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Originally published In Press as doi:10.1074/jbc.M208184200 on August 23, 2002

J. Biol. Chem., Vol. 277, Issue 44, 41563-41570, November 1, 2002
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Erythroid Expression of the Human alpha -Spectrin Gene Promoter Is Mediated by GATA-1- and NF-E2-binding Proteins*

Laurent BoulangerDagger , Denise E. Sabatino§, Ellice Y. Wong, Amanda P. Cline§, Lisa J. Garrett§, Michel GarbarzDagger , Didier DhermyDagger , David M. Bodine§, and Patrick G. Gallagher||

From Dagger  INSERM U409, Association Claude Bernard, Universite Paris 7, Faculte X. Bichat, 75870 Paris Cedex 18, France,  Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8021, and § Hematopoiesis Section, Genetics and Molecular Biology Branch, NHGRI, National Institutes of Health, Bethesda, Maryland 20892-4442

Received for publication, August 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha -Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha -spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha -spectrin gene. We mapped the 5' end of the alpha -spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha -spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha -spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha -spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha -spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Spectrin, the most abundant protein of the erythrocyte membrane skeleton, is composed of two structurally similar but nonidentical proteins, alpha - and beta -spectrin, encoded by separate genes (1, 2). alpha - and beta -spectrin are composed primarily of homologous 106-amino acid repeats that fold into three antiparallel alpha -helices connected by short nonhelical segments (3-7). alpha - and beta -spectrin combine to form heterodimers, which in turn self-associate to form tetramers and higher order oligomers to form a lattice-like structure that is critical for erythrocyte membrane stability, as well as erythrocyte shape and deformability (8-12). In the red cell, spectrin maintains cellular shape, regulates the lateral mobility of integral membrane proteins, and provides structural support for the lipid bilayer (2, 13). Quantitative and qualitative disorders of alpha -spectrin have been associated with abnormalities of erythrocyte shape including hereditary spherocytosis, elliptocytosis, and pyropoikilocytosis (12, 14-19).

In erythropoiesis, differentiation of early erythroid progenitor cells into morphologically defined erythroblasts is associated with significant changes in the synthesis and expression of membrane proteins. In splenic erythroblasts isolated from mice early after Friend virus infection, there is marked synthesis of spectrin with a significant excess of alpha -spectrin over beta -spectrin (20, 21). However, at this stage, only a small fraction of this newly synthesized spectrin is incorporated into the membrane skeleton (21). During terminal differentiation, alpha - and beta -spectrin synthesis are decreased, but a markedly increased amount of spectrin is incorporated into the membrane. Studies of erythropoiesis in avian and rat cells have shown that the increased alpha -spectrin synthesis in early erythropoiesis is controlled at the transcriptional level (21-23). The molecular mechanisms that regulate the tissue or developmental stage-specific expression of alpha -spectrin, including the mechanisms that control the increase in alpha -spectrin gene transcription to high levels during the early stages of erythropoiesis, are unknown.

The identification and characterization of the regulatory elements that control alpha -spectrin gene expression have important implications for several biological processes including the pathogenesis of alpha -spectrin-linked hemolytic anemia and erythrocyte membrane protein biosynthesis and assembly. Furthermore, because alpha -spectrin is synthesized in large amounts in erythroid cells, identification of the regulatory elements directing this high level, tissue-specific expression may provide important tools in directing other erythroid-specific genes in gene therapy applications.

To provide insight into the regulation of the human alpha -spectrin gene, we have identified and characterized the human alpha -spectrin gene promoter. Our results demonstrate that the human alpha -spectrin gene promoter requires GATA-1- and NF-E2-binding proteins to direct high level expression in erythroid cells in vitro. In transgenic mice, a reporter gene directed by the alpha -spectrin promoter directed expression exclusively in erythroid progenitor cells at early stages of erythroid differentiation. These results suggest that the minimal alpha -spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells and that additional regulatory elements are required for late developmental stage expression of the alpha -spectrin gene in erythroid cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RNA Preparation and 5' Rapid Amplification of cDNA Ends-- Total RNA was prepared from human tissues or from the human tissue culture cell lines K562 (chronic myelogenous leukemia in blast crisis with erythroid characteristics; ATCC, CCL 243) or HeLa (epithelial-like carcinoma; cervix, CCL 2) as described (24). 1 µg of total human fetal liver RNA was reverse-transcribed using Primer A (see Table I) and avian myeloblastosis virus reverse transcriptase (Promega). Single-stranded oligonucleotide ligation and PCR amplification were carried out as described using Primers A + C and B + C (25, 26) (see Fig. 1). Amplification products were subcloned and sequenced.

