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Erythrocyte Ankyrin Promoter Mutations Associated with Recessive Hereditary Spherocytosis Cause Significant Abnormalities in Ankyrin Expression
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m105844200
ISSN1083-351X
AutoresPatrick G. Gallagher, Denise E. Sabatino, Daniela S. Daniela Sanchez Bassères, Douglas M. Nilson, Clara Wong, Amanda P. Cline, Lisa Garrett, David M. Bodine,
Tópico(s)Pancreatic function and diabetes
ResumoAnkyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, −108T to C and −108T to C in cis with −153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a humanAγ-globin gene expressed γ-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant −108 promoter demonstrated variegated γ-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked −108/−153 mutations. Three transgenic lines had undetectable levels of Aγ-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of Aγ-globin mRNA than wild type. Two of four expressing lines showed variegated γ-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy number-independent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations. Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, −108T to C and −108T to C in cis with −153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a humanAγ-globin gene expressed γ-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant −108 promoter demonstrated variegated γ-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked −108/−153 mutations. Three transgenic lines had undetectable levels of Aγ-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of Aγ-globin mRNA than wild type. Two of four expressing lines showed variegated γ-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy number-independent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations. hereditary spherocytosis polymerase chain reaction base pair(s) ankyrin Hereditary spherocytosis (HS)1 is a common hemolytic anemia characterized by the presence of spherically shaped erythrocytes on peripheral blood smear. The principal cellular defect in HS is loss of erythrocyte membrane surface area relative to intracellular volume, accounting for the spherical shape as well as decreased deformability of the erythrocyte (1Gallagher P.G. Jarolim P. Hematology: Basic Principles and Practice. W. B. Saunders Co., Philadelphia1999: 576-610Google Scholar). The primary biochemical defects in HS reside in the proteins of the erythrocyte membrane, particularly those proteins involved in the interactions between the membrane skeleton and the lipid bilayer: ankyrin, α and β spectrin, band 3, and protein 4.2 (2Iolascon A. Miraglia del Giudice E. Perrotta S. Alloisio N. Morle L. Delaunay J. Haematologica. 1998; 83: 240-257PubMed Google Scholar, 3Tse W.T. Lux S.E. Br. J. Haematol. 1999; 104: 2-13Crossref PubMed Scopus (240) Google Scholar). In two thirds to three quarters of cases, HS is inherited in an autosomal dominant fashion (1Gallagher P.G. Jarolim P. Hematology: Basic Principles and Practice. W. B. Saunders Co., Philadelphia1999: 576-610Google Scholar, 2Iolascon A. Miraglia del Giudice E. Perrotta S. Alloisio N. Morle L. Delaunay J. Haematologica. 1998; 83: 240-257PubMed Google Scholar, 3Tse W.T. Lux S.E. Br. J. Haematol. 1999; 104: 2-13Crossref PubMed Scopus (240) Google Scholar, 4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar). In the remaining patients, HS is inherited in a recessive fashion or is the result of a de novo mutation. Ankyrin-1 (ANK1, Mendelian Inheritance in Man 182900) deficiency is one of the most common abnormalities found in the erythrocyte membranes of HS patients (5Savvides P. Shalev O. John K.M. Lux S.E. Blood. 1993; 82: 2953-2960Crossref PubMed Google Scholar, 6Saad S.T. Costa F.F. Vicentim D.L. Salles T.S. Pranke P.H. Br. J. Haematol. 1994; 88: 295-299Crossref PubMed Scopus (33) Google Scholar). First identified in preparations of erythrocyte membranes, ankyrin provides the primary linkage between the spectrin-actin-based erythrocyte membrane skeleton and the plasma membrane by attaching tetramers of spectrin to the cytoplasmic domain of band 3 (7Bennett V. J. Biol. Chem. 1992; 267: 8703-8706Abstract Full Text PDF PubMed Google Scholar, 8Peters L.L. Lux S.E. Semin. Hematol. 1993; 30: 85-118PubMed Google Scholar). Studies have revealed that abnormalities of the ankyrin gene, primarily frameshift or nonsense mutations, are the most common cause of typical, dominant HS (3Tse W.T. Lux S.E. Br. J. Haematol. 