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• W2092133078 abstract "α-Spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the α-spectrin gene. Previously, we demonstrated that the core promoter of the human α-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3′ of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the α-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3′ of the α-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased α-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis. α-Spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the α-spectrin gene. Previously, we demonstrated that the core promoter of the human α-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3′ of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the α-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3′ of the α-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased α-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis. Spectrin, the most abundant protein of the erythrocyte membrane skeleton, exists in the erythrocyte as a heterodimer of two homologous proteins, α-spectrin and β-spectrin (1Winkelmann J.C. Forget B.G. Blood. 1993; 81: 3173-3185Crossref PubMed Google Scholar, 2Morrow, J. S., Rimm, D. L., Kennedy, S. P., Cianci, C. D., Sinard, J. H., and Weed, S. A. (1997) in Handbook of Physiology (Hoffman, J., and Jamieson, J., eds) pp. 485–540, Oxford, LondonGoogle Scholar). α- and β-spectrin are composed primarily of 106-amino acid repeats that fold into three antiparallel α-helices connected by short non-helical segments (3Speicher D.W. Marchesi V.T. Nature. 1984; 311: 177-180Crossref PubMed Scopus (309) Google Scholar, 4Sahr K.E. Laurila P. Kotula L. Scarpa A.L. Coupal E. Leto T.L. Linnenbach A.J. Winkelmann J.C. Speicher D.W. Marchesi V.T. Curtis P.J. Forget B.G. J. Biol. Chem. 1990; 265: 4434-4443Abstract Full Text PDF PubMed Google Scholar, 5Winkelmann J.C. Costa F.F. Linzie B.L. Forget B.G. J. Biol. Chem. 1990; 265: 20449-20454Abstract Full Text PDF PubMed Google Scholar, 6Wandersee N.J. Birkenmeier C.S. Gifford E.J. Mohandas N. Barker J.E. Hematol. J. 2000; 1: 235-242Crossref PubMed Scopus (27) Google Scholar, 7Bloom M.L. Birkenmeier C.S. Barker J.E. Blood. 1993; 82: 2906-2914Crossref PubMed Google Scholar). αβ-Spectrin heterodimers self-associate to form tetramers and higher order oligomers, forming a lattice-like structure that provides stability and deformability to the erythrocyte membrane (8Morrow J.S. Speicher D.W. Knowles W.J. Hsu C.J. Marchesi V.T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6592-6596Crossref PubMed Scopus (109) Google Scholar, 9Speicher D.W. Morrow J.S. Knowles W.J. Marchesi V.T. J. Biol. Chem. 1982; 257: 9093-9101Abstract Full Text PDF PubMed Google Scholar, 10Liu S.C. Palek J. Prchal J. Castleberry R.P. J. Clin. Investig. 1981; 68: 597-605Crossref PubMed Scopus (84) Google Scholar, 11Liu S.C. Palek J. Prchal J.T. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2072-2076Crossref PubMed Scopus (52) Google Scholar, 12Chasis J.A. Agre P. Mohandas N. J. Clin. Investig. 1988; 82: 617-623Crossref PubMed Scopus (68) Google Scholar). In the erythrocyte, spectrin functions include maintenance of cellular shape, regulation of the lateral mobility of integral membrane proteins, and provision of structural support for the lipid bi-layer (2Morrow, J. S., Rimm, D. L., Kennedy, S. P., Cianci, C. D., Sinard, J. H., and Weed, S. A. (1997) in Handbook of Physiology (Hoffman, J., and Jamieson, J., eds) pp. 485–540, Oxford, LondonGoogle Scholar, 13Bennett V. Baines A.J. Physiol. Rev. 2001; 81: 1353-1392Crossref PubMed Scopus (794) Google Scholar). Quantitative and qualitative disorders of spectrin have been associated with abnormalities of erythrocyte shape including hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis (12Chasis J.A. Agre P. Mohandas N. J. Clin. Investig. 1988; 82: 617-623Crossref PubMed Scopus (68) Google Scholar, 14Tse W.T. Lux S.E. Br. J. Haematol. 1999; 104: 2-13Crossref PubMed Scopus (245) Google Scholar, 15Walensky L.D. Narla M. Lux S.E. Handin R.I. Lux S.E. Stossel T.P. Blood: Principles and Practice of Hematology. 