Mapping the Transcription Initiation Site-- The transcription initiation site of the erythroid alpha -spectrin cDNA was determined using a primer extension assay. Primer D (see Table I) was 5' end-labeled with [32P]ATP and T4 polynucleotide kinase and then ethanol was precipitated with 20 µg of total K562 cell RNA. The pellets were resuspended in hybridization buffer, heated to 60 °C for 90 min, and then precipitated. The pellets were resuspended, and the primer was extended with avian myeloblastosis virus reverse transcriptase at 42 °C for 1 h. The RNA was then digested with RNase A. Extension products were ethanol-precipitated, dissolved in loading buffer, and analyzed on a 6% denaturing acrylamide gel.

Genomic Cloning-- A human alpha -spectrin cDNA fragment corresponding to the 5' end of the coding region, alpha 19 (see Fig. 1A) (4, 27), was used as hybridization probe to screen a human genomic DNA library. The library is a Charon 4A bacteriophage library containing fragments of genomic DNA partially digested with AluI and HaeIII with EcoRI linkers added. Selected recombinants that hybridized to alpha 19 were purified and subcloned into pGEM-7Z plasmid vectors (Promega). Subcloned fragments were analyzed by restriction endonuclease digestion, Southern blotting, and nucleotide sequencing.

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 (28) or Dignam et al. (29).

DNase I Footprinting in Vitro-- Probes for DNase I footprinting were produced by PCR amplification of an alpha -spectrin genomic fragment as template and a pair of oligonucleotide primers, E and F (see Table I). One oligonucleotide, either E or F, was 5' end-labeled with [32P]ATP using polynucleotide kinase prior to 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.

Preparation of Promoter-Reporter Plasmids for Transfection Assays-- A 793-bp fragment corresponding to 5' flanking alpha -spectrin genomic DNA was amplified using primers G + H (see Table I), which contain sites for KpnI and XhoI, respectively. The 793-bp amplification product was digested with KpnI and XhoI and then subcloned upstream of the firefly luciferase reporter gene in the plasmid pGL2B (Promega). Serial truncations of this 793-bp fragment in the pGL2B plasmid were constructed using PCR amplification. Integrity of all test plasmids was confirmed by sequencing.

Transient Transfection Analyses-- All plasmids tested were purified using Qiagen columns or cesium chloride plasmid purification, and at least two preparations of each plasmid were tested in triplicate. 107 K562 cells were transfected by electroporation with a single pulse of 300 V at 960 microfarad with 20 µg of test plasmid and 0.5 µg of pCMVbeta , a mammalian reporter plasmid expressing beta -galactosidase driven by the human cytomegalovirus immediate early gene promoter (Clontech) (30). 105 HeLa cells were transfected with 2.0 µg of test plasmid and 0.25 µg of the pCMVbeta plasmid by lipofection using 4 µl of LipofectAMINE (Invitrogen). Twenty-four h after transfection, cells were harvested and lysed, and the activity of both luciferase and beta -galactosidase activity was determined in cell extracts. All assays were performed in triplicate. Differences in transfection efficiency were determined by co-transfection with the pCMVbeta plasmid.

COS cells (107) were transfected with 20 µg of the expression plasmids pMT/BKLF (31) (a kind gift of Drs. M. Crossley and S. Orkin) or pSG5/EKLF (32) (a kind gift of Dr. J. Bieker) as described above. Forty-eight h after transfection, nuclear extracts were prepared for use in gel shift analyses.