1999; 104: 2-13Crossref PubMed Scopus (240) Google Scholar, 9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar, 10Gallagher P.G. Forget B.G. Blood Cells Mol. Dis. 1998; 24: 539-543Crossref PubMed Scopus (60) Google Scholar, 11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar). Ankyrin-1 is transcribed in erythroid cells from a compact, erythroid-specific promoter (12Gallagher P.G. Romana M. Tse W.T. Lux S.E. Forget B.G. Blood. 2000; 96: 1136-1143Crossref PubMed Google Scholar). One molecular mechanism that could lead to ankyrin deficiency is a mutation in the ankyrin erythroid promoter leading to decreased ankyrin synthesis. Several reports have identified sequence variations in the ankyrin gene promoter in individuals with recessively inherited, ankyrin-deficient HS (4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar, 9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar,11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar). Whether these are disease causing mutations or are merely polymorphisms in linkage disequilibrium with as yet unidentified mutations is unknown, as it is difficult to assess the effect of promoter mutations on ankyrin gene expression in affected HS patients. To address this question, we have analyzed the effect of two HS-associated, ankyrin gene promoter mutations in vitro and in vivo. The first mutation, a T to C substitution at position −108, was discovered in the heterozygous state in four of seven German families with ankyrin-deficient, recessive HS (9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar). In two of the four kindreds, mutations in the coding region of the ankyrin gene were discovered in trans, and the mutation was silent in the heterozygous state. In this mostly German HS population, the allele frequency was estimated to be 29% in HS patients and 2.0% in normal individuals. This mutation has also been associated with ankyrin-deficient, nondominantly inherited HS in Italy (4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar). The second mutation, a G to A substitution at position −153, was discovered in the heterozygous state in a Brazilian kindred with ankyrin-deficient, recessive HS (11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar). The −153 substitution was always found in cis to the previously reported −108T to C ankyrin promoter mutation. These linked substitutions were silent in the heterozygous state. No individuals with the −153 mutation alone were detected. In a control Brazilian population, the allelic frequency of the −108/−153 allele was 2.4% and the −108 allele was 1.4%. Previously, we have shown the wild type ankyrin promoter directed expression of a linked Aγ-globin reporter gene in an erythroid-specific, position-independent, copy number-dependent fashion in transgenic mice (13Sabatino D.E. Wong C. Cline A.P. Pyle L. Garrett L.J. Gallagher P.G. Bodine D.M. J. Biol. Chem. 2000; 275: 28549-28554Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Levels ofAγ-globin mRNA correlated directly with levels of functional γ-globin protein in erythrocytes of these transgenic mice (14Persons D.A. Allay E.R. Sabatino D.E. Kelly P. Bodine D.M. Nienhuis A.W. Blood. 2001; 97: 3275-3282Crossref PubMed Scopus (86) Google Scholar). We used this ankyrin promoter/human Aγ-globin reporter transgenic mouse model as a system to study the effect of the spherocytosis-associated promoter mutations in vivo.Significant abnormalities in reporter gene mRNA and protein expression were seen, the first demonstration of functional defects caused by HS-related ankyrin gene promoter mutations. These promoter mutations may represent a common pathogenetic mechanism of recessive HS in ankyrin-deficient patients. The mutations described in this report have been reported previously and are schematically shown in Fig.1 (4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar, 9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar, 11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar). The tissue culture cell lines K562 (chronic myelogenous leukemia in blast crisis with erythroid characteristics, ATCC CCl243) and HEL (human erythroleukemia, ATCC TIB 180) were used to study expression of the mutant ankyrin promoters. Cells were maintained in RPMI 1640 medium containing 10% fetal calf serum. We have shown previously that a 286-bp minimal ankyrin gene promoter fragment directs high level expression of a luciferase reporter gene in erythroid cells (plasmid p296 in Ref. 12Gallagher P.G. Romana M. Tse W.T. Lux S.E. Forget B.G. Blood. 