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• W2062068633 abstract "The human α-globin gene complex includes three functional globin genes (5′-ζ2-α2-α1–3′) regulated by a common positive regulatory element named HS-40 displaying strong erythroid-specific enhancer activity. How this enhancer activity can be shared between different promoters present at different positions in the same complex is poorly understood. To address this question, we used homologous recombination to target the insertion of marker genes driven by cytomegalovirus or long terminal repeat promoters in both possible orientations either upstream or downstream from the HS-40 region into the single human α-globin gene locus present in hybrid mouse erythroleukemia cells. We also used CRE recombinase-mediated cassette exchange to target the insertion of a tagged α-globin gene at the same position downstream from HS-40. All these insertions led to a similar decrease in the HS-40-dependent transcription of downstream human α-globin genes in differentiated cells. Interestingly, this decrease is associated with the strong activation of the proximal newly inserted α-globin gene, whereas in marked contrast, the transcription of the non-erythroid marker genes remains insensitive to HS-40. Taken together, these results indicate that the enhancer activity of HS-40 can be trapped by non-erythroid promoters in both upstream and downstream directions without necessarily leading to their own activation. The human α-globin gene complex includes three functional globin genes (5′-ζ2-α2-α1–3′) regulated by a common positive regulatory element named HS-40 displaying strong erythroid-specific enhancer activity. How this enhancer activity can be shared between different promoters present at different positions in the same complex is poorly understood. To address this question, we used homologous recombination to target the insertion of marker genes driven by cytomegalovirus or long terminal repeat promoters in both possible orientations either upstream or downstream from the HS-40 region into the single human α-globin gene locus present in hybrid mouse erythroleukemia cells. We also used CRE recombinase-mediated cassette exchange to target the insertion of a tagged α-globin gene at the same position downstream from HS-40. All these insertions led to a similar decrease in the HS-40-dependent transcription of downstream human α-globin genes in differentiated cells. Interestingly, this decrease is associated with the strong activation of the proximal newly inserted α-globin gene, whereas in marked contrast, the transcription of the non-erythroid marker genes remains insensitive to HS-40. Taken together, these results indicate that the enhancer activity of HS-40 can be trapped by non-erythroid promoters in both upstream and downstream directions without necessarily leading to their own activation. kilobase pair(s) mouse erythroleukemia hexamethylenebisacetamide long terminal repeat cytomegalovirus Louis control region phosphoglycerate kinase Human α-globin genes are clustered on a single complex located in the telomeric region of the short arm of chromosome 16. This complex includes three functional genes, the embryonic ζ2 gene and the two fetal/adult α2 and α1 genes, which are arranged in the order 5′-ζ2-α2-α1–3′ of their expression during development (1Higgs D.R. Higgs D.R. Weatherall D.J. Bailliere's Clinical Haematology. 6. Bailliere Tindall Ltd., London1993: 117-150Google Scholar). Although the α-globin genes are transcribed exclusively in erythroid cells, the whole complex is located in a GC-rich isochore, within an early replicating and constitutively DNase I-sensitive chromatin domain in both erythroid and non-erythroid cells (2Craddock C.F. Vyas P. Sharpe J.A. Ayyub H. Wood W.G. Higgs D.R. EMBO J. 1995; 14: 1718-1726Crossref PubMed Scopus (91) Google Scholar, 3Fischel-Ghodsian N. Nicholls R.D. Higgs D.R. Nucleic Acids Res. 1987; 15: 9215-9225Crossref PubMed Scopus (24) Google Scholar, 4Smith Z.E. Higgs D.R. Hum. Mol. Genet. 1999; 8: 1373-1386Crossref PubMed Scopus (72) Google Scholar). The human α-globin gene complex is also surrounded by several widely expressed genes, including the −14 gene of unknown function, which is located in the opposite transcriptional orientation, 14 kb1 upstream from the ζ2 gene (5Vickers M.A. Vyas P. Harris P.C. Simmons D.L. Higgs D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3437-3441Crossref PubMed Scopus (59) Google Scholar, 6Vyas P. Vickers M.A. Picketts D.J. Higgs D.R. Genomics. 