Gel Mobility Shift Analyses-- Binding reactions were carried out as described (33). Competitor oligonucleotides were added at molar excesses of 100-fold. Antibodies to GATA-1, p45 NF-E2, and Sp1 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Reaction mixes electrophoresed through 6% nondenaturing polyacrylamide gels in 0.5× Tris borate-EDTA at 21 °C at 200 watts for 2 h. Gels were dried and subjected to autoradiography.

Preparation of Promoter-Reporter Plasmids for Transgenic Mice-- A 793-bp alpha -spectrin gene promoter fragment was excised from plasmid p793 as a SmaI/BglII fragment and ligated into the EcoRV/BamHI sites of pSP72. A 2266-bp EcoRV/AatII fragment containing the human Agamma -globin gene was excised from a pSP72 plasmid containing a 1909-bp BsaHI/HindIII fragment of the human Agamma -globin gene (34) and ligated into the AatII/PvuII sites of the pSP72 plasmid to generate alpha Sp/Agamma . This plasmid construct was sequenced to confirm that the alpha -spectrin promoter was correctly fused to the gamma -globin gene. Finally, a 2694-bp alpha -spectrin promoter/Agamma -globin gene fragment (see Fig. 8) was excised from this plasmid with KpnI and HindIII for microinjection.

Generation of Transgenic Mice-- Transgenic mice were generated as described by Hogan et al. (35) and Sabatino et al. (34). Fertilized eggs were collected from superovulated FVB/N female mice ~9 h after mating to CB6F1 male mice. After purification, alpha -spectrin promoter/Agamma -globin DNA fragments were microinjected into the male pronucleus of fertilized eggs. The injected eggs were transferred into pseudopregnant CB6F1 foster mothers. Founders were identified by Southern blotting of genomic DNA obtained from tail biopsies. Founder animals were crossed to FVB/N mice for propagation.

Transgene Copy Number Analysis-- Copy number was determined by comparing the gamma -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.

Preparation of Murine RNA-- Total cellular RNA was extracted from mouse tissues, including adult reticulocytes and separated splenic cells, using TRIZOL reagent (Invitrogen). Splenic cells were separated into erythroid, myeloid, or lymphoid fractions using the following method. Total spleen cells were dissociated by passage through 16- and then 21-gauge needles. The single cell suspension was washed in phosphate-buffered saline with 5% fetal calf serum two times and suspended at 107 cells per ml in phosphate-buffered saline with 5% fetal calf serum. Ten ml of this was used to prepare RNA by TRIZOL. To the remaining 10 ml, 250 µl of TER 119 antibody was added. The cells were incubated for 25 min on ice with mixing every 5 min. The cells were underlayed with 1 ml of phosphate-buffered saline with 10% fetal calf serum and spun at 1500 rpm for 5 min at 4 °C. The cells were resuspended in 10-ml goat and rat magnetic beads in 5% fetal calf serum for 30 min on ice with mixing every 5 min. The cells were applied to a magnet for 10 min. The beads and adherent cells were collected and processed directly to yield erythroid RNA. The nonadherent cells were collected and placed in a new tube to which 50 µl of anti-CD4 and 50 µl of anti-CD8 were added, and the cells were incubated on ice for 30 min with mixing. CD4/8-positive cells were collected as above from the magnet and processed for lymphoid RNA. The remaining nonadherent cells were treated with antibodies against MAC-1 (50 µl) and Gr-1 (50 µl), and myeloid cells were collected for myeloid RNA.

Ribonuclease Protection Analysis of Transgene Expression-- Expression of the erythroid alpha -spectrin promoter/Agamma -globin reporter transgene was analyzed using an RNase protection assay. Linear DNA templates for the RNase protection assay were prepared by EcoRI digestion of a human alpha  spectrin/Agamma -globin plasmid or by HindIII digestion of a murine alpha -globin plasmid. Templates were purified by agarose gel electrophoresis. The 32P-labeled antisense RNA probe was synthesized by transcription with SP6 RNA polymerase (MAXIscript in vitro transcription kit; Ambion Incorporated, Austin, TX). The probe (1 × 105 cpm per assay) was hybridized to template RNA at 42 °C overnight. Template RNAs in these reactions were 1 µg of total RNA from yolk sac, fetal liver, adult reticulocyte, bone marrow, muscle, brain, heart, kidney, and splenic cells separated into erythroid, myeloid, or lymphoid cells or 10 µg of tRNA. Hybrids were digested with a mixture of the nucleases RNase A and RNase T1. After digestion, protected fragments were detected by autoradiography after electrophoresis in 8% denaturing polyacrylamide gels.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of Chromosomal Gene, Isolation and Analysis of Recombinant Clones-- Primary screening of a human genomic DNA library with the alpha -spectrin cDNA probe alpha 19 (Fig. 1A) yielded multiple hybridization-positive plaques. Selected recombinants were analyzed, and one clone was identified, lambda 3021, that spanned ~16.5 kb of DNA containing the alpha -spectrin gene. A limited restriction map of this region is shown in Fig. 1B.


