A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer
2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês
10.1074/jbc.m700867200
ISSN1083-351X
AutoresCarmelo Ferrai, Davide Munari, Paolo Luraghi, Lorenza Pecciarini, Maria Giulia Cangi, Claudio Doglioni, Francesco Blasi, Massimo P. Crippa,
Tópico(s)Chromosomal and Genetic Variations
ResumoWe show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon α-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene. We show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon α-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene. Transcription regulation in eukaryotic cells is a multistep process that involves the assembly of multiprotein complexes on gene regulatory regions (1Lemon B. Tjian R. Genes Dev. 2000; 14: 2551-2569Crossref PubMed Scopus (606) Google Scholar). These regulatory regions contain two types of sequences: enhancers/silencers, which recruit a complex array of transcription factors and chromatin-modifying activities, and core promoter elements to which the general transcriptional machinery, including RNAP II, 2The abbreviations used are: RNAP II, RNA polymerase II; uPA, urokinase-type plasminogen activator; MNase, micrococcal nuclease; ChIP, chromatin immunoprecipitation; ChIP-ready chromatin, cross-linked sonicated chromatin; DAF, distinctive amplification fragment; CTD, C-terminal domain of RNAP II; CTD-P-S2/S5, C-terminal domain phosphorylated in serine 2/serine 5; HMGN1/2, high mobility group protein N1/N2; MP, minimal promoter; IVS, intervening sequence. is recruited. Understanding the molecular mechanism(s) involved in transcriptional regulation over long distances is of fundamental importance, because in most cases the activities of remote control elements are essential in turning on or off specific subsets of genes in a temporally and spatially regulated manner (2Li Q. Harju S. Peterson K.R. Trends Genet. 1999; 15: 403-408Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The main models to explain distal enhancer function invoke enhancer-promoter communication, either through protein-protein interactions resulting in the formation of DNA/chromatin loops (looping model), the free sliding of proteins recruited on the enhancer along the DNA (scanning model), or the establishment of modified chromatin domains between the enhancer and the promoter by facilitator proteins, which generate a progressive chain of higher order complexes along the chromatin fiber (3Ptashne M. 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However it is not yet clarified if the activation of transcription stems from the transient interaction of regulatory elements or through a more stable structure working as a single control unit. The uPA gene codes for a serine protease involved in the degradation of the extracellular matrix and in cell motility (16Mondino A. Blasi F. Trends Immunol. 2004; 25: 450-455Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The expression of this gene is normally dependent on the presence of inducers, but in cancer cells it is often constitutive. The regulatory region of the uPA gene contains an MP and an enhancer located 2 kb upstream (17Verde P. Boast S. Franzè A. Robbiati F. Blasi F. Nucleic Acids Res. 1988; 16: 10699-10716Crossref PubMed Scopus (103) Google Scholar). In highly invasive human prostate adenocarcinoma (PC3) cells this gene is constitutively expressed (18Ibañez-Tallon I. Ferrai C. Longobardi E. Facetti I. Blasi F. Crippa M.P. Blood. 2002; 100: 3325-3332Crossref PubMed Scopus (42) Google Scholar) and is present in multiple copies, not all of which are expressed (19Helenius M.A. Saramaki O.R. Linja M.J. Tammela T.L. Visakorpi T. Cancer Res. 2001; 61: 5340-5344PubMed Google Scholar), and the activity of the MP is modulated by Sp1 (18Ibañez-Tallon I. Ferrai C. Longobardi E. Facetti I. Blasi F. Crippa M.P. Blood. 