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• W2082113631 abstract "Sequences containing the matrix recognition signature were identified adjacent to theLMP/TAP gene cluster in the human and mouse major histocompatibility complex class II region. These sequences were shown to function as nuclear matrix attachment regions (MARs). Three of the five human MARs and the single mouse MAR recruit heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) in vivo during transcriptional up-regulation of the major histocompatibility complex class II genes. The timing of this recruitment correlates with a rise in mature TAP1 mRNA. Two of the human MARs bind hnRNP-A1 in vitro directly within a 35-bp sequence that shows over 90% similarity to certain Alu repeat sequences. This study shows that MARs recruit and bind hnRNP-A1 upon transcriptional up-regulation. Sequences containing the matrix recognition signature were identified adjacent to theLMP/TAP gene cluster in the human and mouse major histocompatibility complex class II region. These sequences were shown to function as nuclear matrix attachment regions (MARs). Three of the five human MARs and the single mouse MAR recruit heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) in vivo during transcriptional up-regulation of the major histocompatibility complex class II genes. The timing of this recruitment correlates with a rise in mature TAP1 mRNA. Two of the human MARs bind hnRNP-A1 in vitro directly within a 35-bp sequence that shows over 90% similarity to certain Alu repeat sequences. This study shows that MARs recruit and bind hnRNP-A1 upon transcriptional up-regulation. Matrix attachment regions (MARs) 1The abbreviations used are: MARs, matrix attachment regions; MHC, major histocompatibility complex; MRS, the matrix recognition signature; hnRNP, heterogeneous nuclear ribonucleoprotein; RT, reverse transcriptase; DTT, dithiothreitol; CHIP, chromatin immunoprecipitation; IFN-γ, interferon-γ; EMSA, electrophoretic mobility shift assay; HREs, hormone-response elements; LIS, lithium 3,5-diiodosalicylate; PGE2, prostaglandin E2; rec, recombinant; AFM, atomic force microscopy are short DNA sequences that bind to the ribonucleoprotein network that is generally referred to as the nuclear matrix or scaffold (1Boulikas T. Int. Rev. Cytol. 1995; 162: 279-388Crossref Scopus (251) Google Scholar). A subset of MARs is believed to mediate the organization of chromatin into a higher order structure consisting of multiple topologically constrained loops attached at their bases to the matrix (2Razin S.V. Cell. Mol. Biol. Lett. 2001; 6: 59-69PubMed Google Scholar). A role for MARs in DNA replication has been proposed because many MARs contain origins of replication, and newly replicated DNA is anchored to the nuclear matrix (3Pemov A. Bavykin S. Hamlin J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14757-14762Crossref PubMed Scopus (50) Google Scholar). The mapping of MARs to 5′-regulatory regions of certain genes, to actively transcribing genes, and to sites for binding activator, repressor, and demethylation proteins suggests that MARs also play a role in gene regulation (1Boulikas T. Int. Rev. Cytol. 1995; 162: 279-388Crossref Scopus (251) Google Scholar, 4Bode J. Benham C. Knopp A. Mielke C. Crit. Rev. Eukaryotic Gene Expr. 2000; 10: 73-90Crossref PubMed Google Scholar). In addition, MARs flanking individual genes or gene clusters in several species have been shown to act as insulator or boundary elements shielding genes within the domain from adjacent regulatory elements (5Namciu S.J. Blochlinger K.B. Fournier R.E.K. Mol. Cell. Biol. 1998; 18: 2382-2391Crossref PubMed Scopus (91) Google Scholar). MARs are identified biochemically by their selective retention in nuclear matrix preparations, which are derived from nuclei depleted of histones and most of the DNA. A set of characteristic motifs has been associated with MARs, including DNA unwinding elements, AT tracts, and DNase I-hypersensitive sites (1Boulikas T. Int. Rev. Cytol. 