2nd Ed. Lippincott Williams & Wilkins, Philadelphia2003: 1709-1858Google Scholar, 16Agre P. Orringer E.P. Bennett V. N. Engl. J. Med. 1982; 306: 1155-1161Crossref PubMed Scopus (131) Google Scholar, 17Agre P. Casella J.F. Zinkham W.H. McMillan C. Bennett V. Nature. 1985; 314: 380-383Crossref PubMed Scopus (142) Google Scholar, 18Agre P. Asimos A. Casella J.F. McMillan C. N. Engl. J. Med. 1986; 315: 1579-1583Crossref PubMed Scopus (113) Google Scholar, 19Coetzer T. Palek J. Lawler J. Liu S.C. Jarolim P. Lahav M. Prchal J.T. Wang W. Alter B.P. Schewitz G. Mankad V. Gallanello R. Cao A. Blood. 1990; 75: 2235-2244Crossref PubMed Google Scholar). Structural abnormalities of α spectrin in the region of the αβ self-association site are the most common defects associated with hereditary elliptocytosis and hereditary pyropoikilocytosis. However, in many patients with α-spectrin-linked hereditary spherocytosis and hereditary pyropoikilocytosis, the precise genetic defect(s) is unknown. Studies suggest that these patients have a defect in α-spectrin mRNA accumulation, which has been termed a “thalassemia-like” defect (20Hanspal M. Hanspal J.S. Sahr K.E. Fibach E. Nachman J. Palek J. Blood. 1993; 82: 1652-1660Crossref PubMed Google Scholar, 21Gallagher P.G. Tse W.T. Marchesi S.L. Zarkowsky H.S. Forget B.G. Trans. Assoc. Am. Physicians. 1991; 104: 32-39PubMed Google Scholar). The identification and characterization of the regulatory elements that control α-spectrin gene expression has important implications for understanding the pathogenesis of α-spectrin-linked hemolytic anemia and erythrocyte membrane protein biosynthesis and assembly. In splenic erythroblasts isolated from mice early after Friend virus infection, there is marked synthesis of spectrin with a significant excess of α-spectrin over β-spectrin (22Hanspal M. Hanspal J.S. Kalraiya R. Liu S.C. Sahr K.E. Howard D. Palek J. Blood. 1992; 80: 530-539Crossref PubMed Google Scholar). Studies in avian and rat cells have shown that the increased α-spectrin synthesis in early erythropoiesis is controlled at the transcriptional level (23Peters L.L. White R.A. Birkenmeier C.S. Bloom M.L. Lux S.E. Barker J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5749-5753Crossref PubMed Scopus (24) Google Scholar, 24Hanspal M. Palek J. J. Cell Biol. 1987; 105: 1417-1424Crossref PubMed Scopus (74) Google Scholar, 25Koury M.J. Bondurant M.C. Rana S.S. J. Cell. Physiol. 1987; 133: 438-448Crossref PubMed Scopus (33) Google Scholar). However, the molecular mechanisms that regulate the erythroid tissue-specific or developmental stage-specific expression of α-spectrin, including the mechanisms that control the increase in α-spectrin gene transcription to high levels during the early stages of erythropoiesis, are unknown. Our previous studies demonstrated that the core promoter of the human α-spectrin gene directed very low levels of erythroid-specific expression only in the early stages of erythroid development, indicating that elements outside the core α-spectrin gene promoter are required for high level erythroid expression (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This report describes the identification and characterization of a region 3′ of the core α-spectrin gene promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in transient and stable reporter gene/transfection assays. This region contains two functional GATA-1 sites, both required for full activity. In transfection assays this region did not display characteristics of a classical enhancer. Chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy of GATA-1 and CBP. 1The abbreviations used are: CBP, cAMP-response element-binding protein (CREB)-binding protein; ChIP, quantitative chromatin immunoprecipitation. These results demonstrate that a region 3′ of the α-spectrin promoter positioned in its proper genomic orientation is required for high level, erythroid-specific, α-spectrin gene expression. This is an excellent candidate region for mutations associated with decreased α-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis. DNase I Hypersensitive Site Mapping—DNase I hypersensitive site mapping was performed as described with minor modifications (27Nemeth M.J. Bodine D.M. Garrett L.J. Lowrey C.H. Blood Cells Mol. Dis. 2001; 27: 767-780Crossref PubMed Scopus (3) Google Scholar, 28Lowrey C.H. Bodine D.M. Nienhuis A.