1995; 29: 679-689Crossref PubMed Scopus (59) Google Scholar). Several studies have shown that the erythroid-specific transcriptional activation of all α-globin genes present in the locus is controlled by a single positive regulatory element, named HS-40, which corresponds to a DNase I-hypersensitive site located 40 kb upstream from the ζ2 gene (7Higgs D.R. Wood W.G. Jarman A.P. Sharpe J. Lida J. Pretorius I.M. Weatherall D.J. Genes Dev. 1990; 4: 1588-1601Crossref PubMed Scopus (258) Google Scholar, 8Jarman A.P. Wood W.G. Sharpe J.A. Gourdon G. Ayyub H. Higgs D.R. Mol. Cell. Biol. 1991; 11: 4679-4689Crossref PubMed Google Scholar). HS-40 is characterized by a high density of DNA-binding sites for ubiquitous and erythroid-specific transcription factors (8Jarman A.P. Wood W.G. Sharpe J.A. Gourdon G. Ayyub H. Higgs D.R. Mol. Cell. Biol. 1991; 11: 4679-4689Crossref PubMed Google Scholar, 9Rombel I. Zhang Q. Papayannopoulou T. Stamatoyannopoulos G. Shen C.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6454-6458Crossref PubMed Scopus (27) Google Scholar, 10Zhang Q. Rombel I. Reddy G.N. Gang J.B. Shen C.K. J. Biol. Chem. 1995; 270: 8501-8505Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is a strong erythroid-specific transcriptional enhancer in cell lines (7Higgs D.R. Wood W.G. Jarman A.P. Sharpe J. Lida J. Pretorius I.M. Weatherall D.J. Genes Dev. 1990; 4: 1588-1601Crossref PubMed Scopus (258) Google Scholar, 11Chen H. Lowrey C.H. Stamatoyannopoulos G. Nucleic Acids Res. 1997; 25: 2917-2922Crossref PubMed Scopus (18) Google Scholar, 12Pondel M.D. George M. Proudfoot N.J. Nucleic Acids Res. 1992; 20: 237-243Crossref PubMed Scopus (37) Google Scholar, 13Ren S. Luo X.N. Atweh G. Blood. 1993; 81: 1058-1066Crossref PubMed Google Scholar, 14Sharpe J.A. Chan-Thomas P.S. Lida J. Ayyub H. Wood W.G. Higgs D.R. EMBO J. 1992; 11: 4565-4572Crossref PubMed Scopus (65) Google Scholar), and it confers erythroid lineage-specific, autonomous, and appropriate developmental patterns of expression of either the ζ2 or α-globin promoter in transgenic mice (14Sharpe J.A. Chan-Thomas P.S. Lida J. Ayyub H. Wood W.G. Higgs D.R. EMBO J. 1992; 11: 4565-4572Crossref PubMed Scopus (65) Google Scholar, 15Gourdon G. Sharpe J.A. Wells D. Wood W.G. Higgs D.R. Nucleic Acids Res. 1994; 22: 4139-4147Crossref PubMed Scopus (42) Google Scholar, 16Robertson G. Garrick D. Wu W. Kearns M. Martin D. Whitelaw E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5371-5375Crossref PubMed Scopus (145) Google Scholar, 17Huang B.L. Fan-Chiang I.R. Wen S.-C. Koo H.C. Kao W.Y. Gavva N.R. Shen C.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14669-14674Crossref PubMed Scopus (18) Google Scholar). Despite this strong enhancer activity, the expression level of globin genes linked to the HS-40 element in transgenic mice remains sensitive to position effects, is not copy number-dependent, and tends to decrease in adults. Natural or targeted deletions of HS-40 lead to a complete loss of globin gene transcriptional activation in erythroid cells, but do not affect the DNase I sensitivity or the replication timing of the whole complex (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar, 19Hatton C.S. Wilkie A.O. Drysdale H.C. Wood W.G. Vickers M.A. Sharpe J. Ayyub H. Pretorius I.M. Buckle V.J. Higgs D.R. Blood. 1990; 76: 221-227Crossref PubMed Google Scholar, 20Liebhaber S.A. Griese E.U. Weiss I. Cash F.E. Ayyub H. Higgs D.R. Horst J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9431-9435Crossref PubMed Scopus (44) Google Scholar, 21Romao L. Osorio-Almeida L. Higgs D.R. Lavinha J. Liebhaber S.A. Blood. 