2002; 100: 3325-3332Crossref PubMed Scopus (42) Google Scholar, 20Benasciutti E. Pagès G. Kenzior O. Folk W. Blasi F. Crippa M.P. Blood. 2004; 104: 256-262Crossref PubMed Scopus (93) Google Scholar). The uPA enhancer contains two AP1 binding sites (21Nerlov C. Rørth P. Blasi F. Johnsen M. Oncogene. 1991; 6: 1583-1592PubMed Google Scholar, 22Nerlov C. De Cesare D. Pergola F. Caracciolo A. Blasi F. Johnsen M. Verde P. EMBO J. 1992; 11: 4573-4582Crossref PubMed Scopus (142) Google Scholar, 23De Cesare D. Vallone D. Caracciolo A. Sassone-Corsi P. Nerlov C. Verde P. Oncogene. 1995; 11: 365-376PubMed Google Scholar, 24De Cesare D. Palazzolo M. Blasi F. Oncogene. 1996; 13: 2551-2562PubMed Google Scholar, 25Cirillo G. Casalino L. Vallone D. Caracciolo A. De Cesare D. Verde P. Mol. Cell. Biol. 1999; 19: 6240-6252Crossref PubMed Scopus (51) Google Scholar) and, together with the MP, is important for high expression levels of the gene (17Verde P. Boast S. Franzè A. Robbiati F. Blasi F. Nucleic Acids Res. 1988; 16: 10699-10716Crossref PubMed Scopus (103) Google Scholar, 18Ibañez-Tallon I. Ferrai C. Longobardi E. Facetti I. Blasi F. Crippa M.P. Blood. 2002; 100: 3325-3332Crossref PubMed Scopus (42) Google Scholar). In an attempt to obtain new insights into the molecular mechanism involved in enhancer-mediated gene activation, we asked if specific DNA fragments in the enhancer and MP of the uPA gene were involved in the interaction between the regulatory elements by using a modified chromatin immunoprecipitation (ChIP) approach, including an MNase digestion step prior to immunoprecipitation. This procedure allows the fine mapping of DNA sequences involved in these interactions, while maintaining the information content on their protein composition. Here we provide experimental evidences for the presence of specific structures in the MP region of the uPA gene that are associated with transcriptional events and for the interaction of one of them with the enhancer. Our results support a model of interaction between the regulatory regions that persists at least through the early steps of elongation. Cell Cultures, α-Amanitin Treatment, and Chromatin Preparation—PC3 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing a final concentration of 10% (v/v) fetal bovine serum, 0.2 mg/ml streptomycin, 20 units/ml penicillin, 2 mm glutamine, and 1 mm sodium pyruvate. α-Amanitin (Sigma) was dissolved in water, and increasing concentrations were added to the culture medium for 24 h, as previously described (26Casse C. Giannoni F. Nguyen V.T. Dubois M.F. Bensaude O. J. Biol. Chem. 1999; 274: 16097-16106Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Inhibition of uPA transcription was estimated by quantitative reverse transcription-PCR of uPA mRNA at different α-amanitin concentrations. At 10 μg/ml uPA transcription was inhibited by >90%; this concentration was used in further experiments as follows. Untreated cells or cells that were treated with α-amanitin (10 μg/ml for 24 h) were cross-linked with 1% formaldehyde for 10 min, and chromatin was prepared essentially as described (27Orlando V. Strutt H. Paro R. Methods. 