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• W2068238048 abstract "We have previously identified a DNase I-hypersensitive site in the T cell receptor β locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Eβ and is induced during CD4−CD8− to CD4+CD8+thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARβ. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARβ represses Eβ-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARβ from the endogenous locus does not change T cell receptor β gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context. We have previously identified a DNase I-hypersensitive site in the T cell receptor β locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Eβ and is induced during CD4−CD8− to CD4+CD8+thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARβ. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARβ represses Eβ-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARβ from the endogenous locus does not change T cell receptor β gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context. T cell receptor β TCRβ gene enhancer immunoglobulin heavy chain immunoglobulin κ light chain nuclear matrix attachment region DNase I-hypersensitive sites electrophoretic mobility shift assay CD4−CD8− double negative CD4+CD8+ double positive base pair(s) kilobase pair(s) dithiothreitol. The rearrangement and expression of the T cell receptor β (TCRβ)1 gene is essential to early T lymphocyte development (1Levelt C.N. Eichmann K. Immunity. 1995; 3: 667-672Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Prior to TCRβ gene rearrangement, germline transcripts are initiated upstream of the Dβ1 gene segment in CD4−CD8−(double negative, DN) thymocytes (2Godfrey D.I. Kennedy J. Mombaerts P. Tonegawa S. Zlotnik A. J. Immunol. 1994; 152: 4783-4792PubMed Google Scholar, 3Malissen M. Gillet A. Ardouin L. Bouvier G. Trucy J. Ferrier P. Vivier E. Malissen B. EMBO J. 1995; 14: 4641-4653Crossref PubMed Google Scholar). Recombination of TCRβ gene occurs exclusively during the DN stage of thymocyte development by an ordered process wherein Dβ-Jβ rearrangement occurs prior to joining of a Vβ gene segment. Allelic exclusion operating at the level of Vβ to DβJβ rearrangement ensures that only one of the two possible TCRβ alleles are expressed by an individual T cell (4Sleckman B.P. Gorman J.R. Alt F.W. Annu. Rev. Immunol. 1996; 14: 459-481Crossref PubMed Scopus (261) Google Scholar, 5Schlissel M.S. Stanhope-Baker P. Semin. Immunol. 1997; 9: 161-170Crossref PubMed Scopus (77) Google Scholar). After VβDβJβrearrangement, a mature transcript is initiated from the Vβ promoter in a T cell-specific manner (6Leiden J.M. Annu. Rev. Immunol. 1993; 11: 539-570Crossref PubMed Scopus (136) Google Scholar). To achieve the lineage-, stage-, and allele-specific TCRβ gene rearrangement and transcription, many cis-acting elements and their associated trans-acting factors are likely to be involved (4Sleckman B.P. Gorman J.R. Alt F.W. Annu. Rev. Immunol. 1996; 14: 459-481Crossref PubMed Scopus (261) Google Scholar, 5Schlissel M.S. Stanhope-Baker P. Semin. Immunol. 1997; 9: 161-170Crossref PubMed Scopus (77) Google Scholar). To date, the TCRβ gene enhancer (Eβ) is the only cis-regulatory element demonstrated to be required for both the lineage- and stage-specific transcription and rearrangement of the TCRβ gene (7McDougall S. Peterson C.L. Calame K. Science. 