2000; 96: 1136-1143Crossref PubMed Google Scholar). Mutant ankyrin promoter fragments corresponding to this 286-bp promoter fragment were generated by PCR amplification and subcloning into the firefly luciferase reporter plasmid pGL2B (Promega Corp., Madison, WI). Test plasmids were sequenced to exclude cloning or PCR-generated artifacts. All plasmids tested were purified using Qiagen columns (Qiagen, Inc., Chatsworth, CA) or cesium chloride plasmid purification, and at least two preparations of each plasmid were tested in triplicate. 107 K562 or HEL cells were transfected by electroporation with a single pulse of 300 V at 960 microfarads with 20 μg of test plasmid and 0.5 μg of pCMVβ, a mammalian reporter plasmid expressing β-galactosidase driven by the human cytomegalovirus immediate early gene promoter (CLONTECH, Palo Alto, CA) to normalize for transfection efficiency. Twenty-four hours after transfection, cells were harvested and lysed, and the activity of both luciferase and β-galactosidase activity determined in cell extracts. All assays were performed in triplicate. Nuclear extracts were prepared by hypotonic lysis followed by high salt extraction of nuclei as described by Andrews and Faller (15Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar). Oligonucleotide primers used in gel mobility shift assays are shown in Table I. Gel mobility shift binding reactions were carried out as described (16Mason P. Enver T. Wilkinson D. Williams J. Gene Transcription: A Practical Approach. IRL Press, Oxford1993: 243-294Google Scholar, 17Gallagher P.G. Sabatino D.E. Romana M. Cline A.P. Garrett L.J. Bodine D.M. Forget B.G. J. Biol. Chem. 1999; 274: 6062-6073Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Competitor oligonucleotides were added at molar excesses of 100-fold. Resulting complexes were separated by electrophoresis through 6% polyacrylamide gels in 0.5× Tris borate-EDTA at 21 °C at 200 watts for 2 h. Gels were dried and subjected to autoradiography.Table IOligonucleotide primers used in electrophoretic mobility shift assays−108T to C Wild typeSense5′-CCCTCGGGGGCCTGGCCCGCACGT-3′Antisense5′-ACGTGCGGGCCAGGCCCCCGAGGG-3′ MutantSense5′-CCCTCGGGGGCCCGGCCCGCACGT-3′Antisense5′-ACGTGCGGGCCGGGCCCCCGAGGG-3′ AP-2 controlSense5′-GATCGAACTGACCGCCCGCGGCCCGT-3′Antisense5′-ACGGGCCGCGGGCGGTCAGTTCGATC-3′−153G to A Wild typeSense5′-GGAGCGCCCGGCCCGACAGCA-3′Antisense5′-TGCTGTCGGGCCGGGCGCTCC-3′ MutantSense5′-GGAGCGCCCGACCCGACAGCA-3′Antisense5′-TGCTGTCGGGTCGGGCGCTCC-3′ Open table in a new tab To generate mutant Ank/Aγ-globin transgenes, 276-bp SmaI/BglII fragments containing mutant ankyrin promoters (−291 to −20 plus polylinker sequence) were excised from the pGL2B luciferase reporter vector (see above). A 1938-bp BglII/HindIII Aγ-globin fragment was excised from plasmid 72βsp+Aγ (described in Sabatino et al. in Ref 18Sabatino D.E. Cline A.P. Gallagher P.G. Garrett L.J. Stamatoyannopoulos G. Forget B.G. Bodine D.M. Mol. Cell. Biol. 1998; 18: 6634-6640Crossref PubMed Google Scholar). Triple ligations consisting of the ankyrin promoter, the Aγ-globin gene, and SmaI/HindIII-digested pSP72 were used to generate the mutant Ank/Aγ-globin plasmids. The ankyrin/Aγ-globin gene fragments (2244 bp) were excised from the plasmids with EcoRV and HindIII for microinjection (Fig. 2 A). All plasmid constructs were sequenced to confirm that the appropriate mutant ankyrin promoter was correctly fused to the Aγ-globin gene. Transgenic mice were generated as described by Hogan et al. (19Hogan B. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar) and Sabatino et al. (18Sabatino D.E. Cline A.P. Gallagher P.G. Garrett L.J. Stamatoyannopoulos G. Forget B.G. Bodine D.M. Mol. Cell. Biol. 1998; 18: 6634-6640Crossref PubMed Google Scholar). Fertilized eggs were collected from superovulated FVB/N female mice (Taconic Farms, Germantown, NY) ∼9 h after mating to CBy6F1 male mice (Jackson Laboratory, Bar Harbor, ME). Fragments for microinjection were separated on an agarose gel, electroeluted, and purified with an Elutip-d minicolumn (Schleicher & Schuell). The fragments were diluted to a concentration of 2 ng/μl in 10 mm Tris, 0.25 mm EDTA (pH 7.5), and ∼ 2 pl was injected into the male pronucleus of fertilized eggs. The injected eggs were transferred to psuedopregnant CByB6/F1 foster mothers. Founder animals were identified by Southern analysis of DNA extracted from tail biopsies by probing with an Ank/Aγ-globin probe. Founder animals were crossed to FVB/N mice for propagation. Copy number was determined by