1995; 162: 279-388Crossref Scopus (251) Google Scholar, 6Bode J. Schlake T. Rios-Ramirez M. Mielke C. Stehgert M. Kay V. Klehr-Wirth D. Int. Rev. Cytol. 1995; 162: 389-454Crossref Scopus (151) Google Scholar). Binding of MARs to the matrix has not been assigned to unique DNA sequences but rather to sequences dispersed over several hundred base pairs. Recently, a unique bipartite sequence element was found in a large proportion of MARs, called the “MAR/SAR recognition signature” (MRS) (7van Drunen C.M. Sewalt R.G. Oosterling R.W. Weisbeek P.J. Smeekens S.C. van Driel R. Nucleic Acids Res. 1999; 27: 2924-2930Crossref PubMed Scopus (61) Google Scholar). The MRS was reported to predict correctly the positions of MARs/SARs in several species from their genomic sequence alone. In this study, we examined the role of MARs adjacent to theLMP/TAP gene cluster in the class II region of the human MHC. This genomic region was chosen as a model as both its sequence, transcription, and function have been well characterized (8The MHC Sequencing Consortium Nature. 1999; 401: 921-923Crossref PubMed Scopus (926) Google Scholar,9van Endert P.M. Curr. Opin. Immunol. 1999; 11: 82-88Crossref PubMed Scopus (67) Google Scholar). The cluster consists of four genes, LMP2 andTAP1 at the centromeric end which share a bidirectional promoter, and LMP7 and TAP2 at the telomeric end (10Beck S. Kelly A. Radley E. Khurshid F. Alderton R.P. Trowsdale J. J. Mol. Biol. 1992; 228: 433-441Crossref PubMed Scopus (148) Google Scholar). The LMP gene products (“low molecular mass polypeptide”) enhance the degradation of cytosolic antigens into peptide fragments in the multisubunit proteasome in preparation for binding at the cell surface to MHC class I molecules (11Driscoll J. Brown M.G. Finley D. Monaco J.J. Nature. 1993; 365: 262-266Crossref PubMed Scopus (402) Google Scholar). TheTAP gene products (“transporter associated with antigen processing”) are involved in the transport of these peptides to the endoplasmic reticulum where they bind newly synthesized MHC class I molecules (12Momburg F. Hämmerling H.G. Adv. Immunol. 1998; 68: 191-256Crossref PubMed Google Scholar). Expression of the LMP and TAPgenes can be up-regulated by interferon-γ (IFN-γ), resulting in an increase in processing and presentation of MHC class I-associated antigens (13Tanaka K. J. Leukocyte Biol. 1994; 56: 571-575Crossref PubMed Scopus (106) Google Scholar). We used the MRS in conjunction with a pattern finding and clustering program to predict the positions of five MARs in non-coding DNA centromeric to the LMP/TAP gene cluster. Biochemical analysis confirmed that they do indeed bind the nuclear matrix and that three of these MARs recruit the mRNA processing protein hnRNP-A1 in vivo during transcriptional up-regulation. We identified a 35-bp sequence in two of these MARs that is able to bind hnRNP-A1 directly in vitro. Similar findings in the homologous region of the mouse genome suggest a conserved role for these MARs in regulation of gene expression. The MRS proposed recently (7van Drunen C.M. Sewalt R.G. Oosterling R.W. Weisbeek P.J. Smeekens S.C. van Driel R. Nucleic Acids Res. 1999; 27: 2924-2930Crossref PubMed Scopus (61) Google Scholar) as a recognition signature for MARs has a pattern of two nucleotide motifs separated by 200 bp or less. The match required for MAR prediction is 15 of 16 bases in motif 1, AWWRTAANNWWGNNNC, and 8 of 8 bases in motif 2, AATAAYAA (IUB-IUPAC incompletely specified nucleotide code (www.chem.qmw.ac.uk/iubmb/misc/naseq.html): W = A or T; R = A or G; Y = C or T; N = A C G or T). Each motif can be present in either forward or reverse orientation, and clusters of more than one example of either motif within the constraint of the pattern are regarded as a single MRS. An iteration of the Nucleotide Interpretation Program (14Staden R. Comput. Appl. Biosci. 