1988; 241: 205-208Crossref PubMed Scopus (61) Google Scholar, 8Krimpenfort P. de Jong R. Uematsu Y. Dmebic Z. Ryser S. von Boehmer H. Steinmetz M. Berns A. EMBO J. 1988; 7: 745-750Crossref PubMed Scopus (112) Google Scholar, 9Capone M. Watrin F. Fernex C. Horvat B. Krippl B. Wu L. Scollay R. Ferrier P. EMBO J. 1993; 12: 4335-4346Crossref PubMed Scopus (119) Google Scholar, 10Okada A. Mendelsohn M. Alt F. J. Exp. Med. 1994; 180: 261-272Crossref PubMed Scopus (74) Google Scholar, 11Bories J.-C. Demengeot J. Davidson L. Alt F.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7871-7876Crossref PubMed Scopus (139) Google Scholar, 12Bouvier G. Watrin F. Naspetti M. Verthuy C. Naquet P. Ferrier P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7877-7881Crossref PubMed Scopus (160) Google Scholar). Although the Vβ promoter is required for lineage-specific TCRβ transcription, its role in regulating Vβ gene rearrangement remains unclear (13Alvarez J.D. Anderson S.J. Loh D.Y. J. Immunol. 1995; 155: 1191-1202PubMed Google Scholar, 14Anderson S.J., S., C.H. Loh D.Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3551-3554Crossref PubMed Scopus (50) Google Scholar, 15Anderson S.J. Miyake S. Loh D.Y. Mol. Cell. Biol. 1989; 9: 4835-4845Crossref PubMed Scopus (36) Google Scholar, 16Lee M.-R. Chung C.S. Liou M.L. Wu M. Li W.F. Hsueh Y.P. Lai M.Z. J. Immunol. 1992; 148: 1906-1912PubMed Google Scholar, 17Halle J.-P. Haus-Seuffert P. Woltering C. Stelzer G. Meisterernst M. Mol. Cell. Biol. 1997; 17: 4220-4229Crossref PubMed Google Scholar). In addition to Vβ promoters and Eβ, there are likely other cis-regulatory elements involved in the control of various aspects of TCRβ gene rearrangement and transcription. In particular, nuclear matrix attachment regions (MAR) are a class of cis-regulatory elements found in many genetic loci that are distinct from transcriptional promoters and enhancers, and yet are often closely associated with these regulatory elements (18Blasquez V.C. Sperry A.O. Cockerill P.N. Garrard W.T. Genome. 1989; 31: 503-509Crossref PubMed Scopus (65) Google Scholar, 19Berezney R. Coffey D.S. Biochem. Biophys. Res. Commun. 1974; 60: 1410-1417Crossref PubMed Scopus (786) Google Scholar, 20Gasser S.M. Laemmli U.K. Trends Genet. 1987; 3: 16-22Abstract Full Text PDF Scopus (353) Google Scholar). MARs are typically AT-rich DNA sequences that bind to the nuclear matrix, often contain topoisomerase II cleavage sites, and exhibit a propensity for base unpairing when subjected to superhelical strain (21Bode J. Kohwi Y. Dickinson L. Joh T. Klehr D. Mielke C. Kohwi-Shigematsu T. Science. 1992; 255: 195-197Crossref PubMed Scopus (383) Google Scholar, 22Cockerill P.N. Garrard W.T. FEBS Lett. 1986; 204: 5-7Crossref PubMed Scopus (102) Google Scholar). They have been proposed to be involved in transcription, DNA recombination, replication, and repair (23Boulikas T. Int. Rev. Cytol. 1995; 162A: 279-388PubMed Google Scholar). In the immunoglobulin heavy chain (IgH) locus, MARs flank the intronic enhancer Eμ and are in close proximity to VH promoters (22Cockerill P.N. Garrard W.T. FEBS Lett. 1986; 204: 5-7Crossref PubMed Scopus (102) Google Scholar, 23Boulikas T. Int. Rev. Cytol. 1995; 162A: 279-388PubMed Google Scholar, 24Cockerill P.N. Nucleic Acids Res. 1990; 18: 2643-2648Crossref PubMed Scopus (61) Google Scholar, 25Webb C.F. Das C. Eneff K.L. Tucker P.W. Mol. Cell. Biol. 1991; 11: 5206-5211Crossref PubMed Scopus (48) Google Scholar, 26Avitahl N. Calame K. Int. Immunol. 