fragment of pM18). RI/Nvalues represent an average of two Southern blots each from a minimum of three separate experiments. The ratio of the mean cross-linking rate in intact verses relaxed domains (RI/N = Xl/kbI/Xl/kbN) reflects the level of unrestrained supercoiling (24Jupe E.R. Sinden R.R. Cartwright I.L. EMBO J. 1993; 12: 1067-1075Crossref PubMed Scopus (67) Google Scholar). An unpaired, two-tailed, ttest was performed with these RI/N values, and a significant difference is indicated if p ≤ 0.05. The frequency of single strand breaks introduced by BrdUrd photolysis was determined as described previously (26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar) with the following modifications: 5 ml of 5–20% alkaline sucrose gradients were centrifuged at 35,000 rpm in a Beckman SW55Ti rotor for 4 h. Thirty equal volume fractions were collected from the bottom of the tube onto 3-cm square sections of a nylon membrane. After baking at 80 °C for 2 h, the membrane was hybridized with the 3.0-kb SpeI fragment32P-labeled probe of pM18D containing the MMTV promoter and v-ras gene (Fig. 1). The membrane was washed twice with 2× SSC at 65 °C for 60 min. The membrane was dried then the 3-cm sections were placed into vials with scintillation fluid and counted for radioactivity. Molecular weights of the single strand DNA fragments (Z) were calculated (41Rupp W.D. Howard-Flanders P. J. Mol. Biol. 1968; 31: 291-304Crossref PubMed Scopus (733) Google Scholar), and the number of single strand breaks per tandem array was calculated using a molecular weight of 338 g/mol bp and 8 × 106 bp per tandem array. Cell line 904.13 was originally established by transformation of C127 cells with a bovine papilloma virus vector containing the MMTV promoter driving expression of the ha-v-ras gene (Fig.1 C). Cell line 3134 was derived from the 904.13 line after a spontaneous event resulted in the integration and amplification of the episome. CHEF gel analysis (Fig.1 A) of DNA from this cell line with three different restriction enzymes, which do not cut the circular DNA construct, resulted in fragments ranging in size from 1.8 to 2.2 × 106 base pairs. Partial digestion with a single-cut enzyme produced a ladder of fragments with a repeat size of 9 kb (Fig.1 B). These data indicate that the cell contains a large tandem array with approximately 200 head to tail copies of the original 9-kb fragment (Fig. 1 C). Details of the 9-kb repeat unit are diagrammed in Fig. 1 D. A 3,026-bp SpeI fragment encompassing the MMTV promoter was used to assess the level of unrestrained supercoiling within the inserts of the tandem array (Fig. 1 D). This fragment contains the entire MMTV LTR, and most of the transcribed region of theras sequence. The percentage of MMTV templates activated by dexamethasone in this cell line was assessed by hypersensitivity toSacI cleavage, which cuts in the nucleosome B region of the promoter (Fig. 1 D) (29Archer T.K. Cordingley M.G. Wolford R.G. Hager G.L. Mol. Cell. Biol. 1991; 11: 688-698Crossref PubMed Scopus (294) Google Scholar, 31Fragoso G. John S. Roberts M.S. Hager G.L. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar, 42Bresnick E.H. Bustin M. Marsaud V. Richard-Foy H. Hager G.L. Nucleic Acids Res. 1992; 20: 273-278Crossref PubMed Scopus (183) Google Scholar). In a representative experiment, about 7–8% of the SacI sites were accessible in control cells or in cells treated with α-amanitin (Fig.2; Control andAmanitin). Treatment with dexamethasone resulted in an increase in the accessibility of this region, approximately 23% of the promoters were cut (Fig. 2; Dex). This represents an increase of 0.15–0.16 in the fractional cleavage, in agreement with previous studies (31Fragoso G. John S. Roberts M.S. Hager G.L. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar, 42Bresnick E.H. Bustin M. Marsaud V. Richard-Foy H. Hager G.L. Nucleic Acids Res. 1992; 20: 273-278Crossref PubMed Scopus (183) Google Scholar) and suggesting that 15–20% of the promoters are active at the time of the assay. In addition, treatment with dexamethasone following a 1-h pretreatment of the cells with α-amanitin resulted in a similar increase in fractional cleavage (Fig. 2; Dex + Amanitin), indicating that remodeling of the Nuc-B region occurs independently of transcription, as suggested by others (43Truss M. Bartsch J. Schelbert A. Hache R.J. Beato M. EMBO J. 1995; 14: 1737-1751Crossref PubMed Scopus (265) Google Scholar). α-Amanitin inhibits MMTV-driven transcription under our incubation conditions as illustrated by a representative experiment in Fig.3. Specific transcription, or more properly RNA accumulation, was examined with an S1 protection assay. 