comparing the γ-globin signals from Southern blot analysis of transgenic mouse and K562 DNA using a Molecular Dynamics PhosphorImager. Statistical analysis of copy number and expression data was analyzed by linear regression using GraphPad Prism® version 2.0 software. Total cellular RNA was extracted from adult reticulocytes, obtained by collecting 200 μl of blood from phlebotomized animals, using TRIZOL reagent according to the manufacturer’s specifications (Life Technologies, Inc.). Linear DNA templates for RNase protection assays were prepared by restriction enzyme digestion of cesium chloride purified plasmid preparations. Riboprobe one, which contains sequences for both exon 2 of the humanAγ-globin gene and exon 2 of the murine α-globin gene, was linearized with BglII (Fig. 2 B). This riboprobe ensures that both the human Aγ-globin and murine α-globin sequences are labeled to equal specific activity, allowing direct comparison of human Aγ-globin and murine α-globin mRNA levels. Riboprobe two, which includes part ofAγ-globin exon 2, all of exon 1, and the ankyrin transcription initiation site, was linearized with EcoRI (Fig. 2 C) (18Sabatino D.E. Cline A.P. Gallagher P.G. Garrett L.J. Stamatoyannopoulos G. Forget B.G. Bodine D.M. Mol. Cell. Biol. 1998; 18: 6634-6640Crossref PubMed Google Scholar). This riboprobe allows detection of the transcription initiation site directed by the mutant ankyrin promoters. Templates were purified by agarose gel electrophoresis and purified using a Geneclean® II kit (Bio 101, Inc.). Linear DNA template for the mouse β-actin gene was obtained from the MAXIscript™ in vitrotranscription kit (Ambion, Inc.). 32P-Labeled RNA probes were transcribed using the MAXIscript™ in vitrotranscription kit (Ambion, Inc.). Hybridization of the probe and RNA (0.1–0.25 μg) was carried out overnight according to standard procedures (RPA II, Ambion, Inc.). RNase digestion was performed using an RNase A/RNase T1 mixture in RNase digestion buffer and the protected fragments separated on an 8% nondenaturing polyacrylamide gel. To quantitate the levels of mRNA, the gel was exposed to a PhosphorImager screen and scanned on a Molecular Dynamics PhosphorImager (Amersham Pharmacia Biotech). The relative amounts of the bands humanAγ-globin exon 2 (223 bp) and mouse α-globin exon 2 (186 bp) were estimated by the following formula: (Aγ-globin RNA/transgene copy number) × (1/mouse α-globin RNA). Detection and measurement of γ-globin protein in red blood cells was performed as described by Thorpe et al. (20Thorpe S.J. Thein S.L. Sampietro M. Craig J.E. Mahon B. Huehns E.R. Br. J. Haematol. 1994; 87: 125-132Crossref PubMed Scopus (88) Google Scholar). Blood cells collected by phlebotomy from the retro-orbital sinus of transgenic mice were washed in cold (4 °C) phosphate-buffered saline and then fixed in ice-cold (4 °C) 4% paraformaldehyde solution. The cells were washed with 1:1 acetone/water (−20 °C), acetone (−20 °C), and 1:1 acetone/water (−20 °C) before resuspension in phosphate-buffered saline plus 2% fetal bovine serum (4 °C). Hemoglobin tetramers containing human γ-globin were identified with a fluorescein isothiocyanate-conjugated human hemoglobin F antibody (PerkinElmer Life Sciences). Analysis was performed on a FACStar instrument (Becton Dickinson, Franklin Lakes, NJ). To prevent leaching of hemoglobin, cells were maintained at 4 °C or lower throughout the procedure. Computer-assisted analyses of mutant ankyrin promoter nucleotide sequences were performed utilizing the sequence analysis software package of the University of Wisconsin Genetics Computer Group (Madison, WI) (21Genetics Computer GroupProgram Manual for the Wisconsin Package, Version 8. Genetics Computer Group, Madison, WI1994Google Scholar) and the BLAST algorithm (National Center for Biotechnology Information, Bethesda, MD) (22Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70715) Google Scholar). Our previous in vitro studies showed that expression of a minimal human ankyrin-1 promoter in erythroid cells was mediated by GATA-1-, CACCC-, and CGCCC-binding proteins (12Gallagher P.G. Romana M. Tse W.T. Lux S.E. Forget B.G. Blood. 2000; 96: 1136-1143Crossref PubMed Google Scholar). Inspection of the ankyrin promoter sequence reveals that neither the −108 nor −153 mutations are located in these binding sites, nor are they located in regions protected during in vitro DNase I footprinting experiments using erythroid nuclear extracts (12Gallagher P.G. Romana M. Tse W.T. Lux S.E. Forget B.G. Blood. 