1996; 8: 1359-1366Crossref PubMed Scopus (9) Google Scholar). Reporter gene assays in cell lines and transgenic mice have suggested that these MARs exert both positive and negative effects on IgH gene transcription and promote long range chromatin accessibility (27Wasylyk C. Wasylyk B. EMBO J. 1986; 5: 553-560Crossref PubMed Scopus (62) Google Scholar, 28Scheuermann R.H. Chen U. Genes Dev. 1989; 3: 1255-1266Crossref PubMed Scopus (89) Google Scholar, 29Forrester W.C. van Genderen C. Jenuwein T. Grosschedl R. Science. 1994; 265: 1221-1225Crossref PubMed Scopus (207) Google Scholar, 30Oancea A.E. Berru M. Shulman M.J. Mol. Cell. Biol. 1997; 17: 2658-2668Crossref PubMed Scopus (37) Google Scholar, 31Jenuwein T. Forrester W.C. Fernandez-Herrero L.A. Laible G. Dull M. Grosschedl R. Nature. 1997; 385: 269-272Crossref PubMed Scopus (226) Google Scholar). A highly conserved MAR is also found 200 base pairs (bp) upstream of the intronic immunoglobulin κ (Igκ) enhancer in mouse, human, and rabbit (22Cockerill P.N. Garrard W.T. FEBS Lett. 1986; 204: 5-7Crossref PubMed Scopus (102) Google Scholar, 32Whitehurst C. Henney H.R. Max E.E. Schroeder H.W.J. Stuber F. Siminovitch K.A. Garrard W.T. Nucleic Acids Res. 1992; 20: 4929-4930Crossref PubMed Scopus (28) Google Scholar). Together, the Igκ MAR and enhancer promote demethylation, transcription, recombination, and somatic hypermutation of the locus although no specific function has been attributed to the MAR alone (33Lichtenstein M. Keini G. Cedar H. Bergman Y. Cell. 1994; 76: 913-923Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 34Betz A.G. Milstein C. González-Fernández A. Pannell R. Larson T. Neuberger M.S. Cell. 1994; 77: 239-248Abstract Full Text PDF PubMed Scopus (361) Google Scholar, 35Xu Y. Davidson L. Alt F.W. Baltimore D. Immunity. 1996; 4: 377-385Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 36Goyenechea B. Klix N. Yélamos J. Williams G.T. Riddell A. Neuberger M.S. Milstein C. EMBO J. 1997; 16: 3987-3994Crossref PubMed Scopus (99) Google Scholar). The presence of MARs at other antigen receptor loci has not been reported. To characterize novel cis-regulatory elements involved in controlling TCRβ gene rearrangement and/or transcription, we previously screened a 100-kb region of the TCRβ locus and identified along with Eβ 10 additional DNase I-hypersensitive sites (HS) (37Chattopadhyay S. Whitehurst C. Schwenk F. Chen J. J. Immunol. 1998; 160: 1256-1267PubMed Google Scholar). HS1 was previously shown to be just 400 bp upstream of Eβand to be strongly induced during DN to DP thymocyte differentiation. In this report, we localize nuclear factor binding sites within HS1, characterize two nuclear factors that bind to HS1, demonstrate that HS1 is a nuclear matrix attachment region, and reveal the potential pitfalls of functional analyses of MARs outside of their natural chromosomal context. A 780-bpBstXI-BglII DNA fragment containing HS1 was blunt-ended by Klenow fragment of DNA polymerase I, gel-purified, and cloned into the SmaI site of Bluescript SK(+) (p780; Stratagene, La Jolla, CA). Probe II (300-bpAccI-BglII fragment), probe II* (300-bpAccI-BglII fragment with a deletion of 30 bp from the BglII end), probe III (128-bpAccI-BsgI fragment), and probe IV (170-bpBsgI-BglII fragment) were isolated from p780 plasmid and subcloned into the SmaI site of pSP72 (Fig. 1 A; Promega, Madison, WI). DNA fragments containing these probes (II, II*, III, and IV) were isolated from the resulting plasmids by XhoI-EcoRI digestion. Probes V and VIII were isolated from plasmid containing probe II* byBsgI-EcoRI or MboII-EcoRI digestion, respectively. Probes VI, VII, and IX were obtained from plasmid containing probe IV by digestion withMboII-EcoRI, BsmAI-EcoRI, or XhoI-BsmAI, respectively. Luciferase reporter