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1143-1147Crossref PubMed Scopus (78) Google Scholar). Approximately 1 × 108 logarithmically growing K562, SH SY5Y (human, neuroblastoma), or Jurkat (human, T lymphocyte) cells were collected by centrifugation, washed in cold phosphate-buffered saline, and resuspended in 14 ml of ice cold RSB (10 mm Tris, pH 7.5, 10 mm NaCl, 3 mm MgCl2) to which 375 μl of 10% Nonidet P-40 was added dropwise with gentle mixing. The nuclei were pelleted by gentle centrifugation and resuspended in ∼1.5 ml of ice-cold RSB. 200 μl of nuclei were placed in tubes containing increasing amounts of DNase I (0–4.0 μg/ml) in a volume of 30 μl. After a 37 °C incubation for 10 min, 230 μl of stop buffer (1% SDS, 20 mm Tris, pH 7.5, 600 mm NaCl, 10 mm EDTA, 500 μg/ml proteinase K) was added. The DNA was digested at 37 °C overnight before extraction with phenol, phenol/CHCl3, and CHCl3 followed by ethanol precipitation. DNA was digested with HindIII for Southern blot analysis using a 600-bp PstI/BglII fragment containing the α-spectrin core promoter as a probe. For fine mapping the migration of the band generated by DNase I and HindIII digestion was compared with the migration of bands generated by the digestion of high molecular weight K562 DNA digested with HindIII and either PstI, PvuII, EcoRI, MfeI, NheI, or EcoRV. Preparation of Promoter-Reporter Plasmids for Transfection Assays—A 794-bp human α-spectrin gene promoter fragment, –793 to +1 (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), subcloned upstream of the firefly luciferase reporter gene in the plasmid pGL2B (Promega Corp.) was used as the parent plasmid. DNA fragments corresponding to exon 1′, intron 1′, and exon 1′ + intron 1′ were amplified by PCR using primers A+B, C+D, and A+D, respectively (Table I), and subcloned between the 3′ end of the α-spectrin promoter and the luciferase reporter gene. Serial truncations of these 793-bp fragment in the pGL2B plasmid were constructed by PCR amplification using the primers described in Boulanger et al. (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Integrity of all test plasmids was confirmed by sequencing.Table IOligonucleotide primersA5′-GCCGGATCCATGTCTTCTAAAGATAATGTCGATTG-3′B5′-GGGCTCGAGCTCTTGCTTGGTCCTAGAATC-3′C5′-GCCGGATCCGTTTTTTTTTTTTCCCCCACATACTTAACGTTT -3′D5′-GGGGGATCCGGTTTAGAACCTGGCAAGATAA-3′E5′-GCCGGTACCGTCACCCAGTATCTGTAAAAC-3′F5′-GGGCTCGAGTTTTCCTAAAGGTTTAGAACC-3′G5′-CTTCTAAAGATAATGTCGAT-3′H5′-ATCGACATTATCTTTAGAAG-3′I5′-CACATTTTATCTTGCCAGGT-3′J5′-ACCTGGCAAGATAAAATGTG-3′K5′-GGTGGGTAAAGAAGATAAGGCCCATCAG-3′L5′-CTGATGGGCCTTATCTTCTTTACCCACC-3′M5′-CAGTTTCCTTTGGAGTTTCC-3′N5′-CTCTGCTCAGCTCAGACTAAGG-3′O5′-GTAATACGACTCACTATAGGGC-3′P5′-ACTATAGGGCACGCGTGGT-3′Q5′-CCTTGAACCTCTGGTACTGGT-3′R5′-GGACCTTTGGTCCGCTGCTCT-3′S5′-ATGTCCCCTGGAGATAATGC-3′ Open table in a new tab Transfection Analyses—Transient transfections were performed exactly as described (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Stable transfections were performed as described (29Lin H.J. Anagnou N.P. Rutherford T.R. Shimada T. Nienhuis A.W. J. Clin. Investig. 1987; 80: 374-380Crossref PubMed Scopus (12) Google Scholar) with minor modifications. 107 K562 cells were transfected by electroporation with a single pulse of 300 V at 960 microfarads with 10 μg of SalI-linearized test plasmid and 1.0 μg of EcoRI-linearized pSV3-neo plasmid (29Lin H.J. Anagnou N.P. Rutherford T.R. Shimada T. Nienhuis A.W. J. Clin. Investig. 