2000; 96: 1136-1143Crossref PubMed Google Scholar). The −108 mutation disrupts a potential AP-2 binding site; it does not create a novel site for any known DNA-binding proteins. The −153 mutation is not contained in a known DNA binding protein site, nor does it create a site for any known DNA-binding proteins. Plasmids containing wild type and mutant −108 and −108/−153 ankyrin promoter fragments linked to a firefly luciferase reporter gene transiently transfected into K562 and HEL cells and luciferase expression assayed. There was no significant difference in luciferase activity directed by wild type or mutant plasmids in either K562 or HEL cells (Fig.3). Oligonucleotides containing the wild type and mutant ankyrin promoter sequences were synthesized, and protein binding activity was analyzed in mobility shift assays using K562 cell nuclear extracts. Wild type and mutant −108 oligonucleotides formed complexes that migrated at the same mobility as a control AP-2 oligonucleotide. In competition assays, the −108 mutant oligonucleotide competed away both the corresponding wild type ankyrin and control AP-2 complexes (data not shown). Neither wild type nor mutant −153 oligonucleotides formed complexes with K562 extracts (data not shown). Previously, we have shown the wild type ankyrin promoter-directed expression of a linked Aγ-globin reporter gene in an erythroid-specific, position-independent, copy number-dependent (p = 0.004) fashion in transgenic mice (18Sabatino D.E. Cline A.P. Gallagher P.G. Garrett L.J. Stamatoyannopoulos G. Forget B.G. Bodine D.M. Mol. Cell. Biol. 1998; 18: 6634-6640Crossref PubMed Google Scholar). We used this ankyrin promoter/humanAγ-globin reporter transgenic mouse model as a system to study the effect of the spherocytosis-associated promoter mutations in vivo. To dissect the effect of the −108 mutation from the linked −153 mutation, we created mutant ankyrin promoter transgenes with the −108 mutation, the linked −108 and −153 mutations, and an experimental control with the −153 mutation (Fig.1), even though the −153 mutation has not been reported solely without the −108 mutation. Wild type or mutant minimal ankyrin-1 promoter fragments were fused to the human Aγ-globin sequence immediately upstream of the ATG initiation codon (Ank WT, −108, −108/−153, or −153, respectively/Aγ-globin; Figs. 1 and 2). Sixteen wild type, eight −108, seven −108/−153, and eight −153 transgenic mouse lines were generated. Southern blot analysis determined that the transgene copy number in these mice ranged between 1 and 15 (TableII).Table IIExpression of ankyrin/Aγ-globin mRNA in transgenic miceLinesTransgene copy no.Aγ-globin mRNA/α-globin mRNAAγ-globin mRNA/transgene copy no.Wild type A10.020.02 B10.0230.023 C20.1120.056 D10.0390.039 E20.0550.0275 F150.40.0267 H10.0470.047 I20.1230.0615 J30.1690.0563 K60.2810.0468 L20.1280.064 M30.1290.043 N50.1980.0396 O40.2440.061 P30.1110.037 Q120.140.017 Mean0.041 ± 0.004−108T to C A10.1120.112 B20.040.02 C10.0120.012 D110.3050.0277 E20.0450.0225 F40.1010.0253 G20.0510.0265 H40.1980.0495 Mean0.03681 ± 0.011p = 0.653, NSaNS, not significant.−108T to C/−153G to A A10.0150.015 B30.0160.0053 C20.0110.0055 D10.005bValues ∼0.005 and lower are indistinguishable from background.0.005 E30.008bValues ∼0.005 and lower are indistinguishable from background.0.003 F40.007bValues ∼0.005 and lower are indistinguishable from background.0.0018 G40.0180.0036 Mean0.006 ± 0.002p < 0.0001−153G to A A110.2730.0248 B110.3380.0307 C30.170.0567 D30.120.04 E60.1770.0295 F10.0380.038 G40.1910.0478 H10.0350.035 Mean0.03781 ± 0.004p = 0.5907, NSa NS, not significant.b Values ∼0.005 and lower are indistinguishable from background. Open table in a new tab The levels of Ank/Aγ-globin mRNA and endogenous murine α-globin mRNA in reticulocytes were analyzed by ribonuclease protection. Riboprobe one (Fig. 2), which ensures that both the human Aγ-globin and murine α-globin sequences are labeled to equal specific activity, allowing comparison of humanAγ-globin and murine α-globin mRNA levels, was used. Wild type mice demonstrated position-independentAγ-globin gene expression in all 16 lines (Fig.4 and Table II). Transgenic mice with the −108 mutation demonstrated position-independent expression (eight of eight transgenic lines expressed γ-globin with Ank/Aγ-globin mRNA levels similar to wild type,p = 0.6533). Severe effects on Ank/Aγ-globin mRNA levels were seen in seven lines of mice with the linked −108/−153 mutations (Fig.5 and Table II). Three lines had undetectable