1997; 11: 205-214Crossref PubMed Scopus (513) Google Scholar) by using 10 sonication cycles (35 s at 60–70 watts, in an Ultrasonic Processor XL Sonicator (Miosonix), followed by a 2-min rest on ice). Cross-linked chromatin-containing fractions were pooled and stored at –80 °C. Cross-linked chromatin was prepared from different batches of PC3 cells (two batches of untreated and two batches of α-amanitin-treated) PC3 cells. All experiments (e.g. MNase digestion and ChIP) were repeated at least twice for each batch. RNA Detection—Total RNA was extracted with an RNeasy mini kit (Qiagen), quantitated by spectrophotometry (Nanodrop), and 5 μg was reverse-transcribed using a SuperScript™ First-Strand kit using random primers (Invitrogen) according to the manufacturer's instructions. For quantitative reverse transcription-PCR, 5 ng of reverse-transcribed RNA were amplified, and the amplification products were detected using the TaqMan gene expression assay (Hs00170182_m1) primers and probes specific for uPA, in an ABI PRISM 7900HT Sequence Detection System. 18 S rRNA gene levels were examined and used to normalize the results (TaqMan gene expression assay 4319413E). To check for the presence of RNA in the enhancer and coding regions, total RNA was extracted from untreated cells, reverse transcribed as above, and 5, 10, and 20 ng of reverse transcribed products were amplified with primers corresponding to different sections of the enhancer and coding region of uPA. The PCR reaction (see below) was performed with 40 amplification cycles. Primer sequences, location with respect to the uPA sequence (17Verde P. Boast S. Franzè A. Robbiati F. Blasi F. Nucleic Acids Res. 1988; 16: 10699-10716Crossref PubMed Scopus (103) Google Scholar), and annealing temperatures used for each primer set are reported in Table 1.TABLE 1List of the primer sets used in this studyPrimer setsPrimers sequences (5′ to 3′ orientation)Primers position in uPA sequencePCR annealing temperature for primer sets°CF8TGTCCAGGAGGAAATGAAGTCATC-1981/-195857R11GAAACTCCCAGGTTAGTTATCAGG-1836/-1859F8TGTCCAGGAGGAAATGAAGTCATC-1981/-195857R12GACCAGAACATAAACAGAGATGCTG-1792/-1816F8TGTCCAGGAGGAAATGAAGTCATC-1981/-195857R14CTCTAGAAGACTGTGGTCAGTTTTG-1731/-1755F5GATTAGCGCATGGATAAGGAAGTTC-2105/-208154R14CTCTAGAAGACTGTGGTCAGTTTTG-1731/-1755F22CAGTAATCTGGCCTTGCCTTTCC-645/-62360R26GAGGAATCGAGAGGCTTGTAAATTC-181/-205F26GAATTTACAAGCCTCTCGATTCCTC-205/-18160R31GGGATCTCAGGACCGCGG+114/+97F22CAGTAATCTGGCCTTGCCTTTCC-645/-62360R31GGGATCTCAGGACCGCGG+114/+97F21CCCAATCCTTATCAAGCCCTGTC-700/-67860R26GAGGAATCGAGAGGCTTGTAAATTC-181/-205F22CAGTAATCTGGCCTTGCCTTTCC-645/-62360R27CGCAACGCTCACAAAGATTTG-114/-134F26GAATTTACAAGCCTCTCGATTCCTC-205/-18160R34ACCAGGCTCCCCAGCTGTC+304/+286F26GAATTTACAAGCCTCTCGATTCCTC-205/-18160R36GAGGTCGGGGCGCTAGACG+420/+402F26GAATTTACAAGCCTCTCGATTCCTC-205/-18160R37CAGGACGCAGAGAAGCAGG+465/+447F25GAGCTGGGCGAGGTAGAGAGTC-313/-29260R31GGGATCTCAGGACCGCGG+114/+97F7GGGAGAAAGGGTGTCACGC-2024/-200657R10GCCGTCATGATTCATGTTGCTCC-1872/-1894F3GAGGACCCCTTGAACCCAGAAG-2192/-217157R6CCGTGCCACCTCTTCACCTAGC-2043/-2064F11CCTGATAACTAACCTGGGAGTTTC-1859/-183657R15CTTCAGAGCCAACCTTGCTACTTC-1707/-1730F6GCTAGGTGAAGAGGTGGCACGG-2064/-204354R10GCCGTCATGATTCATGTTGCTCC-1872/-1894F9GCATGACAGCCTCCAGCCAAG-1942/-192257R12GACCAGAACATAAACAGAGATGCTG-1816/-1792F7GGGAGAAAGGGTGTCACGC-2024/-200654R11GAAACTCCCAGGTTAGTTATCAGG-1836/-1859F14CAAAACTGACCACAGTCTTCTAGAG-1755/-173160R26GAGGAATCGAGAGGCTTGTAAATTC-181/-205F27CAAATCTTTGTGAGCGTTGCG-134/-11457R31GGGATCTCAGGACCGCGG+114/+97F32GGGATCTCAGGACCGCGG+194/+21357R36GAGGTCGGGGCGCTAGACG+420/+402F29GCTGCAAGACAGGGGAGGGAG-85/-6560R31GGGATCTCAGGACCGCGG+114/+97F26GAATTTACAAGCCTCTCGATTCCTC-205/-18160R29CTCCCTCCCCTGTCTTGCAGC-65/-85 Open table in a new tab Micrococcal Nuclease Digestion—Cross-linked chromatin fractions were first dialyzed against 25 mm KCl, 50 mm Tris-HCl, pH 8, and then diluted with dialysis buffer to a concentration of 200 μg/ml. MNase digestions were performed in bulk by adding CaCl2 to a final concentration of 2 mm and MNase (2 units/ml, Sigma) and incubating at 37 °C. Aliquots (1 ml) were withdrawn from the digestion mixture at each time point and directly added to the digestion stop solution (1% SDS, 0.1 m NaCl, 10 mm EDTA, 10 mm EGTA, 50 mm Tris-HCl, pH 