constructs were prepared using the pGL2 promoter vector (Promega, Madison, WI). The existing SV40 promoter in pGL2 was deleted by BglII-HindIII digestion, and a 424-bpEcoRI-NcoI fragment containing the Vβ13 promoter was inserted to generate construct 1 (Fig. 8 A). Construct 1 was then used to generate constructs 2 and 3. An 830-bp BglII-NcoI fragment containing Eβ was cloned into the BamHI site located downstream of the poly(A) site of the luciferase gene, generating construct 2. A 1000-bp BsgI-NcoI fragment containing HS1 and Eβ in their natural configuration was cloned into the same position of construct 1 to generate construct 3. Therefore, construct 3 differed from construct 2 only by having the 170-bp HS1 (Fig. 8 A). Nuclear extracts were prepared from DN and DP thymocytes of RAG-deficient mice and RAG-deficient mice complemented with an activated lck transgene, respectively (37Chattopadhyay S. Whitehurst C. Schwenk F. Chen J. J. Immunol. 1998; 160: 1256-1267PubMed Google Scholar). Nuclei from thymocytes were prepared following the method of Forresteret al. (38Forrester W.C. Epner E. Driscoll M.C. Enver T. Brice M. Papayannopoulou T. Groudine M. Genes Dev. 1990; 4: 1637-1649Crossref PubMed Scopus (389) Google Scholar). Nuclei were resuspended in 0.2 ml of buffer A containing 10 mm Tris-Cl, pH 7.5, 10 mm HEPES, 10 mm MgCl2, 1 mm DTT, 50 mm NaCl, and 20% glycerol. An equal volume of buffer A supplemented with 420 mm NaCl was then added slowly to the nuclei suspension and mixed immediately. The resulting mixture was incubated on ice for 10 min and then centrifuged at 14,000 ×g for 5 min at 4 °C. Supernatants were then dialyzed against a low salt buffer containing 20 mm Tris-Cl, pH 7.5, 10 mm MgCl2, 1 mm DTT, 50 mm NaCl, and 10% glycerol, and were dispersed into small aliquots and stored at −80 °C. Protein concentrations of nuclear extracts were determined using a protein assay kit from Bio-Rad. All DNA probes for EMSA were end-labeled with [α-32P]dCTP using Klenow fragment of DNA polymerase I. Reaction mixtures were passed through a Nuc-trap column (Stratagene, La Jolla, CA) and precipitated. The labeled DNA fragments were further purified on a 5% polyacrylamide gel. For EMSA, 0.2–20 μg of nuclear extract or 1.25–5 ng of purified Cux/CDP protein were incubated with 2 ng of labeled DNA probes (∼20,000 cpm) in 24 μl of reaction buffer containing 10 mm HEPES-KOH, 10 mm Tris glutamate, pH 8.0, 100 mm NaCl, 10 mm magnesium glutamate, 50 mm potassium glutamate, 10% glycerol, 2 mmDTT, and 100 μg/ml of poly(dI-dC) (Sigma) at room temperature for 10 min. For EMSA in the presence of antiserum, probes were first incubated with the nuclear extracts as above and then 5 μl of diluted antiserum or control preimmune serum were added and incubated for another 10 min. Anti-Cux/CDP antiserum was kindly provided by Dr. Ellis Neufeld of Children's Hospital (Boston, MA), anti-SATB1 antiserum by Dr. Terumi Kohwi-Shigematsu of Lawrence Berkeley Laboratory (Berkeley, CA), and purified Cux/CDP protein by Dr. Richard Scheuermann of the University of Texas Southwestern Medical Center (Houston, TX). For EMSA in the presence of competing oligonucleotides, probes were mixed with competing oligonucleotides first and then nuclear extract was added into the mixture and incubated as above. DIST (5′-GCTTTTCAGTTGACCGGTGATTATTAGCCAATTTCTGATAAAAAGAAAAGGAAACCGATTGC-3′) and γ-globin (5′-TGCCTTGACCAATAGCCTTGACAAGGCAAACTTGACCAATAGTCTTAGAGTATCCAGTG-3′) oligonucleotides were from Dr. Ellis Neufeld, and CD8α MAR was isolated as a 200-bp PstI-EcoRI fragment from a