levels of Ank/Aγ-globin mRNA, indicating position-dependent expression, and in four lines the level of Ank/Aγ-globin mRNA was significantly lower than wild type (−108/−153 mean 0.006 ± 0.002% versuswild type 0.041 ± 0.004% Aγ-globin RNA/copy number, p < 0.0001). Transgenic mice with the −153 mutation demonstrated position-independent expression (8 of 8 transgenic lines expressed γ-globin with Ank/Aγ-globin mRNA levels similar to wild type, p = 0.5907).Figure 5Correlation of transgene copy number with the levels of Ank/Aγ-globin mRNA. Linear regression analysis of the transgene copy number with the corrected mRNA expression level was performed.A, there is a linear relationship in wild type mice, indicating copy number-dependent expression. Correlation of corrected mRNA expression versus copy number in −108/−153 mutant transgenic mice (r 2 = 0.1505,p = not significant) demonstrates complete loss of copy number-dependent expression. B and C, expression is nearly identical to wild type in −108 (r 2 = 0.6533, p = 0.0028) and −153 (r 2 = 0.8755, p = 0.0006) transgenic mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previously, we have shown that the ankyrin promoter/Aγ-globin transgene initiates at position −81 in the corresponding ankyrin promoter/5′-flanking genomic DNA sequence (12Gallagher P.G. Romana M. Tse W.T. Lux S.E. Forget B.G. Blood. 2000; 96: 1136-1143Crossref PubMed Google Scholar, 18Sabatino D.E. Cline A.P. Gallagher P.G. Garrett L.J. Stamatoyannopoulos G. Forget B.G. Bodine D.M. Mol. Cell. Biol. 1998; 18: 6634-6640Crossref PubMed Google Scholar). To ensure that transcription was properly initiated in transgenic mice with mutant ankyrin promoters, we analyzedAγ-globin mRNA expression in reticulocyte RNA using riboprobe two (Fig. 2). All three mutant promoter transgenes initiated transcription at the same location as wild type (data not shown). Transgenic mice with the wild type ankyrin promoter expressed theAγ-globin transgene in a copy number-dependent fashion (copy number correlation with mRNA level r 2 = 0.5890, p = 0.0005 for all 16 strains, linear relationship, Table II and Fig. 5). Reporter gene expression relative to copy number in the −108 mice was similar to wild type, i.e. copy number-dependent (copy number correlation with mRNA level r 2= 0.7991, p = 0.0028, linear relationship). In the four −108/−153 mutant-expressing mice, there was no correlation between transgene copy number and mRNA level, indicating copy number-independent expression (copy number correlation with mRNA level r 2 = 0.1505, p = not significant; Table II and Fig. 5). The −153 experimental control mice expressed the Aγ-globin reporter gene relative to copy number in a copy number-dependent fashion (copy number correlation with mRNA level r 2 = 0.8755,p = 0.0006, linear relationship). To determine whether humanAγ-globin protein was present in the red cells of transgenic animals, we used an anti-human γ-globin monoclonal antibody for fluorescence-activated cell sorting analyses. Transgenic mice with the wild type promoter expressed human γ-globin in a uniform pattern, i.e. in 100% of erythrocytes (TableIII, Fig.6). Similar analyses demonstrated different results in the mutant promoter transgenic lines (Table III, Fig. 6). γ-Globin expression was variegated in all eight lines of −108 mice, with between 10 and 80% of erythrocytes expressing γ-globin. In two of the four expressing transgenic lines with the −108/−153 mutation and in five of eight −153 mutant control lines, γ-globin gene expression was variegated (Table III, Fig. 6).Table IIIγ-Globin protein expression in erythrocytes of transgenic miceLinesRed blood cells expressing γ-globin%Wild type A100 C100 D100 H100 J100 Q100−108T to C A50 B50 C10 D50 E40 F40 G30 H80−108T to C/−153G to A A100 B60 C80 D—aRNA undetectable. E—aRNA undetectable. F—aRNA undetectable. G100−153G to A A100 B60 C100 D50 E50 F100 G50 H60a RNA undetectable. Open table in a new tab Nondominant inheritance is the term assigned to patients with clinical and laboratory features of HS whose parents do not demonstrate clinical features of the disease. Nondominant inheritance is relatively common, seen in one quarter to one third of HS patients (1Gallagher P.G. Jarolim P. Hematology: Basic Principles and Practice. W. B. Saunders Co., Philadelphia1999: 576-610Google Scholar, 2Iolascon A. Miraglia del Giudice E. Perrotta S. Alloisio N. Morle L. Delaunay J. Haematologica. 1998; 83: 240-257PubMed Google Scholar, 3Tse W.T. Lux S.E. Br. J. Haematol. 