Insulation of the Ubiquitous Rxrb Promoter from the Cartilage-specific Adjacent Gene, Col11a2
2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês
10.1074/jbc.m803657200
ISSN1083-351X
AutoresJunko Murai, Daisuke Ikegami, Mina Okamoto, Hideki Yoshikawa, Noriyuki Tsumaki,
Tópico(s)interferon and immune responses
ResumoThe retinoid X receptor β gene (Rxrb) is located just upstream of the α2(XI) collagen chain gene (Col11a2) in a head-to-tail manner. However, the domain structures of these genes are unknown. Col11a2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. Col11a2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the Col11a2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and Col11a2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from Col11a2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF. The retinoid X receptor β gene (Rxrb) is located just upstream of the α2(XI) collagen chain gene (Col11a2) in a head-to-tail manner. However, the domain structures of these genes are unknown. Col11a2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. Col11a2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the Col11a2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and Col11a2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from Col11a2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF. Cartilage is a highly specialized tissue that serves as the template for skeletal development and lines the joint surface. Cartilage consists of an abundant extracellular matrix maintained by chondrocytes. The collagen network provides scaffolding for proteoglycans in the extracellular matrix and confers tensile strength important for resisting compression and shearing loads in cartilage. Cartilage collagen fibrils are heterotypic fibrils composed of types II, IX, and XI collagens. The type XI collagen molecules co-assemble stoichiometrically with the major collagen, type II collagen, to form cartilage fibrils, whereas type IX collagen is associated with the surface of the fibrils (1Mendler M. Eich-Bender S.G. Vaughan L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (401) Google Scholar, 2Vaughan L. Mendler M. Huber S. Bruckner P. Winterhalter K.H. Irwin M.I. Mayne R. J. Cell Biol. 1988; 106: 991-997Crossref PubMed Scopus (258) Google Scholar). The type XI collagen molecule is composed of three distinct subunits: α1(XI), α2(XI), and α3(XI) (3Morris N.P. Bachinger H.P. J. Biol. Chem. 1987; 262: 11345-11350Abstract Full Text PDF PubMed Google Scholar). Type XI collagen likely regulates the diameter of cartilage collagen fibrils (4Li Y. Lacerda D.A. Warman M.L. Beier D.R. Yoshioka H. Ninomiya Y. Oxford J.T. Morris N.P. Andrikopoulos K. Ramirez F. Wardell B.B. Lifferth G.D. Teuscher C. Woodward S.R. Taylor B.A. Seegmiller R.E. Olsen B.R. Cell. 1995; 80: 423-430Abstract Full Text PDF PubMed Scopus (297) Google Scholar). Expression of the α2(XI) collagen chain gene is highly specific to cartilage (5Sugimoto M. Kimura T. Tsumaki N. Matsui Y. Nakata K. Kawahata H. Yasui N. Kitamura Y. Nomura S. Ochi T. Cell Tissue Res. 1998; 292: 325-332Crossref PubMed Scopus (27) Google Scholar). Mutations in the α2(XI) collagen chain gene are associated with certain forms of human chondrodysplasia, Stickler syndrome and otospondylomegaepiphyseal dysplasia, indicating that type XI collagen is intimately involved in skeletal morphogenesis (6Vikkula M. Mariman E.C. Lui V.C. Zhidkova N.I. Tiller G.E. Goldring M.B. van Beersum S.E. de Waal Malefijt M.C. van den Hoogen F.H. Ropers H.H. Mayne R. Cheah K.S.E. Olsen B.R. Warman M.L. Brunner H.G. Cell. 1995; 80: 431-437Abstract Full Text PDF PubMed Scopus (305) Google Scholar).We previously cloned the mouse α2(XI) collagen chain gene (Col11a2) (7Tsumaki N. Kimura T. J. Biol. Chem. 1995; 270: 