plasmid kindly provided by Dr. Paul Gottlieb of the University of Texas (Austin, TX). Fifteen μl of the incubation mixture were electrophoresed in a 4.5% polyacrylamide gel (acrylamide:bisacrylamide, 40:1) in 0.5× TBE (Tris borate-EDTA) buffer at 15 V/cm for 4 h in the cold room. Gels were dried and autoradiographed. Soluble nuclear matrix was isolated from DP thymocytes following the standard protocol (22Cockerill P.N. Garrard W.T. FEBS Lett. 1986; 204: 5-7Crossref PubMed Scopus (102) Google Scholar). The matrix binding assay was performed following the method described by Zong and Scheuermann (39Zong R.-T. Scheuermann R.H. J. Biol. Chem. 1995; 270: 24010-24018Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) with minor changes. Briefly, 10 μl of nuclear matrix (corresponding to 1 × 107 nuclei) was incubated with 2 ng of each labeled probe, and 20 or 40 μg of sonicatedEscherichia coli DNA at the room temperature for 1 h in a total volume of 100 μl containing 10 mm Tris-Cl, pH 7.4, 50 mm NaCl, 2 mm EDTA, 0.5 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 0.25 mg/ml bovine serum albumin. The mixture was centrifuged for 5 min and the pellet was washed in 1 ml washing buffer containing 10 mm Tris-Cl, pH 7.4, 50 mm NaCl, 2 mm EDTA, and 0.25 mg/ml bovine serum albumin. The nucleoprotein complex was treated with proteinase K in the presence of 10 mm Tris-Cl, pH 7.4, 2 mm EDTA, and 5 μg/ml salmon sperm DNA at 37 °C overnight. The mixture was extracted with phenol-chloroform, and DNA was recovered by alcohol precipitation. Bound DNA was resolved on a 4.5% acrylamide gel and visualized by autoradiography. The expression cDNA library was kindly provided by Dr. Linda Clayton of the Dana-Farber Cancer Institute (Boston, MA). The library was constructed in λ ZAP Express vector (Stratagene, La Jolla, CA) using cDNA prepared from mRNA of DP thymocytes. To identify cDNA clones that encode proteins capable of binding probe IV (40Singh H. LeBowitz J.H. Baldwin A.S. P. A. S. Cell. 1988; 52: 415-423Abstract Full Text PDF PubMed Scopus (419) Google Scholar), 5 × 104 phages were plated per 150-mm plate (a total of 13 plates were screened). Plates were incubated for 3 h at 42 °C until tiny plaques were visible. Plates were then moved to a 37 °C incubator, overlaid with nitrocellulose filters that have been soaked in 10 mmisopropyl-1-thio-β-d-galactopyranoside, and incubated for 6 h. Filters were blocked with BLOTTO containing 5% Carnation nonfat milk powder, 50 mm Tris-Cl, pH 7.5, 50 mm MaCl, 1 mm EDTA, and 1 mm DTT and then hybridized with 32P-labeled probe IV in the binding buffer containing 50 mm Tris-Cl, pH 7.5, 50 mm NaCl, 1 mm EDTA, 1 mm DTT, and 5 μg/ml sonicated calf thymus DNA. Filters were washed in the binding buffer and exposed to x-ray film. Putative positive plaques were isolated and screened for two more rounds as above until single positive plaques were obtained. Thymoma line P4890 and P4833 were derived from DN thymocytes of mice deficient in both p53 and RAG1. Thymoma line θ4b was derived from a DP thymocyte of a mouse deficient in both p53 and TCRα (41Mombaerts P. Terhorst C. Jacks T. Tonegawa S. Sancho J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7420-7424Crossref PubMed Scopus (47) Google Scholar). Thymoma EL4 was derived from a CD4+ thymocyte. M12, a murine B cell lymphoma, was also used in the assays. Cells were maintained in Dulbecco's modified Eagle's medium containing 10 mmHEPES, 10% heat-inactivated fetal bovine serum, 50 μmβ-mercaptoethanol, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. For