1988; 4: 53-60PubMed Google Scholar) was used to identify such MRS patterns, the output being parsed by a simple PERL script, pars_nip, to identify clusters of motifs. Entering the output into the chromosome 6 data base (6ace) allowed visualization of the MRS patterns within their genomic environment and the determination of the positions shown in Figs. 1 and 9. This procedure successfully confirmed the seven MRS patterns described in the human globin locus (GenBankTMaccession number U01317) (7van Drunen C.M. Sewalt R.G. Oosterling R.W. Weisbeek P.J. Smeekens S.C. van Driel R. Nucleic Acids Res. 1999; 27: 2924-2930Crossref PubMed Scopus (61) Google Scholar).Figure 9Scheme of theLmp/Tap region of the mouse MHC.A, candidate MAR in the Lmp/Tap gene region containing the MRS pattern (black box 1).B, L- and M-DNA fractions were isolated from NIH3T3 and IFN-γ up-regulated NIH3T3 mouse cells by 25 mm LIS extraction. These fractions were used in the subsequent PCR assay as templates. Positive and negative internal controls were carried out with total genomic DNA and pBR322 templates, respectively, for each sequence. Two non-MAR sequences from the same gene region were tested for their nuclear matrix binding ability as a control for assay specificity. The PCR products were separated in a 1.5% agarose gel.C, R values for the sequences binding to the nuclear matrix were estimated as in Fig. 2 B. Values show the means ± S.E. for three independent PCR assays.View Large Image Figure ViewerDownload (PPT) MRC5 human lung fibroblast cells were grown as a monolayer to confluence in RPMI 1640 supplemented with 10% fetal calf serum at 37 °C in a 5% CO2 atmosphere. In experiments with up-regulated MRC5 cells, 200 units/ml IFN-γ (recombinant human IFN-γ, R & D Systems, Oxon, UK) was added to the medium for 24 h (15Pederson T. J. Mol. Biol. 1998; 277: 147-159Crossref PubMed Scopus (132) Google Scholar). In some experiments mouse embryo monolayer NIH3T3 cells were used. These were grown and treated with IFN-γ as for MRC5 cells; however, E4 medium was used instead of RPMI 1640. The B-lymphoblastoid cell line AHB, which constitutively expresses the classical MHC and the LMP/TAP genes at high levels, was grown in suspension in RPMI 1640 medium supplemented with 10% fetal calf serum at 37 °C in a 5% CO2 atmosphere. In experiments to inhibit MHC class II gene expression, PGE2 was added into the culture medium for 20 h as described previously (16Roper R.L. Ludlow J.W. Phips R.P. Cell. Immunol. 1994; 154: 296-308Crossref PubMed Scopus (52) Google Scholar). The nuclear matrix-attached (M) and the matrix-independent or loop (L) DNA fractions from MRC5 cells, IFN-γ up-regulated MRC5 cells, AHB and NIH3T3 cells were isolated as described previously (17Donev R.M. Mol. Cell. Biochem. 2000; 214: 103-110Crossref PubMed Scopus (16) Google Scholar). Extractions with 25 mm LIS, 0.65m ammonium sulfate, or 2 m NaCl were employed in these separations. A PCR assay was used to identify specific DNA sequences in M- and L-fraction chromatin (18Kramer J.A. Krawetz S.A. BioTechniques. 