2372-2378Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We found that when the 1st intron sequence of Col11a2 was linked to the Col11a2 autologous basal promoter, it directed cartilage-specific expression in transgenic mice, indicating that the 1st intron sequence is a cartilage-specific enhancer (8Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). We also found that the retinoid X receptor β gene (Rxrb) 2The abbreviations used are: Rxrb, retinoid X receptor β gene; BAC, bacterial artificial chromosome; BORIS, Brother of the Regulator of Imprinted Sites; ChIP, chromatin immunoprecipitation; Col11a2, α2(XI) collagen chain gene (Col11a2); CTCF, CCCTC-binding factor; EMSA, electrophoretic mobility shift assay; RCS, rat chondrosarcoma; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Nf, neurofilament; RXR, retinoid X receptor; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AD, adenovirus. 2The abbreviations used are: Rxrb, retinoid X receptor β gene; BAC, bacterial artificial chromosome; BORIS, Brother of the Regulator of Imprinted Sites; ChIP, chromatin immunoprecipitation; Col11a2, α2(XI) collagen chain gene (Col11a2); CTCF, CCCTC-binding factor; EMSA, electrophoretic mobility shift assay; RCS, rat chondrosarcoma; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Nf, neurofilament; RXR, retinoid X receptor; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AD, adenovirus. is located just upstream of Col11a2 in a head-to-tail manner. This alignment is conserved in organisms ranging from humans to zebrafish. The distance between the termination codon of Rxrb and the ATG codon of Col11a2 is 2,441 bp in humans, 2,161 bp in rats, 2,123 bp in mice, and 13,800 bp in zebrafish. The COL11A2 is 30-kb in size, consisting of more than 50 exons and RXRB is 7-kb in size, consisting of 10 exons. The three retinoid X receptors, RXRα, RXRβ and RXRγ, belong to the family of nuclear hormone receptors that are ligand-activated transcription factors (9Germain P. Staels B. Dacquet C. Spedding M. Laudet V. Pharmacol. Rev. 2006; 58: 685-704Crossref PubMed Scopus (449) Google Scholar). RXR is an obligatory component of various nuclear receptor heterodimers such as the retinoic acid receptor, vitamin D receptor, thyroid hormone receptor, and peroxisome proliferator activated receptor RXRs play an integrative role in a number of nuclear receptor-mediated pathways. RXRα and RXRβ transcripts are widely expressed in embryo and adult tissues, whereas the distribution of RXRγ transcripts is more restricted (10Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1061) Google Scholar). Approximately 50% of mice lacking RXRβ died before birth or at birth. Male mice that survived were sterile due to oligoasthenoteratozoospermia (11Kastner P. Mark M. Leid M. Gansmuller A. Chin W. Grondona J.M. Decimo D. Krezel W. Dierich A. Chambon P. Genes Dev. 1996; 10: 80-92Crossref PubMed Scopus (270) Google Scholar). RXRβ and RXRγ are expressed in the striatum and control the function of the dopaminergic mesolimbic pathway (12Krezel W. Ghyselinck N. Samad T.A. Dupe V. Kastner P. Borrelli E. Chambon P. Science. 1998; 279: 863-867Crossref PubMed Scopus (298) Google Scholar). RXR forms heterodimers with liver X receptor and farnesoid X-activated receptor and regulates cholesterol balance (13Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1144) Google Scholar). Thus, the fidelity of the spatiotemporal expression of RXRβ is crucial for the normal development of a wide variety of tissues.The organization of eukaryotic genomes necessarily results in the proximity of domains with distinct functions. The identity of domains is maintained by classical transcriptional regulatory elements, such as enhancers, silencers, and upstream activating sequences. In some cases, specific DNA sequences, referred to