1987; 80: 374-380Crossref PubMed Scopus (12) Google Scholar). After 48 h of culture, cells were propagated in RPMI with 400 μg/ml G418 sulfate for 2–4 weeks. Single cell clones were isolated by serial dilution and expanded. Luciferase assays were performed as described (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Genomic DNA was isolated from clonal cell lines and analyzed by Southern blot analysis. A human α-spectrin gene promoter fragment (–793 to +1) (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) was used as probe. The relative copy number of the integrated plasmid constructs was determined by blot analysis with a PhosphorImager (Amersham Biosciences) using control human genomic DNA, copy number = 2. Preparation of Nuclear Extracts—Nuclear extracts were prepared from K562 and HeLa cells by hypotonic lysis followed by high salt extraction of nuclei as described by Andrews and Faller (30Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2214) Google Scholar) or Dignam et al. (31Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (746) Google Scholar). In Vitro DNase I Footprinting—Probes for in vitro DNase I footprinting were produced by PCR amplification using an α-spectrin genomic fragment, λ3021 (wild type probe) (32Sahr K.E. Tobe T. Scarpa A. Laughinghouse K. Marchesi S.L. Agre P. Linnenbach A.J. Marchesi V.T. Forget B.G. J. Clin. Investig. 1989; 84: 1243-1252Crossref PubMed Scopus (37) Google Scholar), or an α-spectrin gene plasmid with mutations of the GATA-1 sites in both exon 1′ and intron 1′ (Fig. 6, mutant exon 1′ + mutant intron 1′) as template and primers E and F (Table I). One oligonucleotide, either E or F, was 5′ end-labeled with [32P]ATP using polynucleotide kinase before use in PCR. Reaction mixes contained K562 cell nuclear extracts, 10,000 cpm of labeled probe, and 1 μg of poly(dI-dC). After digestion with DNase I, samples were electrophoresed in 6% denaturing polyacrylamide gels, and the gels were dried and subjected to autoradiography. Gel Mobility Shift Analyses—Binding reactions were carried out as described (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Competitor oligonucleotides were added at molar excesses of 100-fold. Antibodies to GATA-1 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Quantitative Chromatin Immunoprecipitation (ChIP) Assay—Anti-diacetylated histone H3 (06-599) and anti-tetraacetylated histone H4 (06-866) antibodies were obtained from Upstate Biotechnology Corp. (Lake Placid, NY). Anti-GATA-1 antibody (N6) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CBP antibody (A-22) was obtained from Santa Cruz. ChIP analysis was performed as described (33Johnson K.D. Bresnick E.H. Methods. 2002; 26: 27-36Crossref PubMed Scopus (107) Google Scholar, 34Im H. Park C. Feng Q. Johnson K.D. Kiekhaefer C.M. Choi K. Zhang Y. Bresnick E.H. J. Biol. Chem. 2003; 278: 18346-18352Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). After formaldehyde fixation, chromatin was fragmented by sonication 10 times for 10 s each. Extracts were precleared with protein G-Sepharose, antibody was added, and incubated was overnight at 4 °C. After elution and extraction, immunoprecipitated DNA was analyzed by quantitative real-time PCR (iCycler, Bio-Rad). PCR primers A and D (Table I) amplify the 183-bp 5′-untranslated region. Samples from at least three independent immunoprecipitations were analyzed. SYBR green fluorescence in 25-μl PCR reactions was determined, and the amount of product was determined relative to a standard curve generated from a titration of input chromatin. Amplification of a single amplification product was confirmed by dissociation curve analysis and agarose gel electrophoresis with ethidium bromide staining. Parallel controls for each experiment included samples of no chromatin, no antibody, nonimmune rabbit IgG, and rabbit nonimmune serum. Identification of a DNase I Hypersensitive Site Downstream from the Core α-Spectrin Gene Promoter—The human α-spectrin gene is >120 kilobases and is encoded by 52 exons. This large size makes a systematic search for regulatory elements difficult by functional assays. To identify important nonpromoter-related regulatory sequences, we examined the DNase I hypersensitivity of the α-spectrin gene in the 5′ and 3′ regions as well in the first few introns using K562 cell nuclei. A hypersensitive site was identified in a 3-kilobase HindIII fragment that mapped 3′ of the core α-spectrin gene promoter (Fig. 1A). Fine