1997; 22: 826-828Crossref PubMed Scopus (16) Google Scholar). The M- and L-DNA fractions, 50 ng each as determined by spectrophotometric measurement, were used as templates for amplification with corresponding PCR primer pairs (Table I). In all assays, the quantity of PCR products was maintained within the linear range (increasing the concentration of template or the number of cycles proportionally increased the signal). Aliquots of the PCR products were then electrophoresed in a 1.5% agarose gel, stained with ethidium bromide, and quantified densitometrically using Labworks 3.0 software. A control ratio between the sum of intensities of the M- plus L-fractions and the band intensity of PCR product using total genomic DNA as an internal control with the same primer pair as used in the M- and L-fractions was estimated. This ratio was always ∼1 ± 0.021, confirming the equivalent efficiency of the PCR in the M- and L-fractions and in total genomic DNA. All PCR experiments were carried out in triplicate for three independent M- and L-fraction separations. Similar results were obtained and summarized in histograms.Table IPrimers, annealing temperature, and expected PCR products for amplified probesSequence namePrimersT annealingProduct length°CbpMAR15′-CCCTGTTGTTTGGTGCATACA-3′ (F)1-aF indicates forward primer, and R indicates reverse primer.522765′-AGCGAGACTCCGTCTCAAAAA-3′ (R)MAR25′-CACATCGAACTATGCCTGACT-3′ (F)521675′-GACCTATTCAAGCTGTCAGCT-3′ (R)MAR35′-ACCACCCTAGTATTCCAAGGA-3′ (F)521445′-TGATTGCACCACTGCATTTCG-3′ (R)MAR45′-CAATGCTGGTCTTGAACTCCT-3′ (F)523105′-CCAGATGGTTTCACAGATGAG-3′ (R)MAR55′-TTTAGGGAGTGGTAGAGGAAG-3′ (F)501525′-CACCCAACCCTATATAGGTAT-3′ (R)Human5′-AAAGGTGGTGACCTCCTTCGG-3′ (F)54464Non-MAR15′-GCTCAGCAGCAGAAGGAGAGA-3′ (R)Human5′-CTTCCTCCGCCTACTGTGAAG-3′ (F)54267Non-MAR25′-CCATGCCAGACTTGGAGTACG-3′ (R)Mouse MAR15′-GGTTCCCTTTGTGTGGATGCT-3′ (F)541085′-CAGATCAGCATGCCCTTCCAT-3′ (R)Mouse5′-GACCGTGAACTGGCAGCTTCA-3′ (F)54216Non-MAR15′-TGAAGGACCGGACTTCCATGG-3′ (R)Mouse5′-AACCACTGGGACTCTGGCTCT-3′ (F)54201Non-MAR25′-GGTTCTGAGCCACAAGGCAGT-3′ (R)Probe 15′-CACATCGAACTATGCCTGACT-3′ (F)53715′-CCTGGGCAACATAATGAGACC-3′ (R)Probe 25′-GCTGGTCTTGAACTC-3′ (F)50355′-GGAGGATCGCTTGAG-3′ (R)Probe 35′-TGCCTGGCCTCCTTCCCAAAA-3′ (F)54615′-GACCTATTCAAGCTGTCAGCT-3′ (R)Probe 45′-GCTGGTCTTGAACTC-3′ (F)50355′-GGAGGATCGCCTGAG-3′ (R)Probe 55′-CACCTCGGCCTCCCAGTATTA-3′ (F)542715′-CCAGATGGTTTCACAGATGAG-3′ (R)a F indicates forward primer, and R indicates reverse primer. Open table in a new tab Nuclear extracts from MRC5 cells, IFN-γ up-regulated MRC5 cells, and AHB cells were prepared. The cells were collected and washed in Buffer A (146 mmsucrose, 100 mm KCl, 10 mm Tris, pH 7.0, 1.5 mm MgCl2). The pellet was then resuspended in Buffer A, and Nonidet P-40 was added to a final concentration of 0.5%. After 5 min of incubation on ice, the nuclei were collected by centrifugation. The pellet was then resuspended in Buffer C (5 mm HEPES, pH 7.9, 26% glycerol, 1.5 mmMgCl2, 0.2 mm EDTA, 0.5 mm DTT) to 1 × 108 nuclei/ml, and NaCl was added to a final concentration of 300 mm. After 30 min of incubation on ice, the nuclear extracts were separated from the debris by centrifugation at 24,000 × g for 20 min at 4 °C. The extracts were aliquoted and stored at −80 °C. The pull-down of proteins specifically binding the MARs of interest was carried out as described previously (19Nordhoff E. Krogsdam A.M. Jorgensen H.F. Kallipolitis B.H. Clark B.F. Roepstorff P. Kristiansen K. Nat. Biotechnol. 