as insulators (14Gaszner M. Felsenfeld G. Nature Rev. 2006; 7: 703-713Crossref Scopus (495) Google Scholar), and their associated binding proteins have a role in establishing or maintaining discrete interdomain boundaries. Insulators have been divided into two classes: enhancer-blocking insulators, which prevent distal enhancers from activating a promoter when placed between an enhancer and promoter, and barrier insulators, which block heterochromatinization and the consequent silencing of a gene (15Wallace J.A. Felsenfeld G. Curr. Opin. Genet. Dev. 2007; 17: 400-407Crossref PubMed Scopus (327) Google Scholar). In vertebrates, the enhancer blocking activity of insulators is associated with a binding site for the CCCTC-binding factor (CTCF), which recognizes long and diverse nucleotide sequences. CTCF is a ubiquitously expressed nuclear protein with 11 zinc finger DNA-binding domains. Thus far, CTCF remains as the only major protein implicated in the establishment of insulators in vertebrates (16Felsenfeld G. Burgess-Beusse B. Farrell C. Gaszner M. Ghirlando R. Huang S. Jin C. Litt M. Magdinier F. Mutskov V. Nakatani Y. Tagami H. West A. Yusufzai T. Cold Spring Harbor Symp. Quant. Biol. 2004; 69: 245-250Crossref PubMed Scopus (81) Google Scholar). There has been great interest in identifying other binding sites for CTCF. Several recent high-throughput ChIP-chip analyses and comparative genomic studies have identified tens of thousands of potential CTCF-binding sites in the human and mouse genomes (17Bao L. Zhou M. Cui Y. Nucleic Acids Res. 2008; 36: D83-D87Crossref PubMed Scopus (92) Google Scholar, 18Kim T.H. Abdullaev Z.K. Smith A.D. Ching K.A. Loukinov D.I. Green R.D. Zhang M.Q. Lobanenkov V.V. Ren B. Cell. 2007; 128: 1231-1245Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 19Xie X. Mikkelsen T.S. Gnirke A. Lindblad-Toh K. Kellis M. Lander E.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7145-7150Crossref PubMed Scopus (245) Google Scholar). The sensitivity of the ChIP-chip analysis for CTCF is 88%; thus, there are some false-negative results (18Kim T.H. Abdullaev Z.K. Smith A.D. Ching K.A. Loukinov D.I. Green R.D. Zhang M.Q. Lobanenkov V.V. Ren B. Cell. 2007; 128: 1231-1245Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). Recently, it was discovered that cohesin binds to many of the CTCF-binding sites, co-localizing with CTCF to insulate promoters from distinct enhancers (20Wendt K.S. Yoshida K. Itoh T. Bando M. Koch B. Schirghuber E. Tsutsumi S. Nagae G. Ishihara K. Mishiro T. Yahata K. Imamoto F. Aburatani H. Nakao M. Imamoto N. Maeshima K. Shirahige K. Peters J.M. Nature. 2008; 451: 796-801Crossref PubMed Scopus (864) Google Scholar, 21Parelho V. Hadjur S. Spivakov M. Leleu M. Sauer S. Gregson H.C. Jarmuz A. Canzonetta C. Webster Z. Nesterova T. Cobb B.S. Yokomori K. Dillon N. Aragon L. Fisher A.G. Merkenschlager M. Cell. 2008; 132: 422-433Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar).Despite the proximity of Rxrb and Col11a2, the dissimilarities in their expression patterns and functions suggest the existence of an intergenic boundary. To investigate this possibility, we first clarified differences in the expression patterns of the two genes and examined whether the cartilage-specific enhancer of Col11a2 could affect transcriptional activities of heterologous promoters. We then searched for a CTCF-binding site between the two genes. We examined whether the intergenic sequence and CTCF-binding site affected the activities of the Rxrb promoter by using bacterial artificial chromosome (BAC) transgene constructs that cover the entire Rxrb and Col11a2 genes.EXPERIMENTAL PROCEDURESNorthern Hybridization—Total RNA was extracted from various mouse tissues at 16.5 days postcoitus and from cell lines by using RNeasy Mini Kits (Qiagen, Santa Clara, CA). Ten micrograms of total RNA was fractionated by electrophoresis through formaldehyde-agarose gels and then transferred onto Nytran membranes (Amersham Biosciences). Complementary DNAs (cDNAs) were labeled with [32P]dCTP by using the Rediprime II Random Prime Labeling System (Amersham Biosciences). The membranes were hybridized with 32P-labeled mouse Col11a2 and Rxrb cDNAs.Real-time RT-PCR—Total RNAs were digested with DNase to eliminate any contaminating genomic DNA before real-time quantitative RT-PCR. One microgram of total RNA was reverse transcribed into first-strand cDNA by using QuantiTect Reverse Transcription (Qiagen). The PCR amplification proceeded in a 20-μl reaction mixture containing 2 μl of cDNA, 10 μl of SYBR PremixExTaq (Takara, Japan), and 4 pmol of primers specific for rat Rxrb and Col11a2. The quantified individual RNA expression levels of rat Col11a2 and rat Rxrb were normalized to the respective rat Gapdh expression levels. The primers used are listed in supplemental Table S1.Cell Lines and Cell Culture—RCS (rat chondrosarcoma) cells (22Mukhopadhyay K. Lefebvre V. Zhou G. Garofalo S. Kimura J.H. de Crombrugghe B. J. Biol. Chem. 1995; 270: 27711-27719Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), L6 (rat skeletal muscle) cells (23Yaffe D. Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 477-483Crossref PubMed Scopus (796) Google Scholar), and FR (rat skin fibroblast) cells (ATCC number CRL-1213) (24Dawes K.E. Gray A.J. Laurent G.J. Eur. J. Cell Biol. 1993; 61: 126-130PubMed Google Scholar) were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin. H4IIE (rat hepatoma) cells (25Pitot H.C. Peraino C. Morse Jr., P.A. Potter V.R. Natl. Cancer Inst. Monogr. 1964; 13: 229-245PubMed Google Scholar) were cultured in α-minimal essential medium with 10% fetal bovine serum and 1% streptomycin/penicillin. PC12 (rat adrenal pheochromocytoma) cells (ATCC number CRL-1721) (26Levi A. Eldridge J.D. Paterson B.M. Science. 1985; 229: 393-395Crossref PubMed Scopus (228) Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 10% horse serum, and 1% streptomycin/penicillin in collagen dishes. L6, FR, and H4IIE cells were obtained from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan).Histology and in Situ Hybridization—Mouse embryos were dissected with a stereomicroscope, fixed in 4% paraformaldehyde, processed, and embedded in paraffin. Serial sections were stained with hematoxylin and eosin. RNA in situ hybridization was performed by using 35S-labeled antisense riboprobes as previously described (40D-Pelton R.W. Dickinson M.E. Moses H.L. Hogan B.L. Development. 1990; 110: 609-620Crossref PubMed Google Scholar). Probes included mouse Col11a2 cDNA (pRAC2-28) (7Tsumaki N. Kimura T. J. Biol. Chem. 1995; 270: 2372-2378Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and Rxrb cDNA (residues 910–1341, GenBank™ accession number M84818).Generation of Transgenic Mice Bearing the Col11a2 Enhancer Linked to Heterologous Promoters—pAD-LacZ was the expression vector pADbeta (Clontech number 6176-1), which contains the adenovirus II major promoter, SV40 RNA splice site, the β-galactosidase reporter gene and the SV40 polyadenylation signal. The fragment of 2.3 kb of the first intron sequence of Col11a2 as an enhancer was cloned into the SalI/PstI polylinker site located downstream of the SV-40 polyadenylation signal of ADbeta to create pAD-LacZ-Int. Nf-LacZ-Int was created by inserting the 2.3-kb fragment of the first intron sequence of Col11a2 as an enhancer into the SalI/PstI polylinker site located downstream of the SV-40 polyadenylation signal of pNf-LacZ (27Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), which contains the neurofilament promoter, SV40 RNA splice site, the β-galactosidase reporter gene, and