transient transfection, 2 × 107 cells were mixed with 10 μg of construct DNA and 10 μg of control CMV-β-galactosidase plasmid in 0.5 ml of RPMI. The mixture was electroporated at 300 V and 960 microfarads at room temperature. Cells were immediately resuspended in 10 ml of Dulbecco's modified Eagle's-HEPES medium and cultured for 48 h. Cell lysates were prepared, and luciferase activity was measured using a luciferase assay kit from Promega (Madison, WI). β-Galactosidase activity was measured byO-nitrophenyl-β-d-galactopyranoside assay. Luciferase activity of individual transfection was normalized to β-galactosidase activity of the same sample. Total RNA was isolated from thymocytes of wild type and homozygous mutant mice that were deleted of HS1 from the TCRβ locus (37Chattopadhyay S. Whitehurst C. Schwenk F. Chen J. J. Immunol. 1998; 160: 1256-1267PubMed Google Scholar). RNA was fractionated on a formaldehyde gel and transferred onto Zeta-probe filters. Filters were hybridized individually with a Cβ2 probe, a Vβ8 probe, or a Dβ1-Jβ1 intronic probe and exposed to x-ray films. The probes used were as follows: Cβ2 probe, a 430-bp cDNA fragment; Vβ8 probe, 190-bpEcoRI-StuI fragment containing the Vβ8 gene segment; and Dβ1-Jβ1 intronic probe, a 663-bp PCR product. HS1 was previously mapped to a 780-bpBstXI-BglII fragment located 400 bp upstream of Eβ (Fig. 1 A) (37Chattopadhyay S. Whitehurst C. Schwenk F. Chen J. J. Immunol. 1998; 160: 1256-1267PubMed Google Scholar, 42Hashimoto Y. J. Exp. Med. 1989; 169: 2097-2107Crossref PubMed Scopus (10) Google Scholar). To identify protein factors that bind to HS1, we performed EMSAs with nuclear extracts from DP thymocytes because HS1 was most prominent in these cells (37Chattopadhyay S. Whitehurst C. Schwenk F. Chen J. J. Immunol. 1998; 160: 1256-1267PubMed Google Scholar). Two subfragments of HS1 were used as probes in EMSA, a 480-bp BstXI-AccI fragment (probe I) and a 300-bp AccI-BglII fragment (probe II, Fig. 1 A). While probe I failed to yield any complex, probe II readily generated two shifted complexes in EMSA (Fig. 1 B, lanes 1–4). The same complexes were also observed when the entire 780-bpBstXI-BglII fragment was used as a probe, although at a substantially reduced level probably due to the large size of the probe (data not shown). We next sought to narrow down the location of the factor binding sites within probe II. Previously, sequence analyses showed the presence of consensus motifs of AP-1, Oct-1, and c-Myb binding sites in the 5′ region of the 300-bp probe II in both mouse and human (37Chattopadhyay S. Whitehurst C. Schwenk F. Chen J. J. Immunol. 1998; 160: 1256-1267PubMed Google Scholar, 42Hashimoto Y. J. Exp. Med. 1989; 169: 2097-2107Crossref PubMed Scopus (10) Google Scholar). Therefore, two subfragments of probe II, a 128-bpAccI-BsgI fragment (probe III) and a 170-bpBsgI-BglII fragment (probe IV), were used in EMSA to test if factor binding occurred at any of these consensus sites. Despite the presence of the consensus nuclear factor binding sites within probe III, it did not generate any complex in EMSA (Fig. 1 B, lanes 5 and 6). Likewise, a slightly larger AccI- BsmAI fragment did not produce any complex (Fig. 1 A and data not shown). In contrast, probe IV detected the two shifted complexes as seen when probe II was used (Fig. 1 B, lanes 7and 8). Thus, although the 128-bp fragment (probe III) contained the various sequence motifs (42Hashimoto Y. J. Exp. Med. 1989; 169: 2097-2107Crossref PubMed Scopus (10) Google Scholar), the complexes detected under our reaction