mapping localized this hypersensitive site to a ∼183-bp region 3′ of a PvuII site (Fig. 1, B and C). As a positive control, the same preparation of K562 DNA was digested with varying concentrations of DNase I followed by digestion with EcoRI and hybridization to a human BamHI/PvuII Gγ-globin probe as described (35Groudine M. Kohwi-Shigematsu T. Gelinas R. Stamatoyannopoulos G. Papayannopoulou T. Proc. Natl. Acad. U. S. A. 1983; 80: 7551-7555Crossref PubMed Scopus (112) Google Scholar). A hypersensitive site in the Gγ-globin promoter generated a new band at 1.5 kilobases, as previously shown by Groudine et al. (35Groudine M. Kohwi-Shigematsu T. Gelinas R. Stamatoyannopoulos G. Papayannopoulou T. Proc. Natl. Acad. U. S. A. 1983; 80: 7551-7555Crossref PubMed Scopus (112) Google Scholar) using K562 cell nuclei (not shown). Digestion of the same preparation of K562 DNA with varying concentrations of DNase I followed by digestion with SacI and hybridization to a human keratin 14 probe (36Sinha S. Degenstein L. Copenhaver C. Fuchs E. Mol. Cell. Biol. 2000; 20: 2543-2555Crossref PubMed Scopus (77) Google Scholar) demonstrated that the DNase I hypersensitivity of the α-spectrin gene and the Gγ-globin gene is not the result of general DNase I digestion of the DNA (not shown). When DNase I hypersensitivity experiments were performed with nuclei from the nonerythroid cells SH SY5Y (neuroblastoma) and Jurkat (T lymphocyte) and the same α-spectrin probe, no DNase I sensitivity was observed (not shown). The Region Corresponding to the Hypersensitive Site Contains Binding Sites for GATA-1- and AP-1-binding Proteins—We inspected the DNA sequence of the region corresponding to the DNase I hypersensitive site. Consensus binding sequences for GATA-1 (two sites) and AP1 binding proteins (one site), both important for expression in other erythroid genes, were identified (Fig. 1D). This 183-bp region encodes an untranslated exon (exon 1′, 61 bp) and an alternately spliced, untranslated intron (intron 1′, 122 bp) (32Sahr K.E. Tobe T. Scarpa A. Laughinghouse K. Marchesi S.L. Agre P. Linnenbach A.J. Marchesi V.T. Forget B.G. J. Clin. Investig. 1989; 84: 1243-1252Crossref PubMed Scopus (37) Google Scholar). Addition of the 183-bp Region to an α-Spectrin Gene Promoter Fragment Significantly Increases Promoter Expression in Erythroid Cells—The 183-bp region was placed immediately 3′ of the α-spectrin gene promoter in its proper genomic orientation upstream of a luciferase reporter gene. This plasmid, p794/183 (Fig. 2A), was transiently transfected into K562 cells. The relative luciferase activity was determined 48 h after transfection and compared with the activity obtained with the core α-spectrin promoter plasmid (p794 (26Boulanger L. Sabatino D.E. Wong E.Y. Cline A.P. Garrett L.J. Garbarz M. Dhermy D. Bodine D.M. Gallagher P.G. J. Biol. Chem. 2002; 277: 41563-41570Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar)), a negative control promoterless luciferase plasmid (pGL2B), and a positive control with the luciferase reporter gene under control of the SV40 early promoter (pGL2P). As shown in Fig. 2, the α-spectrin gene promoter + downstream sequence plasmid, p794/183, directed 10-fold high level expression of the luciferase reporter gene in erythroid cells than the promoter alone. The addition of the 183-bp region to deletion mutants of the core α-spectrin gene promoter directed significantly higher levels of expression than the corresponding promoter fragment alone (Fig. 2A). These sequences even conferred activity to a minimal α-spectrin gene promoter fragment, p44/183, which is inactive in the absence of this element. The α-Spectrin Gene Promoter Requires Both Exon 1′ and Intron 1′ for Full Expression in Erythroid Cells—To determine the relative importance of exon 1′ and intron 1′ in increasing expression directed by the α-spectrin gene promoter, luciferase promoter reporter plasmids with exon 1′, intron 1′, and exon 1′ + intron 1′ in forward (genomic) and reverse orientations were transfected into K562 and HeLa cells. In K562 cells, plasmids with exon 1′ + intron 1′ in reverse