the SV40 polyadenylation signal.The plasmids AD-LacZ, AD-LacZ-Int, and Nf-LacZ-Int were digested with EcoRI and PstI to release the inserts from their vector sequences. Transgenic mice were produced by microinjecting each of the inserts into the pronuclei of fertilized eggs from F1 hybrid mice (C57BL/6x DBA) as described previously (8Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). Generation 0 (G0) embryos were sacrificed at 13.5 days postcoitus and processed for expression analysis of the reporter gene. Transgenic embryos were identified by PCR analysis of genomic DNA extracted from the placenta or tail as described previously (8Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). X-Gal staining of mouse bodies and sections was performed as previously described (28Nagy A. Gertsenstein M. Vintersten K. Behringer R. Manipulating the Mouse Embryo., 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2003Google Scholar).Electrophoretic Mobility Shift Assays—The full-length coding region of mouse CTCF cDNA was PCR amplified with primers NT364 and NT365 (supplemental Table S2) and cloned into pCR-BluntII-TOPO using the Zero Blunt TOPO PCR cloning kit (Invitrogen catalog number K2800-20). CTCF cDNA was transferred to pTNT vector and used for in vitro synthesis of CTCF protein with the TnT T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI, catalog number L1170). Nuclear extracts from RCS cells were prepared using CelLytic NuCLEAR Extraction kits (Sigma) according to the manufacturer's instructions. Probes were prepared as described under supplementary data.DNA fragments were denatured, end-labeled at the 5′-end with [γ-32P]ATP and T4 polynucleotide kinase, and annealed to prepare radiolabeled probes. The radiolabeled probes were gel purified and combined with equal amounts of in vitro-synthesized CTCF protein or equal amounts of nuclear extracts. For binding reactions, we used a buffer containing phosphate-buffered saline with 5 mm MgCl2, 0.1 mm ZnSO4, 1 mm dithiothreitol, 0.1% Nonidet P-40, and 10% glycerol in the presence of 10 ng/μl poly(dI-dC) and unlabeled 10 nm double-strand synthesized oligonucleotides containing an Sp1-binding site, as SP1-like factors can bind to GC-rich segments and obscure the binding of CTCF (29Awad T.A. Bigler J. Ulmer J.E. Hu Y.J. Moore J.M. Lutz M. Neiman P.E. Collins S.J. Renkawitz R. Lobanenkov V.V. Filippova G.N. J. Biol. Chem. 1999; 274: 27092-27098Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The reaction mixtures of a 10-μl final volume were incubated for 20 min at room temperature followed by electrophoresis on 6% non-denaturing polyacrylamide gels. We performed electrophoretic mobility shift assay (EMSA) with nuclear extracts in the presence of 0.2 ng/μl salmon sperm DNA. For super-shift assays, the reaction mixture was combined with 2 μl of anti-CTCF antibody (Upstate number 07-729) or normal rabbit IgG and incubated for 2 h at 4 °C before the addition of radiolabeled probes. The primers and synthesized oligonucleotides are listed in supplemental Table S2.ChiP Assay—We used the ChIP Assay Kit (Upstate number 17-295), according to the manufacturer's protocol as described under supplementary data.We performed quantitative real-time PCR in triplicate with DNA prepared with anti-acetylated histone H3 antibody and input DNA. We used the LightCycler FastStart DNA Masterplus HybProbe kit (Roche number 03-515-575001) in a LightCycler (Roche). Amplification reactions were performed in a volume of 20 μl with 20 pm (each) primers and FRET probes (0.4 μl of each primer per reaction), 5 μl of template DNA, and 1× Master mixture (4 μl/reaction).We performed quantitative real-time PCR in triplicate with DNA prepared with anti-CTCF antibody, DNA prepared with anti-Rad21 antibody, DNA prepared with anti-BORIS