8). Digested chromatin was treated with proteinase K (500 μg/ml) for 5 h at 37 °C, cross-links were reverted by heating overnight at 65 °C, and the DNA was purified using phenol extraction and resuspended in distilled water. The DNA was then treated with RNase A (50 μg/ml) for 30 min at 37 °C, again with proteinase K (500 μg/ml) for 5 h at 55 °C, phenol-extracted, and precipitated. For the experiments shown in Figs. 2, 3, 4 and 6, the DNA was resuspended in distilled water and quantitated at the spectrophotometer (A260), and equal amounts (100 ng) of material, for each time point, were used as template in PCR reactions.FIGURE 3Detection and characterization of DAF amplicons in the MP region of the uPA gene. A, scheme (not to scale) of the amplified regions and primers used. Full arrowheads, primers (Table 1) and their orientation. Empty arrowhead, transcription start site. White and hatched boxes, amplified fragments; MP, minimal promoter; I, first, untranslated exon of the uPA gene; white and black boxes with II: untranslated and translated portions, respectively, of the second exon of the uPA gene. B, equal amounts of genomic DNA from each MNase digestion time point were amplified with the appropriate primers (panel A), and PCR products revealed on a 2% agarose gel in 0.5× TBE stained with EtBr. Amplicons F22/R26 and F26/R31 show a loss of PCR signal using material originated from early and intermediate MNase digestion time points. The signal is then recovered at later time points revealing the presence of amplicons resistant to MNase digestion. C, amplification products of the MP region using the F22/R31 primer set were fractionated and visualized as above. Amplification products are visible only at the 0- and 5-min time points, indicating that amplicons F22/R26 and F26/R31 do not belong to the same genomic fragment but represent different chromatin populations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Defining the borders of DAF-A and -B. Genomic DNA prepared as in Fig. 2 was amplified with primers located upstream and downstream of DAF-B (F25 and R34, R36, and R37, respectively) and -A (F21 and R27, respectively) in combination with the upstream or downstream primers used in Fig. 3 that define the amplification pattern of DAF-A and -B. The PCR products were fractionated on a 2% agarose gel in 0.5× TBE and visualized by EtBr staining. A, scheme of the primer sets used and of the amplified regions (not to scale). White and striped boxes are as in Fig. 3. Black box, DAF-Bx amplicon. E, enhancer; MP, promoter. I, first exon and II, second exon of the uPA gene (white box, untranslated region). B, the amplification pattern of DAF-B is extended in the coding region of the uPA gene by using primers R34 and R36 in combination with F26 and is lost using primer R37. The pattern could not be extended 5′ of DAF-B, using primer F25 (upstream of F26) in combination with R31. C, loss of the amplification pattern of DAF-A by using primer F21 in combination with R26 and F22 in combination with R27.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6The presence of DAF-A, -B, and -Bx depends on active transcription. The concentration of α-amanitin to be used in PC3 cells treatment (10 μg/ml for 24 h) was determined in a dose-response experiment in which the endogenous levels of uPA mRNA were determined by quantitative reverse transcription-PCR at eachα-amanitin concentration (data not shown). A, the scheme of the amplified regions and the primers used for DAF amplicons are as in Fig. 4. Squared box, the 199-bp (nucleosome size) fragment amplified with the F29/R31 primer set is contained in the DAF-B amplicon. B, MNase digestion of cross-linked chromatin from α-amanitin-treated PC3 cells generates a genomic DNA