orientation (p794/183Rev) directed low levels of expression at levels comparable with those directed by the promoter alone (p794, p = not significant, unpaired t test) or the promoter + exon 1′ (p794/Exon, p = not significant, unpaired t test) (Fig. 2B). Promoter + intron 1′ plasmids (p794/Intron) directed higher levels of expression, approximately half that directed by p794/183 (p = 0.0046, unpaired t test). In HeLa cells there was no change in levels of luciferase expression of any of the plasmids. These results suggest that exon 1′ and intron 1′ in their appropriate genomic orientation are required for full expression in erythroid cells. The 183-bp Region of the α-Spectrin Gene Contains Binding Sites for GATA-1- and AP-1-binding Proteins—To identify sites for DNA-binding proteins within the 183-bp region, DNase I footprinting analysis with nuclear extracts from K562 cells was performed. Two footprints were observed. One site was in exon 1′ on the sense strand (5′-AAGATAA-3′) (Fig. 3A), and the other was in intron 1′ on the antisense strand (5′-AGATAAA-3′) (Fig. 3B). Both contained consensus binding sequences for the erythroid transcription factor GATA-1. The footprinted region in intron 1′ did not extend into the AP-1 consensus sequence. GATA-1 Binds Both Exon 1′ and Intron 1′ GATA Sites in the 183-bp Region in Electrophoretic Mobility Shift Assays—To determine which nuclear proteins bound to the GATA-1 sites present in exon 1′ and intron 1′, double-stranded oligonucleotides containing the corresponding α-spectrin promoter GATA-1 sequences (exon 1′ G + H; intron 1′, I + J; Table I) or control GATA-1 sequences (K + L; Table I) (37Mignotte V. Wall L. deBoer E. Grosveld F. Romeo P.H. Nucleic Acids Res. 1989; 17: 37-54Crossref PubMed Scopus (216) Google Scholar) were prepared and used in gel shift analyses. A single complex was observed in K562 (erythroid) extracts when probes corresponding to either site were used (Figs. 4, A and B). This complex migrated at the same location as a control oligonucleotide containing a GATA-1 consensus sequence. This species was effectively competed both by an excess of unlabeled homologous oligonucleotide and by an excess of unlabeled control GATA-1 oligonucleotide (not shown). The inclusion of GATA-1 antisera abolished most or all of the DNA binding. Mutation of the GATA-1 Sites in the 183-bp Region Disrupts GATA-1 Binding in Vitro—To further assess the ability of nuclear proteins to bind the α-spectrin GATA-1 sites in vitro, DNase I footprinting and gel mobility shift assays were performed using probes with the GATA-1 sites mutated (GATA to GTTA) (38Zon L.I. Youssoufian H. Mather C. Lodish H.F. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10638-10641Crossref PubMed Scopus (172) Google Scholar). For in vitro DNase I footprinting, mutations were introduced into both exon 1′ and intron 1′ GATA-1 sites of the α-spectrin gene promoter + the 183-bp probe described above. When footprinting was performed with this double mutant GATA-1 probe and K562 cell nuclear extracts, no protected regions at the mutant GATA-1 sites were observed (Fig. 3C). When oligonucleotides with mutation of the consensus GATA-1 binding sequences (GATA to GTTA) (38Zon L.I. Youssoufian H. Mather C. Lodish H.F. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10638-10641Crossref PubMed Scopus (172) Google Scholar) were used in gel mobility shift assays, complex formation was nearly or completely abolished (Fig. 4, C and D). These data indicate that GATA-1 binds to the exon 1′ and intron 1′ sites 3′ of the α-spectrin gene promoter in vitro. Mutation of the 183-bp Region GATA-1 Sites Significantly Decreases Promoter Function—To assess the relative importance of these GATA-1 binding sites in promoter function, mutations were introduced into the GATA-1 sites protected in DNase I footprinting experiments, both individually and in combination. Mutation of the exon 1′ GATA-1 site (GATA to GTTA) (37Mignotte V. Wall L. deBoer E. Grosveld F. Romeo P.H. Nucleic Acids Res. 1989; 17: 37-54Crossref PubMed Scopus (216) Google Scholar) decreased activity by approximately