antibody, and input DNA by using primer pairs designed for 11P and RX4. We used LightCycler and SYBR Green reagent (Takara), according to the manufacturer's instructions.Data were analyzed with LightCycler software by the Fit Point method to minimize noise and obtain the best possible correlation coefficient between standards. The -fold difference for a particular target sequence was determined by calculating the ratio of the amount of the target sequence in the immunoprecipitation to the amount of the target sequence in the input DNA. Primers and FRET probes were selected from human and rat retinoid X receptor and α2(XI) collagen chain gene sequences using Roche Primer design software. Primers and FRET probes were obtained from Sigma and Nihon Gene Research Laboratories Inc. (Sendai, Japan), respectively. The primers and FRET probes are listed in supplemental Table S3.BAC Transgene Construction—A human BAC clone, CTD-2054I15 (WT), which contains RXRB and COL11A2 in the middle, was purchased from Invitrogen. Modifications of a BAC clone were performed as described under supplementary data. Constructs were checked with digestion with various restriction enzymes and PCR analysis. The primers used in BAC modifications are listed in supplemental Table S4.Colony Assays—Two micrograms of the BAC transgene constructs (WT-Pur, 11Psub-Pur, 11Pdel-Pur, and RX4sub-Pur) were transfected into 1 × 106 L6 cells using Lipofectamine 2000 (Invitrogen) in 10-cm dishes (supplemental Fig. S2). After 24 h, cells were selected by puromycin (5 μg/ml). After 10 days of selection, dishes were stained with crystal violet and the colonies were counted.Establishment of Transgenic RCS Cells—The BAC transgene constructs (SV40-Pur/WT, SV40-Pur/11Psub, and SV40-Pur/11Pdel) were transfected into 1 × 106 RCS cells in 10-cm dishes using Lipofectamine 2000 (Invitrogen) (supplemental Fig. S2). After 24 h, cells were selected by puromycin (3 μg/ml). After 14 days of selection, colonies were picked-up and replated in 24-well dishes. We established 12, 14, and 18 independent RCS cell lines for SV40-Pur/WT, SV40-Pur/11Psub, and SV40-Pur/11Pdel transgenes, respectively. Total RNAs were extracted from each transgenic cell and subjected to real-time RT-PCR to analyze the relative expression levels of rat Col11a2, rat Rxrb, human COL11A2, and human RXRB mRNAs. We normalized the quantified individual RNA expression levels of rat Col11a2 and rat Rxrb to the respective rat Gapdh expression levels, and those of human COL11A2 and human RXRB were normalized to the respective puromycin expression levels. The primers used are listed in supplemental Table S1.Production and Genotyping of Transgenic Mouse Lines— BAC transgene constructs (WT-LacZ, 11Psub-LacZ, 11Pdel-LacZ, and RX4sub-LacZ) were purified with the Qiagen large construct kit (Qiagen, number 12462), following the manufacturer's instructions. The BAC DNA constructs were linearized by digestion with PacI. The linearized transgenes (2–5 ng/μl) in injection buffer (10 mm Tris-HCl, pH 7.5, and 0.1 mm EDTA, pH 8.0) were microinjected into fertilized ova (supplemental Fig. S2). Founder embryos at E13.5 were sacrificed and genotyped with three sets of PCR primers (JM281/JM282, JM283/JM284, and NT418/419) located at both ends and the middle of the linearized BAC transgene DNA. The primers are listed in supplemental Table S4.RESULTSDifferent Expression Patterns between the Rxrb and the Col11a2 Genes—To delineate differences in the transcriptional activities of Rxrb and Col11a2 in various tissues, we analyzed the expression of mRNAs of these genes in samples from identical tissues or cells. Northern hybridization analysis on tissue samples showed that Col11a2 mRNAs were abundant in limb buds but not in the intestine, brain, liver, or lung of mouse