1999; 17: 884-888Crossref PubMed Scopus (63) Google Scholar) with slight modifications. The MARs were amplified from total genomic DNA by PCR using sets of specific primers (Table I) where the reverse primers were previously biotinylated by Biotin-Chem-Link kit (Roche Diagnostics). Approximately 80 pmol of each MAR was immobilized onto 400 μg of streptavidin magnetic particles (Roche Diagnostics) following the protocol supplied by the manufacturer. The pull-down was performed in a final volume of 550 μl containing 100 μg of nuclear protein extract, 10% glycerol, 20 mm HEPES, pH 7.9, 1 mm MgCl2, 1 mm DTT, 50 mm NaCl, 0.1 μg/μl poly[d(I-C)] (Roche Diagnostics), and 1 μg/μl sonicated salmon sperm DNA, protease inhibitor (Sigma). The reaction mixture was preincubated for 10 min at room temperature; 400 μg of streptavidin magnetic particles with attached MARs was added and the mixture incubated for a further 25 min on a rotator. After magnetic separation, the particles were washed three times with 400 μl of washing buffer (50 mm KCl, 20 mmHEPES, pH 7.9, 1 mm MgCl2, 0.5 mmDTT). The first washing buffer included 0.1 μg/μl poly[d(I-C)]. The magnetic particles were resuspended in 30 μl of Laemmli sample buffer containing 7 m urea, and the eluted proteins were separated in a 12% Tris glycine pre-cast gel (Invitrogen). The gels were either silver-stained (Silver Xpress, Invitrogen) or Coomassie-stained, and proteins of interest were cut out and processed further for mass spectrometry as described previously (20Pappin D.J.C. Hojrup P. Bleasby A.J. Curr. Biol. 1993; 3: 327-332Abstract Full Text PDF PubMed Scopus (1419) Google Scholar). By using specific primer pairs (Table II) for RT-PCR, hnRNP-A1 and the cdk inhibitor, p21, were amplified from their start to stop codons from cytoplasmic RNAs isolated from MRC5 fibroblasts and MRC5 treated with 50 ng/ml phorbol 12-myristate 13-acetate for 1 h, respectively. Phorbol 12-myristate 13-acetate treatment of fibroblasts was necessary to up-regulate the p21 expression level in MRC5. The amplified cDNAs were cloned in the pcDNA4/HisMax-TOPO expression vector (Invitrogen). They were then transfected transiently into MRC5 fibroblast cells by SuperFect Transfection Reagent (Qiagen Ltd., UK) as suggested by the supplier. The expressed recombinant hnRNP-A1 was purified by Xpress System Protein Purification (Invitrogen), and the N-terminal fusion tag was removed by Enterokinase Max (Invitrogen).Table IIPrimers, annealing temperature, and expected RT-PCR products for amplified probesSequence namePrimersT annealingProduct length°CbphnRNP-A1 cDNA for cloning5′-ATGTCTAAGTCAGAGTCTCCTAAAGAG-3′ (F)2-aF indicates forward primer, and R indicates reverse primer.569635′-TTAAAATCTTCTGCCACTGCCATAGCT-3′ (R)p21cDNA for cloning5′-ATGTCAGAACCGGCTGGGGAT-3′ (F)534955′-TTAGGGCTTCCTCTTGGATAAGA-3′ (R)hnRNP-A1mature mRNA5′-AAAGCTTAGCAGGAGAGGAGAGCCAGA-3′ (F)562795′-GCAATCAACAGCATGGGGTGC-3′ (R)rec-hnRNP-A15′-AATACGACTCACTATAG-3′ (F) (T7 primer)422325′-ATGAGAACCCCCCATGGTTT-3′ (R)TAP1 mature mRNA5′-GGGATCCTGTGACCATTGGGGCTGTAAGC-3′ (F)569995′-CTCTAGAGGTTATTCTGGAGCATCTGCAGGAGC-3′ (R)LMP2mature mRNA5′-CCGGTACCCATGCTGCGCGCGGGAGCACCAACCGGG-3′ (F)567965′-CTCTAGAGGTTACTCATCATAGAATTTTGG-3′ (R)TAP1 (intron 1) pre-mRNA5′-ACACAAGGGGACTGGGACAG-3′ (F)551705′-ATGGAGCCCAGAACCTCTGG-3′ (R)LMP2 (intron 1) pre-mRNA5′-GGCCAGAGCTGACTGTCAGT-3′ (F)551905′-CCTGTAGGAGCCTCATCTCC-3′ (R)Mouse Lmp2mature5′-TTTTGGCAGCTCATCTCCCAG-3′ (F)541115′-ATCACTCTGGCCATGAACCGA-3′ (R)Mouse Tap1mature5′-GGGAATCTTCTCCCTGTTGGT-3′ (F)542645′-GGACGACTGTTCCAGTACAGT-3′ (R)a F indicates forward primer, and R indicates reverse primer. Open table in a new tab Nuclear protein extracts and nuclear matrix protein extracts from hnRNP-A1 transfected MRC5 cells were isolated at 24-h intervals up to 96 h. The proteins were separated in a 12% Tris glycine pre-cast gel (Invitrogen) and transferred onto a nitrocellulose membrane using an electroblotting apparatus XCell SureLockTM (Invitrogen) according to the manufacturer's protocols. Further procedures for detecting the