1991; 78: 1589-1595Crossref PubMed Google Scholar). Furthermore, the expression level of the −14 gene is independent of HS-40 despite the location of HS-40 in its fifth intron (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar). The HS-40 regulatory element thus appears to be involved in the erythroid-specific and selective transcriptional activation of all globin genes belonging to the complex, but the mechanisms responsible for this selective activation are still poorly understood. One way to approach an understanding of these mechanisms is to investigate how this HS-40-mediated transcriptional activation of human α-globin genes can be affected by the insertion of new genes into the complex. In this study, we addressed these questions by using homologous recombination and CRE recombinase-mediated cassette exchange to target the insertion of either an extra α-globin gene or a marker gene driven by the non-erythroid promoter immediately upstream or downstream from HS-40 into the single chromosome 16 present in hybrid mouse erythroleukemia cells. We found that all of these insertions led to a similar drastic reduction of the HS-40-dependent transcription of resident α-globin genes, regardless of the position, the orientation, or the identity of the newly inserted gene. Although this down-regulation is associated with the strong activation of the newly inserted α-globin gene, this is not the case for newly inserted non-erythroid marker genes, the transcription of which appears to be independent of HS-40. Taken together, these results suggest that the enhancer activity of HS-40 spreads in both upstream and downstream directions and can be trapped by non-erythroid promoters without necessarily leading to their own activation. All experiments were performed in the mouse erythroleukemia (MEL) hybrid cell line LT585P3, which contains a single copy of normal human chromosome 16 (18, 22). Cells were cultured as already described (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar). Erythroid terminal differentiation of the cells was induced by the addition of 5 mm hexamethylenebisacetamide (HMBA; Sigma) to the culture medium for 4 days. All four plasmids used to target the insertion of the LTR-neo gene are derived from a single starting plasmid, pHS-40. This pHS-40 plasmid is based on pUC18 in which 9.2 kb of isogenic genomic DNA overlapping the HS-40 regulatory region has been cloned. This isogenic DNA was cloned as two cassettes: a 4.1-kb HindIII genomic fragment including HS-40 was directly cloned in pUC18, and the downstream adjacent 5.1-kbHindIII genomic fragment was cloned as aSalI-XhoI fragment to leave a uniqueSalI site for inserting the LTR-neo gene between the two isogenic cassettes. In addition, pHS-40 contains a herpes simplex virus thymidine kinase XhoI-HindIII gene cassette, derived from a pIC19R/MCI-tk plasmid (23Mansour S.L. Thomas K.R. Capecchi M.R. Cell. 1988; 44: 319-328Google Scholar), which was cloned immediately downstream from the 3′-homology arm. The LTR-neogene derives from the pGEM-I-FLP-neo plasmid (24Fiering S. Kim S.G. Epner E.E. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8469-8473Crossref PubMed Scopus (90) Google Scholar). It is driven by the enhancer/promoter of the Friend retrovirus long terminal repeat and is flanked on both sides by FLP recombinase targets (24Fiering S. Kim S.G. Epner E.E. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8469-8473Crossref PubMed Scopus (90) Google Scholar). The pHS-neoS targeting plasmid was thus obtained by subcloning the LTR-neo gene (taken as aSalI-XhoI fragment) into the singleSalI site of pHS-40 and in the same transcriptional orientation as that of resident α-globin genes. The pHS-neoAS targeting plasmid was obtained similarly by subcloning the LTR-neo gene cassette in reverse orientation. The other two plasmids used to target the insertion of the LTR-neo gene upstream from HS-40 were obtained from the same pHS-40 starting plasmid in which a 1.1-kb HpaI fragment including the HS-40 site was first removed and replaced by the LTR-neo gene (taken as a HincII fragment) in either orientation. The HpaI fragment containing HS-40 was then reinserted in the correct orientation into the singleSalI site remaining downstream from the LTR-neogene. The pneoS-HS targeting plasmid thus contains the LTR-neo gene in the same transcriptional orientation as that of the resident α-globin genes, whereas the pneoAS-HS targeting plasmid contains the LTR-neo gene in reverse orientation. The CMV-hygroTKgene, which is a hygroTK gene fusion driven by the cytomegalovirus promoter, was first isolated as a XhoI fragment from a plasmid kindly provided by Dr. P. Greenberg (Fred Hutchinson Research Center) and cloned using BamHI linkers between two inverted Lox sequences L1 and 1L (25Bouhassira E.E. Westerman K. Leboulch P. Blood. 