embryos at 16.5 days postcoitus (Fig. 1A). The Rxrb mRNAs were clearly detected in all of these tissues. Northern hybridization analysis on cultured cells showed that Col11a2 mRNA was abundant in RCS but not in L6 (rat skeletal muscle), FR (rat fibroblast), PC12 (rat adrenal pheochromocytoma), and H4IIE (rat hepatoma) cells. The Rxrb mRNAs were clearly detected in all these cells except for the RCS cells. Real-time RT-PCR analysis on cultured cells revealed that relative mRNA expression levels of Col11a2 to Gapdh were much higher in RCS as compared with the levels in L6, FR, PC12, and H4IIE cells (Fig. 1B). The relative mRNA expression levels of Rxrb were low in RCS cells as compared with the levels in other cells. In situ hybridization performed on semi-serial sections showed that Col11a2 mRNA was highly and specifically expressed in cartilage (Fig. 1C). Rxrb mRNA was detected in various tissues, with abundant expression in muscle. Signals for Rxrb mRNA were present in cartilage; these signals were weak compared with the signals in other tissues. In summary, we confirmed the previous reports that the Col11a2 gene was transcribed specifically in cartilage. We found that Rxrb was expressed in a variety of tissues; interestingly, Rxrb was weakly expressed in cartilage.Lysine residues on the N-terminal tails of histones H3 and H4 are more highly acetylated in the neighborhood of transcriptionally active promoters and enhancers than those in regions of transcriptional inactivity (30Kuo M.H. Zhou J. Jambeck P. Churchill M.E. Allis C.D. Genes Dev. 1998; 12: 627-639Crossref PubMed Scopus (395) Google Scholar). We examined the acetylation levels and patterns of histone H3 from the Rxrb to Col11a2 genes using RCS and L6 cells (Fig. 2A). A ChIP assay with anti-acetylated histone H3 antibody showed that the promoter region of Rxrb and the promoter and first intron regions of Col11a2 were hyperacetylated as compared with other regions in RCS cells. Acetylation levels of histone H3 were low except for the promoter region of Rxrb in L6 cells; these results are consistent with the expression levels of Rxrb and Col11a2 in L6 cells.FIGURE 2Histone H3 acetylation at Rxrb/Col11a2 loci and the enhancer activities of the 1st intron of Col11a2 to heterologous promoters. A, histone H3 acetylation across the Rxrb/Col11a2 genes. The graphs show the relative chromatin immunoprecipitation of acetylated histone H3 from RCS cells (top) and L6 cells (middle). Co-immunoprecipitating sequences were detected by using real-time RT-PCR. The bottom section shows the name and position of primer pairs and FRET probes used for analysis. To locate the primer position, the translational start site of Col11a2 was set as +1. Error bars indicate mean ± S.D. (n = 3). B–D, transgenic mice bearing the LacZ reporter gene constructs driven by the Col11a2 enhancer linked to a heterologous promoter. B, four of eight AD-LacZ transgenic founder embryos with LacZ expression are shown. C, we obtained six AD-LacZ-Int transgenic founder embryos with LacZ expression. Four embryos showed cartilage-specific LacZ expression (4, 8, 19, and 22), and 2 embryos showed LacZ expression in other tissues (9 and 27). D, we obtained 4 Nf-LacZ-Int transgenic founder embryos with LacZ expression. Three embryos showed cartilage-specific LacZ expression (2, 17, and 19), and 1 embryo showed LacZ expression in various tissues including cartilage (12). Scale bars, 2 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of the First Intron Sequence of Col11a2 on Transcriptional Activities of Heterologous Promoters— Some enhancers are specific for certain promoters (31Butler J.E. Kadonaga J.T. Genes Dev. 2001; 15: 2515-2519Crossref PubMed Scopus (196) Google Scholar, 32Smale S.T. Genes Dev. 2001; 15: 2503-2508Crossref P
Referência(s)