recombinant hnRNP-A1 containing Xpress tag followed instructions of the Anti-XpressTM-horseradish peroxidase antibody supplier (Invitrogen). Signal detection was performed by membrane incubation in Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Inc.). The MRC5 cells were transfected transiently with rec-hnRNP-A1 as described above. After 48 h of growth in culture, half of the cells were treated with IFN-γ at 200 units/ml for a further 24 h to up-regulated MHC gene expression. IFN-γ-treated and -untreated cells were collected (∼5 × 108 cells each) and fixed in tissue culture medium containing 1% formaldehyde for 10 min at room temperature. All further steps of this assay were as described previously (21Orlando V. Paro R. Cell. 1993; 75: 1187-1198Abstract Full Text PDF PubMed Scopus (301) Google Scholar). Chromatin sonication was performed to produce DNA fragments in the range of 400–700 bp (electrophoretically determined in 1.5% agarose). The immunoprecipitation was performed with Anti-XpressTM antibody recognizing the Xpress tag in rec-hnRNP-A1. CHIP was similarly performed on AHB cells, MRC5 cells, IFN-γ up-regulated MRC5 cells, and mouse NIH3T3 with and without IFN-γ treatment for 24 h, using goat polyclonal antibody against endogenous hnRNP-A1 (Santa Cruz Biotechnology, Inc.). In control CHIP experiments, normal goat IgG (Santa Cruz Biotechnology, Inc.) replaced the anti-hnRNP-A1 antibody. The naked co-immunoprecipitated DNAs were then used as templates in semi-quantitative PCR assays (50 ng of DNA/reaction) for MAR sequence detection. Primer pairs used for MAR sequence detection are given in Table I. The quantity of PCR products was again maintained within the linear range. Subsequently, aliquots of the PCR products were electrophoresed in a 1.5% agarose gel, stained with ethidium bromide, and quantified densitometrically using Labworks 3.0 software. The intensities of MAR fragments amplified from co-immunoprecipitated DNA were divided by the intensity of MAR fragments amplified from total genomic DNA (50 ng of DNA/reaction) using the same primer pairs. The calculated ratio (relative binding) indicates the enrichment of co-immunoprecipitated DNA in corresponding MAR sequences, compared with their number in the same amount of genomic DNA. All PCR experiments were carried out in quadruplicate for three independent CHIP analyses. Similar results were obtained and summarized in histograms. Control real time quantitative PCR experiments were performed for human MAR4 and the single mouse MAR1 as described below. Primers used in quantification experiments are given in Table III.Table IIIPrimer pairs used for real time quantitative PCR/RT-PCRSequence namePrimersPrimer concentrationsProductμmbpβ-Actin5′-GGATCAGCAAGCAGGAGTATGA-3′ (F)3-aF indicates forward primer, and R indicates reverse primer.900 /501155′-GCGCAAGTTAGGTTTTGTCAAG-3′ (R)LMP25′-GACAGCCTTTTGCCATTGG-3′ (F)900 /501175′-GCAATAGCGTCTGTGGTGAAG-3′ (R)TAP15′-TGCCTACAGTTCGAAGCTTTGC-3′ (F)300 /3001235′-GTGGTCCAGGAGTTGACTGCAT-3′ (R)Pre-LMP25′-CCCCTCCATCCATATGCCTACT-3′ (F)300 /501445′-GGAGCATTTACACCACGGACAT-3′ (R)Pre-TAP15′-CTGATGCCCTCCAGGATAATG-3′ (F)900 /3001035′-GAGAAGAGAGGTCACGCACAAA-3′ (R)Human MAR45′-TTGTAATATTGTTGAATTCGGTTCATAAAT-3′ (F)900 /300625′-ACCTTGAACACAGATGGGAGAAA-3′ (R)Mouse MAR15′-GGGTGCTCCGCTCTCTTTCT-3′ (F)300 /300615′-TCTTTATCCCCATGCCAAACAC-3′ (R)a F indicates forward primer, and R indicates reverse primer. Open table in a new tab Cytoplasmic RNAs isolated from different cell types (MRC5 and NIH3T3, MRC5 and NIH3T3 IFN-γ treated for 24 h, MRC5/rec-hnRNP-A1 transfected for 72 h, and MRC5/rec-hnRNP-A1 transfected for 72 h, and IFN-γ treated for 24 h, and AHB and AHB cells PGE2-treated for 