1999; 104: 2-13Crossref PubMed Scopus (240) Google Scholar, 4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar). In these cases, the underlying genetic defects are heterogeneous. In some cases, true recessive inheritance as a result of abnormalities in either α spectrin or protein 4.2 has been suspected (1Gallagher P.G. Jarolim P. Hematology: Basic Principles and Practice. W. B. Saunders Co., Philadelphia1999: 576-610Google Scholar, 2Iolascon A. Miraglia del Giudice E. Perrotta S. Alloisio N. Morle L. Delaunay J. Haematologica. 1998; 83: 240-257PubMed Google Scholar, 3Tse W.T. Lux S.E. Br. J. Haematol. 1999; 104: 2-13Crossref PubMed Scopus (240) Google Scholar, 23Agre P. Orringer E.P. Bennett V. N. Engl. J. Med. 1982; 306: 1155-1161Crossref PubMed Scopus (131) Google Scholar, 24Agre P. Casella J.F. Zinkham W.H. McMillan C. Bennett V. Nature. 1985; 314: 380-383Crossref PubMed Scopus (142) Google Scholar, 25Agre P. Asimos A. Casella J.F. McMillan C. N. Engl. J. Med. 1986; 315: 1579-1583Crossref PubMed Scopus (113) Google Scholar, 26Whitfield C.F. Follweiler J.B. Lopresti-Morrow L. Miller B.A. Blood. 1991; 78: 3043-3051Crossref PubMed Google Scholar, 27Wichterle H. Hanspal M. Palek J. Jarolim P. J. Clin. Invest. 1996; 98: 2300-2307Crossref PubMed Scopus (76) Google Scholar, 28Yawata Y. Kanzaki A. Yawata A. Doerfler W. Ozcan R. Eber S.W. Int. J. Hematol. 2000; 71: 118-135PubMed Google Scholar), but precise genetic defects have been identified in only a few (1Gallagher P.G. Jarolim P. Hematology: Basic Principles and Practice. W. B. Saunders Co., Philadelphia1999: 576-610Google Scholar, 27Wichterle H. Hanspal M. Palek J. Jarolim P. J. Clin. Invest. 1996; 98: 2300-2307Crossref PubMed Scopus (76) Google Scholar,28Yawata Y. Kanzaki A. Yawata A. Doerfler W. Ozcan R. Eber S.W. Int. J. Hematol. 2000; 71: 118-135PubMed Google Scholar). In other cases, a de novo dominant mutation in ankyrin or β-spectrin has been identified (29Becker P.S. Tse W.T. Lux S.E. Forget B.G. J. Clin. Invest. 1993; 92: 612-616Crossref PubMed Scopus (56) Google Scholar, 30Miraglia del Giudice E. Hayette S. Bozon M. Perrotta S. Alloisio N. Vallier A. Iolascon A. Delaunay J. Morle L. Br. J. Haematol. 1996; 93: 828-834Crossref PubMed Scopus (25) Google Scholar, 31Morle L. Bozon M. Alloisio N. Vallier A. Hayette S. Pascal O. Monier D. Philippe N. Forget B.G. Delaunay J. Am. J. Hematol. 1997; 54: 242-248Crossref PubMed Scopus (18) Google Scholar, 32Randon J. Miraglia del Giudice E. Bozon M. Perrotta S. De Vivo M. Iolascon A. Delaunay J. Morle L. Br. J. Haematol. 1997; 96: 500-506Crossref PubMed Scopus (26) Google Scholar, 33Dhermy D. Galand C. Bournier O. Cynober T. Mechinaud F. Tchemia G. Garbarz M. Blood Cells Mol. Dis. 1998; 24: 251-261Crossref PubMed Scopus (12) Google Scholar). However, in most cases, the underlying genetic defect is unknown (4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar, 9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar, 11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar, 23Agre P. Orringer E.P. Bennett V. N. Engl. J. Med. 1982; 306: 1155-1161Crossref PubMed Scopus (131) Google Scholar, 24Agre P. Casella J.F. Zinkham W.H. McMillan C. Bennett V. Nature. 1985; 314: 380-383Crossref PubMed Scopus (142) Google Scholar, 25Agre P. Asimos A. Casella J.F. McMillan C. N. Engl. J. Med. 1986; 315: 1579-1583Crossref PubMed Scopus (113) Google Scholar, 28Yawata Y. Kanzaki A. Yawata A. Doerfler W. Ozcan R. Eber S.W. Int. J. Hematol. 2000; 71: 118-135PubMed Google Scholar). These cases present a challenging and unsolved problem in genetic counseling. Large genetic screens of ankyrin-deficient HS patients identified the ankyrin promoter mutations studied in this report (4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar, 9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar, 11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar). In several of these cases, additional mutations in the coding region were found in the ankyrin gene in trans to the allele with the promoter mutation (9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar, 11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar). As the inheritance is recessive, it is anticipated that all patients heterozygous for ankyrin gene promoter mutations have an additional ankyrin gene mutation in trans.These promoter mutations are common in the general population, with a frequency of ∼2–3% (9Eber S.W. Gonzalez J.M. Lux M.L. Scarpa A.L. Tse W.T. Dornwell M. Herbers J. Kugler W. Ozcan R. Pekrun A. Gallagher P.G. Schroter W. Forget B.G. Lux S.E. Nat. Genet. 