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  		Fig. 1.   The 5' end of the human alpha -spectrin gene. A, structure of the 5' end of the human alpha -spectrin-1 erythroid cDNA. A diagram of the 5' end of the human alpha -spectrin cDNA is shown. The location of the initiation codon is shown, as is the location of intron/exon boundaries. The location of the probe used in genomic library screening, alpha 19, the 5' most alpha -spectrin cDNA clone (4), is shown. B, genomic organization of the 5' end of the human alpha -spectrin gene. One clone containing the alpha -spectrin gene was isolated from a human genomic DNA library. This clone, lambda 3021, spanned a distance of ~16.5 kb and contained exon 1 of the alpha -spectrin erythroid cDNA in a 14-kb EcoRI fragment. A partial restriction map of clone lambda 3021 with EcoRI (E) and XbaI (X) is shown.

Mapping the Human alpha -Spectrin Erythroid mRNA Transcription Initiation Site and Identification of 5' cDNA Sequences-- To identify the 5' end of the human alpha -spectrin cDNA, primer extension was performed using total RNA from K562 cells. These experiments identified a single transcription initiation site (Fig. 2) and predicted the presence of an additional 17 bp in the mRNA upstream of the 5' end of the sequence obtained from cDNA cloning. These additional 17 bp of upstream 5' untranslated sequence were obtained by 5' rapid amplification of cDNA ends. Sequences obtained by rapid amplification of cDNA ends (Fig. 1A) were verified by comparison to corresponding genomic DNA sequences (Fig. 3). The sequences around the transcription start site, GTA+1TGTC, closely match transcription initiation recognition sequences, YYA+1NWYY (36). No additional ATGs were present in the 5' untranslated sequences. Taken together, these data suggest that this sequence is at or very near the 5' end of the human alpha -spectrin erythroid cDNA.


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  		Fig. 2.   Mapping the 5' end of the human alpha -spectrin cDNA. Primer extension was carried out using 20 µg of K562 total RNA as template. ACGT, nucleotide markers; M, MspI-digested pBR322 marker; lane 1, labeled primer plus RNA; lane 2, labeled primer alone. The size of the extension products (lane 1, arrow) indicates that the 5' end of the mRNA is located 40 bp upstream relative to the 3' end of the primer. The cDNA sequence of this additional 5' untranslated cDNA was determined by 5' rapid amplification of cDNA ends and is shown in Fig. 3.


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  		Fig. 3.   5' flanking genomic DNA sequence. The nucleotide sequence of the 5' flanking genomic DNA of the human alpha -spectrin gene is shown. Consensus sequences for potential DNA-protein binding sites are underlined. The transcription initiation site, +1, is denoted by the arrow above the A. The sequences beginning intron 1 are shown in lowercase.

The 5' Flanking Genomic DNA Sequence of the Human alpha -Spectrin-1 Gene Exhibits Features of an Erythroid Gene Promoter-- The nucleotide sequence of the 5' flanking genomic DNA upstream of the human alpha -spectrin cDNA transcription start site is shown in Fig. 3. Inspection of the sequence reveals features lack consensus TATA or CCAAT sequences. Consensus sequences for a number of potential DNA-binding proteins, including GATA-1 (three sites), NF-E2, AP-1, and Sp1/CACCC-related proteins are present in the 5' flanking sequences.