3134 cells treated with dexamethasone for 1 h displayed a 7–8-fold increase in RNA detected relative to uninduced cells (Fig. 3;Control and Dex), comparable with previous determinations in the parental 904.13 cell line (44Archer T.K. Lee H.L. Cordingley M.G. Mymryk J.S. Fragoso G. Berard D.S. Hager G.L. Mol. Endocrinol. 1994; 8: 568-576Crossref PubMed Scopus (85) Google Scholar). Incubation of the cells with 50 μg/ml α-amanitin for 1 h before dexamethasone treatment resulted in approximately a 1.5–1.7-fold increase in transcript levels relative to control cells (Fig. 3;Amanitin + Dex). This represents a substantial 90–95% decrease in the steady state RNA level induced by dexamethasone after 1 h. A 50–70% decrease in the basal transcription of uninduced templates was also observed in cells treated with α-amanitin without dexamethasone (Fig. 3;Amanitin); however, the quantitation in this case is not as accurate because of the low RNA levels. The rate of Me3-psoralen cross-linking to DNA depends on two parameters: the accessibility of the DNA and the level of unrestrained supercoiling in the DNA. Accessibility is dependent upon the extent of association of DNA with nucleosomes or other proteins that prevent Me3-psoralen binding (37Sinden R.R. Ussery D.W. Methods Enzymol. 1992; 212: 319-335Crossref PubMed Scopus (28) Google Scholar). Psoralen accessibility of the MMTV promoter in this mouse cell was analyzed in four sets of cells: untreated (control) cells where theha-v-ras gene was inactive; dexamethasone-treated cells that were transcriptionally active; cells treated with dexamethasone followed by α-amanitin, in which transcription elongation was allowed to proceed and then was blocked; and cells treated with α-amanitin followed by dexamethasone, in which the promoter was activated but transcription elongation was prevented. Each set of cells was treated with psoralen and exposed to increasing doses of 360-nm light. The DNA was irreversibly denatured as described under “Experimental Procedures,” and the cross-linked and noncross-linked molecules were separated on a native agarose gel (Fig.4 A). The cross-links per kilobase (Xl/kb), calculated from the intensities of the cross-linked (Xl) and noncross-linked (non-Xl) SpeI fragments (Fig.4 A), showed a linear relationship as a function of light exposure for each set of cells (Fig. 4 B). As shown in Fig.4 B, the cross-linking rates for the cell lines were linear to at least 12 kJ/m2. The molecular bases for the slight decrease in rate of binding in cells treated with dexamethasone (permeability or accessibility changes) was not investigated. In the experiments to determine the level of unrestrained negative superhelical energy, a dose of 6 kJ/m2 of 360-nm light was chosen because it is within the linear range of the cross-linking reaction. Unrestrained supercoiling was determined by comparing the Xl/kb at a constant dose of psoralen and light within the SpeI fragment before and after the chromosomal DNA was nicked by BrdUrd photolysis. In Fig. 5 A the intensity of both the Xl and non-Xl species decreased at higher doses of nicking but proportionally the Xl band decreases faster (determined by PhosphorImager analysis). Thus, the rate of cross-linking (or Xl/kbversus dose) decreased at higher doses of 313-nm light indicating the presence of unrestrained supercoiling. The measurement of supercoiling is presented as RI/N as described previously (24Jupe E.R. Sinden R.R. Cartwright I.L. EMBO J. 1993; 12: 1067-1075Crossref PubMed Scopus (67) Google Scholar, 26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar)). A value of RI/N = 1 indicates that no unrestrained supercoiling was detected, whereas anRI/N > 1 indicates the presence of negative supercoiling. All RI/N values in Fig.5 B represent the average from at least six independent determinations from three separate experiments. A moderately high level of unrestrained superhelical tension (RI/N = 1.55) was present in the promoter region in cells with an uninduced basal level of transcription of the MMTV promoter (Fig. 5 B,Control). This level of supercoiling was lower than that observed in the Drosophila hsp70 or rRNA genes (24Jupe E.R. Sinden R.R. Cartwright I.L. EMBO J. 1993; 12: 1067-1075Crossref PubMed Scopus (67) Google Scholar), but higher than that seen in a hygromycin gene in human cells (26Kramer P.R. Sinden R.R. Biochemistry. 1997; 36: 3151-3158Crossref PubMed Scopus (66) Google Scholar). After dexamethasone treatment for 1 h, the overall level of negative superhelical energy
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