conditions resulted from nuclear factor binding to cis elements in the 170-bp BsgI-BglII region of HS1 (probe IV). The observed nuclear protein-DNA interactions were specific. First, the two complexes were stable in the presence of an excess amount of nonspecific competitor poly(dI-dC) (100 μg/ml). Second, a greater amount of complexes was formed with increasing amounts of DP thymocyte nuclear extract (Fig. 1 B). Finally, the formation of both complexes was specifically abolished by non-radiolabeled probe IV but not probe III in a competition experiment (Fig. 2). To further delineate the location of the nuclear factor binding sites within probe IV, smaller probes were generated by deleting sequences from the 5′ and 3′ flanks of probe IV and used in EMSA (Fig. 1). Probe V, which was 30 bp shorter than probe IV at the 3′ end formed the lower complex at a level comparable to that of probe IV, but did not efficiently form the upper complex, as only a miniscule amount was detected (Fig. 1 B, lanes 9 and 10). This observation suggested that the upper and lower complexes were formed by factor binding to two different regions of probe IV. Factor binding at the far 3′ region of the probe produces the upper complex and factor binding somewhere else in the probe produces the lower complex. Supporting this notion, probe VI, which has 21 bp removed from the 5′ end of probe IV, formed only approximately a third as much of the lower complex as probe IV (Fig. 1 B, lanes 11 and 12). An additional 3-fold reduction in the formation of the lower complex resulted with probe VII, which had additional 33 bp removed from the 5′ end of probe VI (Fig. 1, A and B, lanes 13and 14). In contrast, in both of these cases, the upper complex was not significantly reduced, confirming its probable binding to the far 3′ region of probe IV. The binding site for the formation of the lower complex was probably broad because deletion from 5′ end of probe IV substantially reduced the amount of the complex and probe IX containing only the 5′ third of probe IV did not form a complex (Fig. 1, A and B, lanes 17 and 18). The 3′ region of probe IV appears to stabilize the lower complex because no complexes were formed using probe VIII that had 30 bp deleted from the 3′ end of probe VI (Fig. 1 B, lanes 15 and 16). In summary, these data suggest that there are two major factor binding sites in the 170-bp BsgI-BglII fragment (probe IV) of HS1. We have previously shown that HS1 is induced during DN to DP thymocyte differentiation. In DN thymocytes of RAG2-deficient mice, HS1 is barely detectable, whereas HS1 is a major DNase I-hypersensitive site in DP thymocytes of RAG2-deficient mice that have been complemented with a functional TCRβ or activatedlck transgene, or have been treated with anti-CD3ε antibody (37Chattopadhyay S. Whitehurst C. Schwenk F. Chen J. J. Immunol. 1998; 160: 1256-1267PubMed Google Scholar). We reasoned that if the formation of the complexes in EMSA correlated with HS1 formation, we should detect less complex formation when nuclear extracts from DN thymocytes are used in EMSA. To test this, we performed EMSA using probe IV and nuclear extracts prepared from DN thymocytes of RAG2-deficient mice and from DP thymocytes of RAG2-deficient mice that have been complemented with an activated lck transgene. As shown in Fig. 3, both the upper and lower complexes were formed using nuclear extracts from DN thymocytes; however, the amounts of complexes yielded were only a quarter of those formed by the equivalent amounts of nuclear extracts from DP thymocytes. Thus, the