An alpha -Spectrin Gene Promoter Fragment Is Active in Erythroid Cells-- To investigate whether the region from -793 to +1 was capable of directing expression of a reporter gene in cultured mammalian cells, test plasmid p793 was transiently transfected into K562 cells. The relative luciferase activity was determined 48 h after transfection and compared with the activity obtained with pGL2B, a negative control, the promoterless plasmid, and pGL2P, a positive control, the luciferase reporter gene under control of the SV40 early promoter. As shown in Fig. 4A, the putative alpha -spectrin gene erythroid promoter plasmid, p793, directed high level expression of the luciferase reporter gene in erythroid cells.


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  		Fig. 4.   Activity of the alpha -spectrin gene erythroid promoter in erythroid and nonerythroid cell lines in transient transfection assays. a, plasmids containing 5' flanking DNA of the alpha -spectrin gene inserted upstream of the firefly luciferase gene were transfected into K562 or HeLa cells as described. Relative luciferase activity was expressed as that obtained from the test plasmids versus the activity obtained from the promoterless plasmid pGL2B plasmid taking into account the transfection efficiency. The data are means ±S.D. of at least six independent transfection experiments. b, mutations in consensus DNA protein binding sites are marked with an X.

Transient transfection analysis of deletions of this alpha -spectrin gene erythroid promoter fragment identified a 195-bp minimal promoter fragment, p194, that directed expression of the reporter gene in erythroid cells (Fig. 4A). This minimal promoter fragment contains potential binding sites for GATA-1, as well as Sp1 and CACCC-related proteins, a combination shown to be adequate for expression of a minimal promoter in other erythroid-specific genes. There was minimal or no reporter gene activity of either p793 or p194 in transfected HeLa cells.

The alpha -Spectrin Erythroid Promoter Contains Binding Sites for GATA-1, NF-E2, and CACCC-related Binding Proteins-- To identify binding sites for transcription factors within the core alpha -spectrin promoter, DNase I footprinting analysis with nuclear extracts from K562 cells was performed (Fig. 5). A single long footprint was observed. This site contains consensus binding sequences for GATA-1, NF-E2, and CACCC binding proteins.


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  		Fig. 5.   In Vitro DNase I footprinting of the human alpha -spectrin promoter. In vitro DNase I footprinting of the human alpha -spectrin gene promoter was performed using K562 extracts as described in the text. A single long protected site was identified corresponding to GATA-1-, NF-E2-, and CACCC-related protein consensus binding sites. M, black-square.

GATA-1 Binds the alpha -Spectrin Gene Promoter in Vitro-- To determine whether nuclear proteins could bind the three GATA-1 sites present in the alpha -spectrin gene promoter in vitro, double-stranded oligonucleotides containing the corresponding alpha -spectrin promoter GATA-1 sequences (site 1, I + J; site 2, K + L; site 3, M + N) (see Table I) or control GATA-1 sequences (O + P; see Table I) (37) were prepared and used in gel shift analyses. When oligonucleotides containing the upstream GATA-1 sequences were used in gel shift analyses, a single retarded species was observed in K562 (erythroid) extracts when probes corresponding to either site 2 (Fig. 6) or site 3 (not shown) but not site 1 (not shown). These species 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 of the site 2 (Fig. 6) and site 3 probes (not shown). When oligonucleotides with mutation of the consensus GATA-1 binding sequences (GATA to GTTA) (38) corresponding to either site 2 or site 3 were used in gel mobility shift assays, complex formation was completely abolished (not shown). These data indicate that GATA-1 binds to the two downstream GATA-1 sites but not the upstream GATA-1 site of the alpha -spectrin gene promoter in vitro.

                              
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Table I
Oligonucleotide primers


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  		Fig. 6.   Gel mobility shift assays of the GATA-1 #2 of the human alpha -spectrin gene promoter. Gel mobility shift assays using oligonucleotides corresponding to the GATA-1 #2 consensus binding site of the human alpha -spectrin promoter were performed using K562 nuclear extracts. A GATA-1 antibody was added to the reaction mixtures where indicated.