half in transiently transfected K562 cells (Fig. 5). Mutation of the intron 1′ GATA-1 site in a similar manner (GATA to GTTA) reduced promoter activity by 66% (Fig. 5). When a reporter plasmid with mutations of both GATA-1 sites was transfected into K562 cells, promoter activity was reduced to background levels (Fig. 5). Variability is frequently observed at high levels of luciferase activity from transfection to transfection. As noted in Figs. 2, A and B, 5, and 7A, this is observed in comparison of the α-spectrin promoter alone compared with the α-spectrin promoter with the downstream element, where relative ratios of luciferase activity range from 1:6 to 1:14.Fig. 7Effect of position and orientation of the 183-bp region 3′ with the α-spectrin gene promoter or a heterologous gene promoter in erythroid cells in transient transfection assays. A, plasmids containing the 183-bp region inserted in both orientations 5′ and 3′ of an α-spectrin gene (ASp) promoter/firefly luciferase gene cassette were transfected into K562 cells as described. B, plasmids containing the 183-bp region inserted in both orientations 5′ and 3′ of a thymidine kinase (TK) promoter/firefly luciferase gene cassette were transfected into K562 cells as described. In A and B, relative luciferase activity was expressed as that obtained from the test plasmids versus the activity obtained from the promoterless" @default.
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• W2092133078 title "Sequences Downstream of the Erythroid Promoter Are Required for High Level Expression of the Human α-Spectrin Gene" @default.
• W2092133078 cites W12952247 @default.
• W2092133078 cites W1511507297 @default.
• W2092133078 cites W1512510903 @default.
• W2092133078 cites W1528180394 @default.
• W2092133078 cites W1528238804 @default.
• W2092133078 cites W1545890666 @default.
• W2092133078 cites W1580937961 @default.
• W2092133078 cites W1601094260 @default.
• W2092133078 cites W1942516902 @default.
• W2092133078 cites W1945580731 @default.
• W2092133078 cites W1969230311 @default.
• W2092133078 cites W1970103367 @default.
• W2092133078 cites W1975376384 @default.
• W2092133078 cites W1978125302 @default.
• W2092133078 cites W1979592575 @default.
• W2092133078 cites W1986231947 @default.
• W2092133078 cites W1986783431 @default.
• W2092133078 cites W1991619600 @default.
• W2092133078 cites W1994583688 @default.
• W2092133078 cites W1998193053 @default.
• W2092133078 cites W2004292212 @default.
• W2092133078 cites W2017516970 @default.
• W2092133078 cites W2021864174 @default.
• W2092133078 cites W2022447132 @default.
• W2092133078 cites W2022804480 @default.
• W2092133078 cites W2024317307 @default.
• W2092133078 cites W2024520070 @default.
• W2092133078 cites W2025589527 @default.
• W2092133078 cites W2036871159 @default.
• W2092133078 cites W2038935839 @default.
• W2092133078 cites W2042067464 @default.
• W2092133078 cites W2045013854 @default.
• W2092133078 cites W2048379517 @default.
• W2092133078 cites W2048954418 @default.
• W2092133078 cites W2054338068 @default.
• W2092133078 cites W2066842884 @default.
• W2092133078 cites W2071238021 @default.
• W2092133078 cites W2073389964 @default.
• W2092133078 cites W2085470445 @default.
• W2092133078 cites W2086548325 @default.
• W2092133078 cites W2087591109 @default.
• W2092133078 cites W2094646101 @default.
• W2092133078 cites W2108373939 @default.
• W2092133078 cites W2108643188 @default.
• W2092133078 cites W2127427514 @default.
• W2092133078 cites W2131255875 @default.
• W2092133078 cites W2142342476 @default.
• W2092133078 cites W2142617678 @default.
• W2092133078 cites W2149078679 @default.
• W2092133078 cites W2150313140 @default.
• W2092133078 cites W2151112211 @default.
• W2092133078 cites W2158931590 @default.
• W2092133078 cites W2284120550 @default.
• W2092133078 cites W2290729516 @default.
• W2092133078 cites W2336754635 @default.
• W2092133078 cites W2399056814 @default.
• W2092133078 cites W4231353777 @default.
• W2092133078 cites W4248486473 @default.
• W2092133078 cites W4254878650 @default.
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