ladder similar to that obtained from untreated cells (compare with Fig. 2A). n, 2n, and 3n: mono-, di-, and trinucleosome size DNA; M, DNA size marker (fragments length is indicated to the right). C, loss of the amplification pattern of DAF-A, -B, and -Bx using MNase-digested, ChIP-ready chromatin from α-amanitin-treated PC3 cells, as detected on a 2% agarose, 0.5× TBE gel stained with EtBr. The results indicate that the structures associated with the DAF amplicons are sensitive to drug treatment of PC3 cells, but the nucleosomal structure of the uPA regulatory region is maintained.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For ChIP assays, cross-linked material was digested for 50 min at 37 °C, and MNase digestion was stopped by adding radioimmune precipitation assay buffer (1 mm EDTA, 0.5 mm EGTA, 10 mm Tris, pH 8, 1% Triton, 0.1% sodium deoxycholate, 0.1% SDS, 140 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride). The resulting material was used directly in ChIP assays. ChIP—Each aliquot of MNase-digested cross-linked chromatin (200 μg) was precleared with 25 μl of Protein A-Sepharose beads (Amersham Biosciences), previously coated with 10 μg/ml each of poly(dI-dC), poly(dG-dC), and poly(dA-dT) and with 100 μg/ml of bovine serum albumin in radioimmune precipitation assay buffer. The aliquots were then incubated overnight with 1 μg of the appropriate antibodies (or without antibodies for the mock controls) in a total volume of 1 ml of radioimmune precipitation assay buffer and immunoprecipitated as described (27Orlando V. Strutt H. Paro R. Methods. 1997; 11: 205-214Crossref PubMed Scopus (513) Google Scholar). Following immunoprecipitation, the material was treated with RNase A (50 μg/ml) for 30 min at 37 °C and by proteinase K (500 μg/ml) in 0.5% SDS at the same temperature overnight. Formaldehyde cross-links were reverted by heating the samples at 65 °C for 5 h, and the DNA was purified with phenol extraction and then resuspended in 250 μl of distilled water. Resuspended material (4 μl) was used as a template in PCR reactions. PCRs were performed as follows: (a) first denaturation 1: 95 °C for 3 min; second denaturation: 95 °C, 1 min; annealing step (see Table 1 for temperatures of the primer sets), 1 min; extension: 72 °C, 1 min; final extension: 72 °C, 3 min. The second denaturation, annealing, and extension steps were normally repeated for 33 cycles. To exclude the presence of a signal from other immunoprecipitated material, the number of cycles in the PCR reactions was raised to 40. Primer sequences, location with respect to the uPA sequence (17Verde P. Boast S. Franzè A. Robbiati F. Blasi F. Nucleic Acids Res. 1988; 16: 10699-10716Crossref PubMed Scopus (103) Google Scholar), and annealing temperatures for each primer set are reported in Table 1. For the amplification of the 1574-bp genomic fragment shown in Fig. 1 (primers F14/R26) an elongation step of 2 min was used. PCR products were analyzed on 2% agarose gels in 0.5× TBE buffer. (45 mm Tris-borate; 1 mm EDTA, pH 7) The specific antibodies used for immunoprecipitations were against the following modifications of histone H3: K4me2 or K9me2 (#07–030 or #07–352, respectively, Upstate Biotechnology); K14ac or K9ac (#07–353 or #07–352, respectively, Upstate Biotechnology). The nomenclature of histone H3 post-translational modifications is according to a previous study (28Turner B.M. Nat. Struct. Mol. Biol. 2005; 12: 110-112Crossref PubMed Scopus (194) Google Scholar). Anti-HMGN1 and -HMGN2 antibodies were a kind gift of Dr. Michael Bustin, NCI, National Institutes of Health, Bethesda, MD. Polyclonal antibodies against c-Jun (sc-1694X, Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal antibodies against CTD-P-S2 (MMS-129-RA, Covance) or CTD-P-S2 (MMS-134-RA, Covance) were also used. In all experiments, polyclonal antibodies against the uPA receptor produced in our laboratory were used as unrelated antibodies. In all the experiments investigating the protein composition of MNase-digested chromatin, material from the same immunoprecipitation was amplified with primers located in the enhancer and minimal promoter regions as indicated in the individual figures. Anti-Sp1 or -p300 Antibodies Immunoprecipitate the uPA MP and Enhancer Sequences—Formaldehyde cross-links molecules with reactive groups at a maximum distance of 2 Å. This may occur between proteins bound to distant regulatory elements, if such groups are close enough, implying their interaction. We tested this hypothesis for the MP and the enhancer of the uPA gene, located 2 kb upstream, by performing conventional ChIP experiments on cross-linked, sonicated chromatin from PC3 cells with antibodies against Sp1, which uniquely binds the MP (18Ibañez-Tallon I. Ferrai C. Longobardi E. Facetti I. Blasi F. Crippa M.P. Blood. 2002; 100: 3325-3332Crossref PubMed Scopus (42) Google Scholar, 21Nerlov C. Rørth P. Blasi F. Johnsen M. Oncogene. 1991; 6: 1583-1592PubMed Google Scholar, 22Nerlov C. De Cesare D. Pergola F. Caracciolo A. Blasi F. Johnsen M. Verde P. EMBO J. 1992; 11: 4573-4582Crossref PubMed Scopus (142) Google Scholar, 25Cirillo G. Casalino L. Vallone D. Caracciolo A. De Cesare D. Verde P. Mol. Cell. Biol. 1999; 19: 6240-6252Crossref PubMed Scopus (51) Google Scholar), and the cofactor p300. We asked if the DNA immunoprecipitated with one or the other antibody contained both enhancer and MP sequences, by amplifying the recovered material with specific primers (F5/R14 and F27/R31 in Fig. 1A). Indeed both genomic DNA sequences were immunoprecipitated with either antibody, whereas the IVS was not detected (Fig. 1B) even by increasing the number of PCR cycles. Thus, the results are consistent with a close physical proximity of the regulatory elements and the extrusion of the IVS. However, amplification of the input DNA with the F14/R26 primer set did show the presence of a PCR product (Fig. 1B), indicating that the IVS was not fully broken by sonication. To obtain independent evidence of the interaction of the two separate genomic fragments we enzymatically cleaved chromatin prior to immunoprecipitation. ChIP-ready Chromatin Is Accessible to MNase Cleavage—We chose to digest ChIP-ready chromatin with MNase, a processive enzyme widely employed in chromatin studies (29Turner B.M. Chromatin and Gene Regulation. Molecular Mechanisms in Epigenetics. Blackwell Science Ltd., Oxford, UK2001Crossref Scopus (6) Google Scholar, 30van Holde K.E. Chromatin. Springer-Verlag, New York1989Crossref Google Scholar). The pattern of MNase digestion obtained in a time-course experiment shows that ChIP-ready chromatin from PC3 cells was readily and increasingly cleaved by the enzyme to poly-, mono-, and sub-nucleosomal particles (Fig. 2A). Next, genomic DNA from the various digestion time-points of Fig. 2A was subjected to PCR reactions with sets of primers that amplified increasingly larger (from 145 to 250 bp) fragments in the uPA enhancer region (Fig. 2B). The results showed that only nucleosome-size genomic fragments could be amplified using material from all the digestion time points (Fig. 2C), thus confirming the accessibility to and the cleavage by MNase of the uPA enhancer region. PCR Reactions with Genomic DNA from MNase-digested ChIP-ready Chromatin Reveal Fragments with a Distinctive Amplification Pattern in the MP Region—We then repeated the PCR amplification using primer sets in the uPA MP region. Because we wanted to exclude the presence of genomic fragments larger than 200 bp (mononucleosome size), we deliberately designed two sets of primers spanning 464 bp and 320 bp, respectively, and expected to be able to amplify genomic DNA only by using material from the early digestion time points (see Fig. 2A). As shown in Fig. 3B, primer sets F22/R26 and F26/R31 showed that the amplification signal decreased by using genomic DNA from early and intermediate MNase digestion time points but was rescued with material from later (20 and 50 min) time points. However, the amplification of two fragments of similar size upstream of F22 and one in the enhancer region displayed the progressive loss of the PCR signal, as expected (data not shown). Amplicons F22/R26 and F26/R31 share a common primer (F/R26 in Fig. 3A); however, they displayed the lowest amplification signal at different time points. We asked if they belonged to the same or different genomic populations by amplifying the region spanning both fragments (using primers F22 and R31 in Fig. 3A). In this case the amplification signal decreased with increasing digestion time (Fig. 3C). Thus amplicons F22/R26 and F26/R31, specifically located at and upstream of the MP region of the uPA gene, belong to different DNA fragments. Because F22/R26 and F26/R31 amplicons have a distinctive amplification pattern they were named DAF-A and DAF-B, respectively. The amplification pattern of DAF-A and -B apparently contradicts the MNase digestion kinetics and the results obtained in the enhancer region, shown in Fig. 2. However, one must recall that MNase is a processive endonuclease (30van Holde K.E. Chromatin. Springer-Verlag, New York1989Crossref Google Scholar), and this mode of action has two main consequences: 1) it causes a progressive and generalized loss of genomic DNA, as indicated by the quantitation of the material recovered at each MNase digestion time point (supplemental Fig. S1C). 2) It enriches genomic fragments that originate from a chromatin population that is more resistant to MNase cleavage than bulk chromatin, whereas it rapidly degrades those from a chromatin population more sensitive to cleavage than bulk chromatin. We therefore speculated that DAF-A, -B, and -Bx originate from such resistant populations. Thus we performed Southern blot experiments to test this point. Overall the results (shown in supplemental Fig. S1) indicated that: 1) both the enhancer and DAF-A regions are more sensitive than bulk chromatin to severing treatments; 2) at early digestion time points mono- and subnucleosomes from DAF-A accumulate more slowly than those form the enhancer region; and 3) DAF-A can only be revealed by the amplification provided by PCR and not by probing a Southern blot. The two latter observations indicate that DAF-A originates from a small, severing-resistant subpopulation of chromatin of the uPA regulatory region. Defining the Borders of DAF-A and DAF-B—Because DAF-A and DAF-B represent different chromatin populations we next established their size by amplifying increasingly longer fragments, assuming that the amplification pattern of amplicons F22/R26 and F26/R31 would be maintained if longer fragments belonged to the same chromatin populations. Indeed we could extend the amplification pattern of DAF-B in the 3′ direction (Fig. 4B), using primers R34 and R36, but not R37, in combination with F26. However, by using primer F25 in combination with R31 the pattern of amplicon DAF-B was lost in the 5′ direction (Fig. 4B). Thus DAF-B could be extended to the untranslated portion of the second exon of the uPA gene (Fig. 4A and Ref. 31Riccio A. Grimaldi G. Verde P. Sebastio G. Boast S. Blasi F. Nucleic Acids Res. 1985; 13: 2759-2771Crossref PubMed Scopus (172) Google Scholar). Amplicon F26
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