1997; 90: 3332-3344Crossref PubMed Google Scholar, 26Feng Y.Q. Seibler J. Alami R. Eisen A. Westerman K. Leboulch P. Fiering S.N. Bouhassira E.E. J. Mol. Biol. 1999; 292: 779-785Crossref PubMed Scopus (169) Google Scholar). The resulting L1-CMV-hygroTK-1L cassette isolated as aXhoI-PvuII fragment was then subcloned into the single SalI site of plasmid pHS-40 in the same transcriptional orientation as that of resident α-globin genes, thus leading to the targeting plasmid pCMV-hygroTK. All targeting plasmids were linearized at the unique ScaI site present in the pUC18 sequence before transfection. The human αT-globin gene was previously cloned from the genomic DNA of an α+-thalassemic patient homozygous for the rightward 3.7-kb deletion that generates an α2/α1 fusion gene (27Morlé F. Lopez B. Henni T. Godet J. EMBO J. 1985; 4: 1245-1250Crossref PubMed Scopus (49) Google Scholar). This αT-globin gene carries a two-nucleotide deletion at positions −2 and −3 preceding the ATG initiation codon, which is responsible for reduced translation efficiency, but which does not affect the transcription of the gene (28Morlé F. Starck J. Godet J. Nucleic Acids Res. 1986; 14: 3279-3292Crossref PubMed Scopus (28) Google Scholar). The αT-globin gene cassette was taken as a 1.5-kb PstI fragment and cloned using BamHI linkers between two inverted Lox sequences L1 and 1L, thus generating the pαT gene exchange plasmid. Hybrid MEL cells (107) were transfected by electroporation using 20 μg of each linearized targeting plasmid as described previously (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar). Twenty-four hours after electroporation, surviving cells were plated in selective medium containing 0.6 mg/ml G418 (Life Technologies, Inc.) and 10 μm ganciclovir (Syntex Research Co.) for cells transfected with the pLTR-neo targeting plasmids or containing 1 mg/ml hygromycin (Life Technologies, Inc.) for cells transfected with the pCMV-hygroTK targeting plasmid. After 15 days, individual resistant clones were analyzed by Southern blotting using an HS-40 probe to identify homologous recombinant clones. A yeast FLP recombinase expression vector (pHook-3-FLP) was obtained by subcloning a XbaI fragment containing the FLP coding sequence driven by a cytomegalovirus promoter and derived from vector pCFIZ (24Fiering S. Kim S.G. Epner E.E. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8469-8473Crossref PubMed Scopus (90) Google Scholar) in the polylinker of the pHook-3 plasmid (Invitrogen), which itself contains a gene encoding Zeocin resistance. Cells (107) from clones harboring insertion of the LTR-neo gene were transfected by electroporation using 30 μg of pHook-3-FLP DNA. After 24 h, cells were placed in selective medium (300 μg/ml Zeocin; CAYLA). Among the Zeocin-resistant clones, G418-sensitive clones were analyzed by Southern blotting to verify excision of the LTR-neogene. Recombinase-mediated inversion of the CMV-hygroTKgene was obtained through the transient expression of CRE recombinase. For this purpose, 106 cells of a clone harboring targeted insertion of the L1-CMV-hygroTK-1L cassette were cotransfected using DAC-30 (Eurogentec), 1 μg of expression plasmid DNA encoding the CRE recombinase (pCMV-CRE), and 1 μg of expression plasmid DNA encoding green fluorescent protein (pSV40-GFP). Forty-eight hours following transfection, cells expressing green fluorescent protein were purified by fluorescence-activated cell sorting and recloned in the presence of hygromycin. Isolated clones were then amplified and analyzed by Southern blotting. Recombinase-mediated exchange of the hygroTK gene by the αT-globin gene was obtained similarly by cotransfecting 106 cells of a clone harboring targeted insertion of the CMV-hygroTK gene using 1 μg of pCMV-CRE, 1 μg of pSV40-GFP, and 2 μg of pαT plasmid carrying the tagged human α-globin gene flanked by two inverted Lox sequences. Purified green fluorescent protein-positive cells were then cloned in medium containing 10 μm ganciclovir. Individual ganciclovir-resistant clones were amplified and analyzed by Southern blotting to verify the insertion of the αT-globin gene. Total cellular RNA