Nuclear Proteins Bind the alpha -Spectrin Gene Promoter NF-E2 Site in Vitro-- To determine whether nuclear proteins could bind the NF-E2 consensus binding sequences in vitro, double-stranded oligonucleotides containing the corresponding alpha -spectrin promoter NF-E2 sequences (Q + R; see Table I) or control sequences (S + T; see Table I) (39, 40) were prepared and used in gel shift analyses. When oligonucleotides containing the footprinted NF-E2 sequences were used in gel shift analyses with K562 cells extracts, a large retarded species was observed (Fig. 7). These species migrated at the same location as a control oligonucleotide containing an NF-E2 consensus sequence. This species was effectively competed both by an excess of unlabeled homologous oligonucleotide and by an excess of unlabeled control NF-E2 oligonucleotide. The inclusion of p45 NF-E2 antisera abolished most or all of the DNA binding (Fig. 7). When a double-stranded oligonucleotide with mutation of the alpha -spectrin promoter consensus NF-E2 binding sequences (GCTGAGTCA to TCTGAGTCA) (39, 40) was used in gel mobility shift assays, complex formation was abolished (not shown). These data indicate that NF-E2-binding proteins bind in vitro to the alpha -spectrin gene promoter.


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  		Fig. 7.   Gel mobility shift assays of the NF-E2 site of the human alpha -spectrin gene promoter A. Gel mobility shift assays using alpha -spectrin promoter oligonucleotides corresponding to the NF-E2 consensus binding sequences and K562 nuclear extracts are shown. The radiolabeled, double-stranded oligonucleotide used in lanes 1-6 corresponds to site 3, and the radiolabeled, double-stranded oligonucleotide used in lanes 7-12 is a CACCC control. Increasing amounts of unlabeled, double-stranded oligonucleotide, site 3 (lanes 3, 4, 11, and 12), CACCC control (lanes 5 and 9), or Sp1 control (lanes 6 and 10) were added to the reactions as competitor. A p45 NF-E2 antibody was added to the reaction mixtures where indicated.

CACCC Box-binding Proteins Do Not Bind to the alpha -Spectrin Gene Promoter in Vitro-- The protected region obtained in DNase I footprinting included the sequence, 5'-CCACCC-3', a consensus binding site for CACCC box-binding proteins. To determine whether the nuclear proteins Sp1, BKLF, or EKLF bind this sequence in vitro, double-stranded oligonucleotides containing the corresponding alpha -spectrin CCACCC site sequences (U + V; see Table I) or control sequences (Sp1, W + X (41, 42); CACCC, Y + Z (32, 43); see Table I) were prepared and used in gel shift analyses. When double-stranded oligonucleotides containing the CACCC site sequences were used in gel shift analyses with K562 extracts, no complexes migrated at the same location as those obtained using control oligonucleotides containing either CACCC or Sp1 consensus sequences (not shown). To determine whether the CACCC box binding transcription factors BKLF or EKLF could bind the alpha -spectrin gene promoter CACCC site in vitro, gel shifts using nuclear extracts prepared from COS cells transfected with expression plasmids containing either BKLF or EKLF cDNAs and either the alpha -spectrin gene promoter CACCC site oligonucleotide or a control beta -globin CACCC oligonucleotide (Y + Z) (43) were performed. No complex was obtained with the alpha -spectrin site CACCC oligonucleotide and extracts from either BKLF- or EKLF-transfected cells (not shown).

GATA-1 and NF-E2 Proteins Are Both Major Activators of the Human Erythroid alpha -Spectrin Gene Promoter-- To assess the relative importance of these transcription factor binding sites in promoter function, mutations were introduced into each of the three sites protected in DNase I footprinting experiments and the two upstream GATA-1 sites. Mutation of the upstream GATA-1 consensus sequence (site 1, GATA to GTTA) had no effect on promoter activity (Fig. 4B). Mutating the site 2 GATA-1 consensus sequence in a similar manner (GATA to GTTA) reduced promoter activity by 25%. Mutation of the most 3' GATA-1 site, site 3, had no effect on promoter activity. Mutation of the NF-E2 site (GCTGAGTCA to TCTGAGTCA) reduced promoter activity by approximately one-half, indicating that this site is of major importance in the alpha -spectrin gene promoter. When a reporter plasmid with mutations of both the GATA-1 site 2 and NF-E2 sites was transfected into K562 cells, promoter activity was reduced to nearly background (Fig. 4B).