20 h) were used as starting templates for RT-PCR to monitor the levels of mature mRNAs. To assess the levels of pre-mRNAs, total nuclear RNAs isolated from the above human cell lines were examined. Cytoplasmic and total nuclear RNAs were isolated as described previously (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.6-7.12Google Scholar). The subsequent semi-quantitative RT-PCR was performed using specific primers shown in Table II and Qiagen® One-step RT-PCR kit (Qiagen) following the supplier's recommendations. Control experiments with β-actin were carried out using standard primers (Promega). Aliquots of the RT-PCR products were separated in a 1.5% agarose gel, ethidium bromide-stained, and quantified densitometrically using Labworks 3.0 software. All RT-PCR experiments were carried out in triplicate for two independent RNA isolations. Similar results were obtained and summarized in histograms. Each relative expression value represents a ratio between the densities of specific mRNA transcripts to corresponding β-actintranscripts. Cytoplasmic and nuclear RNAs were isolated from MRC5 cells and MRC5 transfected with p21 for 20 h and treated with IFN-γ for 0, 2, 4, 6, and 8 h. They were used as starting templates for real time quantitative RT-PCR. Aliquots of the RNAs were reverse-transcribed using random hexamers and Multiscribe reverse transcriptase according to the manufacturer's instructions (Applied Biosystems). Primers were designed using Primer Express software (Applied Biosystems), taking into account intron/exon boundaries to ensure specific amplification of cDNA (Table III). Primers were designed to β-actin as an internal control for normalization of starting cDNA levels. Quantitative PCR was performed using SYBR Green PCR Master Mix according to the manufacturer's instructions (Applied Biosystems), with the exception that 25-μl reaction volumes were used, with 45 cycles of amplification. Each of the primer pairs was optimized to ensure amplification of the specific product and absence of primer dimers (Table III). PCR was performed on the Taqman 7700 (Applied Biosystems). Following amplification, RT-PCR products were electrophoresed on agarose gels to check for the correctly sized product. The real time PCR results were analyzed using the sequence detection system software version 1.9 (Applied Biosystems). Gene expression levels were calculated using the comparative Ct method (ΔΔCt). ΔΔCt validation experiments showed similar amplification efficiency for all templates used (difference between line slopes for all templates less than 0.1). Expression levels of the genes were normalized to those in MRC5 cells without IFN-γ treatment. At least two independent experiments were performed for each gene. Similar results were obtained and summarized in histograms. hnRNP-A1 recruitment in vivo to human MAR4 was examined by CHIP at 0, 2, 4, 6, 8, and 24 h after the start of IFN-γ treatment of MRC5 cells and p21-transfected MRC5 cells. Immunoprecipitated DNAs were used as templates in subsequent real time quantitative PCR experiments. Levels of protein recruitment were calculated according to the ΔΔCt method. Similar amplification efficiencies were shown for all templates used. Recruitment levels to MAR4 were normalized to that in MRC5 cells without IFN-γ treatment. Two independent experiments were carried out. Similar results were obtained and summarized in histograms. Recruitment of hnRNP-A1 to the single mouse MAR1 was examined in NIH3T3 cells 0 and 24 h after IFN-γ treatment. Results were analyzed as for the human MAR4. Direct hnRNP-A1 binding to the MARs was investigated by examining DNA-protein complexes by AFM topography imaging as described previously (23Donev R.M. Doneva T.A. Bowen W.R. Sheer D. Mol. Cell. Biochem. 