was prepared using RNA-plusTM(Quantum Biotechnologies) according to the manufacturer's instructions. RNase protection assays were performed as described previously (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar) using 8 μg of total RNA and the following labeled antisense RNA probes: (i) a human riboprobe (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar) that is expected to produce a single protected fragment of 133 nucleotides with normal α-globin mRNA and two protected fragments of 97 and 34 nucleotides with αT-globin mRNA carrying a deletion of two nucleotides at positions −2 and −3 preceding the AUG initiation codon; (ii) a mouse α-globin riboprobe that includes 180 nucleotides complementary to the 3′-end of the first exon of the mouse α-globin gene and that gives a protected fragment of 75 nucleotides with mouse α-globin mRNA; (iii) a neo riboprobe (pT3TKN) that is expected to produce a protected fragment of 260 nucleotides with transcripts of the LTR-neo gene (24Fiering S. Kim S.G. Epner E.E. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8469-8473Crossref PubMed Scopus (90) Google Scholar); (iv) a hygromycin riboprobe (pT7HY) that contains theBamHI-EcoRI fragment including the hygromycin coding sequence from the L1-CMV-hygroTK-1L gene and that is expected to produce a protected fragment of 258 nucleotides with transcripts of the CMV-hygroTK gene. Radioactive signals corresponding to each specific protected fragment were quantified using a GS-525 Molecular Imager (Bio-Rad) and Molecular Analyst software (Bio-Rad). Cells (108) from a 2-day culture in the presence of 5 mm HMBA were harvested by centrifugation, washed with phosphate-buffered saline, and lysed for 5 min on ice in buffer A (10 mm Tris, pH 7.5, 10 mm NaCl, and 2.5 mm MgCl2) containing 0.3% Nonidet P-40. Nuclei were pelleted by centrifugation through a 30% sucrose cushion made in buffer A, resuspended in glycerol buffer (50 mm Tris, pH 7.9, 75 mm NaCl, 0.1 mm EDTA, 50% glycerol, 0.5 mm dithiothreitol, and 0.5 mmphenylmethylsulfonyl fluoride), frozen, and stored in liquid nitrogen in 100-μl aliquots containing 5 × 107 nuclei. Nuclei were thawed on ice by adding an equal volume of transcription buffer (10 mm Tris-HCl, pH 8.0, 10 mmMgCl2, 10 mm MnCl2, and 300 mm KCl) supplemented with 5 mm each ATP, GTP and CTP; 100 μCi of [α-32P]UTP (3000 Ci/mm; Amersham Pharmacia Biotech), and 100 units/ml RNasin (Promega). Transcription reactions were carried out at 30 °C for 15 min and terminated by centrifugation for 30 s. Labeled nuclei were resuspended in 500 μl of 10 mm Tris, pH 7.5, 0.5m NaCl, and 10 mm MgCl2 containing 40 units of RNase-free DNase (Roche Molecular Biochemicals) and incubated at 30 °C for 15 min. Deproteinization was performed for 30 min at 37 °C after the addition of proteinase K (500 μg/ml) and SDS (0.5%). Labeled RNA was extracted with phenol/chloroform, ethanol-precipitated in the presence of 2 m ammonium acetate, and incubated for 15 min at 37 °C in DNase buffer (10 mm Tris, pH 7.5, and 10 mm MgCl2) containing 10 units of RNase-free DNase. RNA was purified again by phenol/chloroform extraction and two rounds of ethanol precipitation, resuspended in water, and hybridized to membranes (Hybond-C Extra, Amersham Pharmacia Biotech) loaded with unlabeled DNA probes. Membranes were loaded using a slot-blot apparatus with a 5 μg of DNA/slot concentrations of the following denatured DNA probes: the pMC1neo plasmid (23Mansour S.L. Thomas K.R. Capecchi M.R. Cell. 1988; 44: 319-328Google Scholar), mouse β-major globin 5-kbEcoRI fragment, and pGEMT plasmid. Membranes were prehybridized for 4 h at 42 °C in 50% formamide, 6× saline/sodium phosphate/EDTA, 5× Denhardt's solution, 0.1% SDS, and 20 μg/ml yeast tRNA and hybridized overnight with labeled RNA under the same conditions. Membranes were washed for 15 min in 1× saline/sodium phosphate/EDTA and 0.1% SDS at room temperature and for 5 min in 0.1× saline/sodium phosphate/EDTA and 0.1% SDS at 65 °C. Hybridization signals were revealed by autoradiography and quantified using the Molecular Imager and Molecular Analyst software. Four different DNA constructs were designed to target the insertion of an LTR-neo gene on either side of HS-40 and in both possible orientations by homologous recombination (Fig.1 A). Each of these DNA constructs was introduced by electroporation into hybrid MEL cells carrying a single copy of human chromosome 16. G418- and ganciclovir-resistant clones obtained were analyzed individually by Southern blotting using a human HS-40 hybridization probe. As schematically presented in Fig. 1 A, the targeted integration of the LTR-neo gene was expected to lead to the conversion of the normal 20-kb BamHI fragment to shorterBamHI fragments of 11.3, 12.3, 9, or 14.7 kb depending on the transfected targeting DNA construct. Five correctly targeted clones were identified among 301 clones obtained after transfection with the pHS-neoS construct (Fig. 1 B, lanes 3–7), and one correctly targeted clone was identified among 300, 150, or 225 clones obtained after transfection with the pneoS-HS (lane 8), pHS-neoAS (lane 9), or pneoAS-HS (lane 10) construct. Further Southern blot analyses using other restriction endonucleases and either an HS-40 or a human α-globin probe confirmed that all eight clones displayed the expected targeted insertions (data not shown). The eight targeted clones described above as well as parental cells and the previously described clone containing a targeted replacement of HS-40 by the same LTR-neo gene (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar) were grown for 4 days in the presence or absence of HMBA, a chemical inducer of differentiation. Equal amounts of total cellular RNA prepared from induced and uninduced cells were then analyzed by RNase protection assay using a mixture of probes allowing the specific detection of human and mouse α-globin and neo gene transcripts. Typical results obtained from three different experiments are shown in Fig. 2 A. These data indicate that the eight clones harboring a targeted insertion of the LTR-neo gene in the vicinity of HS-40 displayed a marked reduction of the HMBA-induced increase in the expression of human α-globin genes (Fig. 2 A, comparelanes 13–20 with lane 12). Quantitative analysis revealed that the levels of human α-globin mRNA in uninduced cells did not significantly differ between parental cells and cells harboring insertions of the LTR-neogene either adjacent to or in place of HS-40 (Fig. 2 B). In contrast, the levels of human α-globin mRNA in induced cells were markedly reduced in all cells harboring insertions of the LTR-neo gene compared with parental cells (Fig.2 C). Interestingly, the levels of human α-globin mRNA in the eight clones harboring insertions of the LTR-neo gene in the vicinity of HS-40 still remained 5–25-fold above the background level observed in clone ΔHS-neo (Fig. 2 C, compare bars 2–9 with bar 10), in which the LTR-neo gene has been inserted in place of HS-40 (18Bernet A. Sabatier S. Picketts D.J. Ouazana R. Morlé F. Higgs D.R. Godet J. Blood. 1995; 86: 1202-1211Crossref PubMed Google Scholar). These levels correspond to a 3–8-fold reduction of the human α-globin mRNA level observed in induced parental cells compared with a >60-fold reduction in clone ΔHS-neo. Since only one example of clones neoS-HS, HS-neoAS, and neoAS-HS could be analyzed, the possibility remained that the reduction of the levels of human α-globin mRNA in these clones was due to clonal variation rather than the direct effect of the inserted LTR-neo gene. To exclude this possibility, we verified that excision of the LTR-neo gene in these three clones was indeed able to rescue a level of human α-globin mRNA in induced cells similar to that in parental cells. For this purpose, each clone was transfected with an expression vector encoding FLP recombinase (24Fiering S. Kim S.G. Epner E.E. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8469-8473Crossref PubMed Scopus (90) Google Scholar); and in each case, one G418-sensitive clone, potentially lacking the LTR-neo gene, was then selected for further analyses. Southern blot analysis using an HS-40 probe revealed the presence of a 20-kb BamHI fragment identical to the fragment present in parental cells (Fig.3 A), thus demonstrating the excision of the LTR-neo gene. As expected, RNase protection analysis using human and mouse α-globin probes performed in induced cells revealed that all three clones that lost the LTR-neo gene recovered a level of human α-globin mRNA similar to that observed in