formation of both the upper and lower complexes was induced in DP thymocytes, correlating with the induction of HS1 during DN to DP thymocyte differentiation. These findings support the possibility that the nuclear factors detected by probe IV in EMSA are directly involved in the formation of HS1. To determine the identity of protein factors in the upper and lower complexes, we screened an expression cDNA library constructed in λ phage using mRNA from DP thymocytes with the 170-bp probe IV. Five positive plaques were obtained from screening 6.5 × 105 phages. DNA was prepared from all five phage clones and analyzed by restriction enzymes. Of the five clones, two contained cDNA inserts that gave distinct restriction patterns (data not shown), indicating that they were unique. The other three clones contained related cDNA inserts, as indicated by the same sizes and restriction patterns (data not shown). Sequence analysis revealed that one of the unique cDNA inserts encoded the deoxycytidine kinase. Considering the function of the deoxycytidine kinase, its binding to probe IV was probably nonspecific. The second unique phage clone contained a 4-kb cDNA insert identical to the 3′ region of the mouse Cux gene (CDP in human) (43Bodmer K. Barbel S. Shepherd S. Jack J.W. Jan L.Y. Jan Y.N. Cell. 1987; 51: 293-307Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 44Ludlow C. Choy R. Blochlinger K. Dev. Biol. 1996; 178: 149-159Crossref PubMed Scopus (47) Google Scholar, 45Valarche I. Tissier-Seya J.-P. Hirsch M.-R. Martiney S. Garidis C. Brunet J.-F. Development. 1993; 119: 881-896Crossref PubMed Google Scholar, 46Neufeld E.J. Skalnik D.G. Lievens P.M.-J. Orkin S.H. Nat. Genet. 1992; 1: 50-55Crossref PubMed Scopus (186) Google Scholar). The other three phage clones contained a cDNA whose sequences did not match any known genes, although identical sequence fragments were found in EST data base. Thus, this cDNA encodes a novel protein; whether it binds to HS1 specifically is currently under investigation. Cux/CDP is a homeodomain protein originally identified in Drosophila (called Cut) and later in mouse and human (43Bodmer K. Barbel S. Shepherd S. Jack J.W. Jan L.Y. Jan Y.N. Cell. 1987; 51: 293-307Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 44Ludlow C. Choy R. Blochlinger K. Dev. Biol. 1996; 178: 149-159Crossref PubMed Scopus (47) Google Scholar, 45Valarche I. Tissier-Seya J.-P. Hirsch M.-R. Martiney S. Garidis C. Brunet J.-F. Development. 1993; 119: 881-896Crossref PubMed Google Scholar, 46Neufeld E.J. Skalnik D.G. Lievens P.M.-J. Orkin S.H. Nat. Genet. 1992; 1: 50-55Crossref PubMed Scopus (186) Google Scholar). In addition to its homeodomain, Cux/CDP also contains three cut repeats that can independently bind DNA of relatively degenerate consensus sequences (47Andrés V. Chiara M.D. Mahdavi V. Genes Dev. 1994; 8: 245-257Crossref PubMed Scopus (96) Google Scholar, 48Aufiero B. Neufeld E.J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7757-7761Crossref PubMed Scopus (105) Google Scholar, 49Harada R. Dufort D. Denis-Laroses C. Nepreu A. J. Biol. Chem. 1994; 269: 2062-2067Abstract Full Text PDF PubMed Google Scholar). To determine whether Cux/CDP binds to HS1 specifically, we first performed a competition assay using DIST oligonucleotide derived from the promoter of cytochrome b heavy chain gene (gp91phox), which was previously characterized to efficiently and specifically bind Cux/CDP in EMSA (50Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 199" @default.
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