2002; 233: 181-185Crossref PubMed Scopus (14) Google Scholar) and by EMSA. Sets of specific primer pairs were used for amplification of the probes for EMSA (Table I). Probes 1–5 were labeled with horseradish peroxidase using the North2South Direct horseradish peroxidase labeling and detection kit (Pierce). Each probe (80 ng) was incubated with 100, 200, or 300 ng of hnRNP-A1 for 50 min in binding buffer (20 mm Tris-HCl, pH 7.4, 0.1 mm EDTA, 0.1% Triton X-100, 1% 2-mercaptoethanol, 2 mm MgCl2, 5% glycerol), containing either 0.1 μg/μl poly[d(I-C)] or 0.1 μg/μl poly[d(I-C)] combined with 1 μg/μl sonicated salmon sperm DNA. Labeled probes 1–5 and those from the incubation reactions were separated in a 2% agarose gel. The DNA signal was detected by chemiluminescence. The coordinates for each human MAR and gene CDS (Fig. 1) in the nucleotide sequence GenBankTMaccession number X87344 are as follows: MAR1 (81997–82190), MAR2 (84791–84899), MAR3 (90295–90323), MAR4 (90593–90800), MAR5 (93872–93977), LMP2 (94885–100219), TAP1(100813–109220), LMP7 (110036–113708), and TAP2(116217–127095). The schematic of the mouseLmp/Tap region (Fig. 9 A) is derived from GenBankTM accession numbers U35323 (24Peleraux A. Karlsson L. Chambers J. Peterson P.A. Immunogenetics. 1996; 43: 204-214Crossref PubMed Google Scholar) and AF027865. The single MAR is located 168 bases downstream of Lmp2 which is transcribed in the negative orientation (telomere to centromere). Sequences used to design primers for cloning of hnRNP-A1 and the cdk inhibitor, p21, from their start to stop codons were NM_002136 and NM_000389, respectively. A total number of 323 clustered MRS-containing genomic fragments were found in the MHC (Fig. 1). Five of these fragments immediately centromeric of theLMP/TAP gene cluster in the MHC class II region were then studied in detail. The ability of these five fragments and two random fragments from the same region, which do not contain the MRS, to bind to the nuclear matrix and therefore to function as MARs was tested by PCR (18Kramer J.A. Krawetz S.A. BioTechniques. 1997; 22: 826-828Crossref PubMed Scopus (16) Google Scholar). The level of relative binding (R) of each DNA fragment to the nuclear matrix was determined as described previously (17Donev R.M. Mol. Cell. Biochem. 2000; 214: 103-110Crossref PubMed Scopus (16) Google Scholar). Cell types with different profiles of MHC class II gene expression were used to determine whether transcriptional status in the region is relevant to the functioning of these MAR sequences. The human fibroblast cell line MRC5 expresses theLMP/TAP genes at low levels and does not express the classical MHC class II genes. Expression of all these genes is greatly increased when MRC5 cells are treated with IFN-γ for 24 h (25Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2493) Google Scholar). The B-lymphoblastoid cell line AHB constitutively expresses these genes at high levels. We found that all five genomic fragments containing the MRS from theLMP/TAP gene region bound to the nuclear matrix, regardless of the extraction method used (Fig. 2). These fragments were thus designated MAR1–5. In all three cell types extracted with lithium 3,5-diiodosalicylate (LIS) and ammonium sulfate, MARs-1, -3, -4, and -5 were detected in nuclear matrix fraction only (R = 100%). In contrast, MAR2 showed substantially different levels of matrix binding in cells with different levels of MHC gene expression. Around 40% of MAR2 sequences were found in the nuclear matrix fra" @default.
• W2082113631 created "2016-06-24" @default.
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