Transgenic Mice Express the alpha -Spectrin/Agamma -Globin Transgene in Erythroid Cells Only at Early Stages of Erythroid Development-- We created transgenic mice with a human alpha -spectrin promoter fragment from -793 to +1 fused to the human Agamma -globin gene (Fig. 8A). We also created a dual riboprobe that detects sequences from both exon 2 of the human gamma -globin gene and exon 2 of the murine alpha -globin gene (Fig. 8B). This riboprobe ensures that both human Agamma -globin and murine alpha -globin sequences are labeled to equal specific activity, allowing direct comparison of human Agamma -globin and murine alpha -globin mRNA levels in tissues from transgenic animals. Seven transgenic lines containing the alpha -spectrin/Agamma -globin transgene were analyzed. RNase protection demonstrated that 0 of 7 alpha -spectrin/Agamma -globin transgenic lines expressed the alpha -spectrin/Agamma -globin transgene in adult reticulocytes (see Table II and Fig. 9). In three lines examined, expression was detected in yolk sac, and lesser amounts were detected in fetal liver (see Table II and Fig. 9). The number of transgenes in each line was estimated by Southern blot analyses to be between 1 and 15 copies per expressing animal. After correction for copy number, the level of alpha -spectrin/Agamma -globin mRNA was compared with the mRNA of murine alpha -globin. Levels of Agamma -globin expression in yolk sac ranged from 0.04 to 0.08% of the levels of murine alpha -globin expression in the same cells.


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  		Fig. 8.   Human alpha -spectrin promoter/Agamma -globin transgene constructs and riboprobes. A, alpha -spectrin promoter/Agamma -globin transgene. A 793-bp alpha -spectrin gene promoter fragment (-793 to +1) was fused to the human Agamma -globin gene (-4 to +1906) to create the transgene construct shown. B, hybrid human Agamma -globin/mouse alpha -globin riboprobe. An Sp6 riboprobe containing sequences for both exon 2 of the human Agamma -globin gene and exon 2 of the murine alpha -globin gene was prepared to ensure that both the human Agamma -globin and murine alpha -globin sequences are labeled to equal specific activity in ribonuclease protection assays.

                              
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Table II
Developmental pattern of gamma -globin mRNA expression in erythroid tissues of alpha -spectrin/Agamma -globin transgenic mice


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  		Fig. 9.   Detection of alpha -spectrin/Agamma -globin mRNA in yolk sac, fetal liver, and adult reticulocytes of transgenic mice containing the alpha -spectrin/human gamma -globin transgene. 1.0 µg of RNA from yolk sac, fetal liver, or adult reticulocytes was hybridized to a 32P-labeled antisense riboprobe that protects exon 2 of the alpha -spectrin/Agamma -globin transgene (top band) and exon 2 of the mouse alpha -globin gene (lower band), digested with RNase, electrophoresed in an 8% nondenaturing gel, dried, and subjected to autoradiography.

The alpha -Spectrin/Agamma -Globin Transgene Does Not Direct Expression in Nonerythroid Tissues-- In three transgenic lines examined, the alpha -spectrin/Agamma -globin transgene was expressed in adult bone marrow (Table III). In these three lines, the level of transgene expression was examined in nonerythroid tissues of mice who were perfused with saline immediately prior to sacrificing. RNase protection did not detect gamma -globin mRNA in kidney, brain, heart, liver, lung, or skeletal muscle mRNA. Low levels of expression were detected in thymus. RNA was isolated from splenic cells harvested from these three transgenic lines and separated into erythroid, myeloid, and lymphoid cells. RNase protection analyses demonstrated low levels of alpha -spectrin/Agamma -globin expression only in erythroid cells (see Table III and Fig. 10).