1996; 13: 214-218Crossref PubMed Scopus (178) Google Scholar, 11Leite R.C. Basseres D.S. Ferreira J.S. Alberto F.L. Costa F.F. Saad S.T. Hum. Mutat. 2000; 16: 529Crossref PubMed Scopus (19) Google Scholar). Thus, the inheritance of one of these promoter mutations could unmask other ankyrin gene mutations, making this a relatively common cause of recessive HS. It is interesting to note that osmotic fragility screening of random blood donors identified an HS “carrier state” in slightly more than 1% of donors (34Eber S.W. Pekrun A. Neufeldt A. Schroter W. Ann. Hematol. 1992; 64: 88-92Crossref PubMed Scopus (81) Google Scholar, 35Godal H.C. Heisto H. Scand. J. Haematol. 1981; 27: 30-34Crossref PubMed Scopus (67) Google Scholar). It has been suggested that these donors were potential parents of patients with recessively inherited HS (4Miraglia del Giudice E. Nobili B. Francese M. D'Urso L. Iolascon A. Eber S. Perrotta S. Br. J. Haematol. 2001; 112: 42-47Crossref PubMed Scopus (52) Google Scholar). It is tempting to speculate that some of these donors may be heterozygous for an ankyrin promoter mutation. We attempted to determine the mechanism(s) by which these mutations exert their influence on ankyrin gene expression. The transgenes tested in these assays initiated mRNA transcription at the correct site, suggesting that a defect in transcript initiation is unlikely. The most severe effects on Ank/Aγ-globin mRNA and protein expression were seen in mice with the linked −108/−153 promoter mutations who demonstrated significantly decreased γ-globin mRNA in a position-dependent, copy number-independent manner, as well as variegated distribution of γ-globin protein in erythrocytes. The −108 mutant promoter transgenic mice demonstrated differences only in the cellular distribution of the γ-globin protein. These are interesting observations, as no differences between these promoter mutations and wild type promoter were seen in in vitrostudies. Together, these data suggest that intact chromatin is necessary to manifest the promoter defects. The −153 mutation does not create or abolish consensus sequences for any known DNA-binding proteins and although the −108 mutation occurs in an AP-2 consensus binding sequence, in vitro studies suggest that this is not a functionally important site in wild type ankyrin gene expression (Ref. 12Gallagher P.G. Romana M. Tse W.T. Lux S.E. Forget B.G. Blood. 2000; 96: 1136-1143Crossref PubMed Google Scholar and this report). It is possible that a previously undescribed DNA-binding protein interacts with either or both of these sites. The upstream −153 mutation is located in a stretch of CpG dinucleotides, a region of potential DNA methylation. DNA methylation controls gene expression by modulating access of regulatory elements to a gene promoter. There is a second ankyrin-1 gene promoter, active in neural and muscle cells, located 40 kilobase pairs upstream of the erythroid promoter (36Gallagher P.G. Wong E. Wong C. Blood. 1998; 92: 300aCrossref PubMed Google Scholar). It is possible that, in wild type cells in vivo, erythroid cell-specific methylation in the region of the −153 mutation prevents binding of a protein with negative regulatory activity or one with enhancer-blocking activity that creates a boundary element (i.e. an “insulator”). Mutation and disruption of methylation in this region could then facilitate DNA-protein binding. The latter has recently been shown at the H19/Igf2 locus, where methylation mediates the binding and enhancer blocking activity of the multifunctional zinc finger protein CTCF (37Bell A.C. Felsenfeld G. Nature. 2000; 405: 482-485Crossref PubMed Scopus (1373) Google Scholar, 38Hark A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. Nature. 2000; 405: 486-489Crossref PubMed Scopus (1230) Google Scholar, 39Bell A.C. West A.G. Felsenfeld G. Cell. 1999; 98: 387-396Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar). Additional evidence for this hypothesis comes from the observations that removal of a long range insulator or boundary element from a region can shut down the locus and lead to position effects (40Kleinjan D.J. van Heyningen V. Hum. Mol. Genet. 1998; 7: 1611-1618Crossref PubMed Scopus (292) Google Scholar). These phenomena, i.e. decreased gene expression and position-dependent expression, were observed in the mutant −153/−108 mice. Future studies will provide insight into how these mutations affect regulation of the ankyrin gene.
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