parental cells (Fig. 3,B and C). Taken together, these data establish that insertions of an LTR-neo gene near the HS-40 regulatory region of the human α-globin gene complex markedly reduce, but do not abolish, the HS-40-mediated transcriptional activation of the downstream human α-globin genes in hybrid MEL cells. This negative effect on the transcription of downstream α-globin genes appears to occur whether the LTR-neo gene is upstream or downstream from HS-40 and regardless of the chromosomal orientation of the inserted LTR-neo gene. One possible explanation for the above observations could be that HS-40 preferentially activates the LTR-neo gene inserted into the human α-globin locus at the expense of downstream human α-globin genes. According to this hypothesis, transcriptional activity of the LTR-neo gene should be higher in induced cells harboring the inserted LTR-neo gene in the vicinity of HS-40 than in induced cells harboring the same LTR-neo gene in place of HS-40. Unexpectedly, both types of induced cells displayed similar levels of neo gene transcripts (Fig. 2 E, comparebars 2–9 with bar 10). However, these levels of neo gene transcripts might not reflect the real transcriptional activities of the genes due to the eventual saturation of the degradation process, which is known to affect selectively non-erythroid gene transcripts during the terminal differentiation of MEL cells (29Krowzynski A. Yenofsky R. Brawerman G. J. Mol. Biol. 1985; 18: 231-239Crossref Scopus (76) Google Scholar). We therefore decided to use a nuclear run-on assay to compare more directly the transcriptional activities of the LTR-neo gene in induced cells from clone Δneo-HS as well as from one example of clones harboring the LTR-neo gene in the four different positions and orientations with respect to HS-40. Induced parental cells were used as a negative control. Briefly, nuclei were prepared from each type of cell and incubated in vitro in the presence of labeled UTP, and labeled nuclear transcripts were hybridized to membranes loaded with neo, mouse β-globin, and empty vector DNA probes (Fig. 4 A). Hybridization signals were quantified, and the neo gene signals were standardized to the β-globin gene signals to allow the direct comparison of the transcriptional activities of the LTR-neogene between the different types of cells (Fig. 4 B). As estimated by this assay, the maximum difference in the transcriptional activities of the LTR-neo gene between the five analyzed clones was 3-fold. The transcriptional activities of the LTR-neo gene in the four clones harboring insertions near HS-40 were alternatively higher, as in clones HS-neoS andneoAS-HS (Fig. 4 B, bars 2 and4), or lower, as in clones neoS-HS and HS-neoAS (Fig. 4 B, bars 3 and 5), than that in the clone harboring the insertion in place of HS-40 (bar 6). Furthermore, the variations in the transcriptional activities of the neogene estimated in the different clones harboring the insertions near HS-40 did not correlate with the variations in the reduction of human α-globin gene expression. Taken together, these data indicate that the transcriptional activity of the LTR-neo gene inserted into the human α-globin gene is not affected by induction of differentiation and therefore does not apparently benefit from HS-40-mediated activation. This unexpected result led us to investigate the effect of the insertion of a new α-globin gene, instead of the LTR-neo gene, at the same downstream position near HS-40. In a previous study, we identified a two-nucleotide deletion at positions −2 and −3 preceding the ATG initiation codon in one human α+-thalasse" @default.
• W2062068633 created "2016-06-24" @default.
• W2062068633 creator A5018391651 @default.
• W2062068633 creator A5023366619 @default.
• W2062068633 creator A5025084942 @default.
• W2062068633 creator A5036924663 @default.
• W2062068633 creator A5044351115 @default.
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• W2062068633 date "2000-08-01" @default.
• W2062068633 modified "2023-09-30" @default.
• W2062068633 title "Non-erythroid Genes Inserted on Either Side of Human HS-40 Impair the Activation of Its Natural α-Globin Gene Targets without Being Themselves Preferentially Activated" @default.
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