                              
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Table III
Pattern of gamma -globin mRNA expression in tissues of alpha -spectrin/Agamma -globin transgenic mice


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  		Fig. 10.   Detection of alpha -spectrin/Agamma -globin mRNA in splenic cells separated into erythroid, myeloid, and lymphoid fractions from transgenic mice containing the alpha -spectrin/human gamma -globin transgene. 1.0 µg of RNA from erythroid, myeloid, or lymphoid cells was hybridized to a 32P-labeled antisense riboprobe which protects exon 2 of the alpha -spectrin/Agamma -globin transgene (top band) and exon 2 of the mouse alpha -globin gene (lower band), digested with RNase, electrophoresed in an 8% nondenaturing gel, dried, and subjected to autoradiography.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Comparison of sequences of the erythroid promoters of ankyrin-1, band 3, and beta -spectrin, other erythrocyte membrane proteins, have been performed (44, 46-48). In vitro characterization of the promoters of ankyrin-1, band 3, and beta -spectrin, other erythrocyte membrane proteins, have been performed previously (44, 46-48). These studies demonstrated that a combination of GATA-1- and CACCC-binding proteins is essential for high level expression of linked reporter genes. We conclude that the promoters of most erythrocyte membrane protein genes share similarities to other erythroid gene promoters where this combination may lead to cooperation between GATA-1- and CACCC-binding proteins to enhance transcription (49-56). Similarly, consensus binding sites for GATA-1- and CACCC-binding proteins are present in close proximity in the alpha -spectrin promoter. Our in vitro studies did not demonstrate a role for the CACCC binding site in the alpha -spectrin gene promoter.

In vitro, the NF-E2 consensus binding sequences in the alpha -spectrin gene promoter appear to play an important role in promoter function. NF-E2, a heterodimer of two basic leucine zipper proteins of 45 and 18 kDa, plays an important role in erythroid gene expression (57). The 45-kDa proteins, identified as proteins that interact with and activate the NF-E2 site in the beta -globin gene locus control region, include p45 NF-E2, the closely related Nrf1, Nrf2, Nrf3, and the more distantly related Bach-1 and Bach-2 (58-64). p45 NF-E2 expression is primarily restricted to hematopoietic cells, erythrocytes, neutrophils, megakaryocytes, and mast cells (57). The 18-kDa protein, a member of the small Maf oncoprotein family, is widely expressed (65, 66). NF-E2 proteins have been shown to bind to consensus sequences in the promoters and enhancers of several erythroid and megakaryocytic genes including the beta -globin locus control region, porphobilinogen deaminase, ferrochelatase, and thromboxane synthase (37, 67-72). NF-E2-binding proteins have not been shown previously to be involved in expression of other erythrocyte membrane skeleton genes. The core erythroid promoters of the ankyrin and beta -spectrin genes do not contain NF-E2 consensus binding sequences (44, 46). The erythrocyte band 3 promoter contains consensus binding sequences for NF-E2 (47, 48), but its role in band 3 promoter function has not been examined.

Our studies show that the core promoter of the human alpha -spectrin gene directs erythroid-specific expression in erythroid cells only in the early stages of erythroid development. This is in contrast to the erythroid promoters of the ankyrin and beta -spectrin genes, which direct expression at all stages of erythroid development (44-46). The overall level of expression directed by the core alpha -spectrin gene promoter was very low, 0.04-0.08% of mouse alpha -globin/transgene copy number in yolk sac compared with ~4 and ~3-9% for ankyrin and beta -spectrin, respectively (45, 46). This observation was surprising as previous studies had demonstrated that alpha -spectrin gene expression is quite high in erythroid cells and that the expression was controlled at the transcriptional level (21-23). These data indicate that elements outside the core alpha -spectrin gene promoter are required for high level expression during erythroid development.

    ACKNOWLEDGEMENTS

We thank Drs. Crossley, Orkin, and Bieker for sharing reagents.

    FOOTNOTES

* This work was supported in part by Grant HL65448 from the NHLBI, National Institutes of Health and by a grant from the March of Dimes Birth Defects Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY138967.

|| To whom correspondence should be addressed: Dept. of Pediatrics, Yale University School of Medicine, 333 Cedar St., P. O. Box 208064, New Haven, CT 06520-8064. Tel.: 203-688-2896; Fax: 203-785-6974; E-mail: patrick.gallagher@yale.edu.

Published, JBC Papers in Press, August 23, 2002, DOI 10.1074/jbc.M208184200

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