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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1136-1143
RED CELLS
From the Departments of Pediatrics, Internal Medicine and Genetics,
Yale University School of Medicine, New Haven, CT, and Division of
Hematology/Oncology, Children's Hospital, Boston, MA.
To begin to study the sequence variations identified in the 5'
flanking genomic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients and to provide additional insight into our understanding of the regulation of genes encoding erythrocyte membrane proteins, we have identified and characterized the erythroid promoter of the human ankyrin-1 gene. This compact promoter has characteristics of a housekeeping gene promoter, including very high
G+C content and enzyme restriction sites characteristic of an
HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple
transcription initiation sites. In vitro DNAseI footprinting analyses
revealed binding sites for GATA-1, CACCC-binding, and CGCCC-binding
proteins. Transfection of ankyrin promoter/reporter plasmids into
tissue culture cell lines yielded expression in erythroid, but not
muscle, neural, or HeLa cells. Electrophoretic mobility shift assays,
including competition and antibody supershift experiments, demonstrated
binding of GATA-1, BKLF, and Sp1 to core ankyrin promoter sequences. In
transfection assays, mutation of the Sp1 site had no effect on reporter
gene expression, mutation of the CACCC site decreased expression by
half, and mutation of the GATA-1 site completely abolished activity.
The ankyrin gene erythroid promoter was transactivated in heterologous
cells by forced expression of GATA-1 and to a lesser degree BKLF.
(Blood. 2000;96:1136-1143)
Ankyrins are a homologous family of multifunctional
proteins involved in the local segregation of integral membrane
proteins within functional domains on the plasma
membrane.1,2 This important cellular localization of
membrane proteins is provided by the relative affinities of the many
different tissue-specific and developmentally regulated isoforms of
ankyrin for target proteins. Ankyrin isoform diversity arises from both
different gene products and from differential, alternative splicing or
alternate polyadenylation of the same gene product. The complementary
DNAs (cDNAs) for 3 different ankyrins, ankyrin-1, -2, and -3, have been
cloned and their gene products studied.3-12
Ankyrin-1, first discovered in preparations of erythrocyte membranes,
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, the anion
exchanger. Ankyrin-1 deficiency is one of the most common abnormalities
found in the erythrocyte membranes of patients with hereditary
spherocytosis (HS).14-16 Genetic studies have revealed that
ankyrin-1 gene defects, primarily frameshift or nonsense mutations, are
the most common cause of typical, dominant HS.17,18 One
molecular mechanism that could lead to ankyrin deficiency is a mutation
in the ankyrin-1 erythroid promoter, leading to decreased ankyrin
synthesis. Sequence variations in the 5' flanking DNA of the
erythrocyte ankyrin gene have been identified in several kindreds with
both dominant and recessively inherited ankyrin-deficient HS.17,19 Whether these are disease-causing mutations or are merely polymorphisms in linkage disequilibrium with the as yet, unidentified mutation is unknown.
To provide additional insight into our understanding of the regulation
of genes encoding erythrocyte membrane proteins and to obtain the tools
necessary for study of the sequence variations identified in the
5' flanking genomic DNA of the ankyrin-1 gene in patients with
ankyrin-deficient HS, we have identified and characterized the
erythroid promoter of the human ankyrin-1 gene. It is a compact,
housekeeping gene-like promoter with a single GATA-1 site responsible
for its erythroid specificity.
Genomic cloning
Nucleotide sequencing
RNA preparation Total RNA was prepared from human fetal liver tissue, human bone marrow, or from the human tissue culture cell lines K562 (chronic myelogenous leukemia in blast crisis with erythroid characteristics, ATCC, CCL 243), HEL (human erythroleukemia, ATCC, TIB 180), HeLa (epithelial carcinoma, cervix, CCL 2), or HL60 (promyelocytic leukemia, CCL 240) as described.21Primer extension analyses and ribonuclease protection assays The transcription initiation site of the ankyrin-1 cDNA was determined using primer extension analysis and RNase protection assays. Primers A or B (Table 1) were used in primer extension reactions as described.22 Templates in these reactions were 10 µg of total human fetal liver RNA or 10 µg of total RNA from the human cell lines K562, HEL, HeLa, and HL60, or 10 µg of transfer RNA (tRNA). For ribonuclease protection assays (RPAs), a 32P-labeled antisense RNA probe was synthesized by transcription with T7 polymerase of a 584-base pair (bp) HindIII-ScaI fragment corresponding to the first exon and 5' flanking sequences of the human ankyrin-1 gene. The probe (1 × 105 cpm per assay) was hybridized to template RNA at 42°C for 16 hours. Templates in these reactions were 20 µg of total human fetal liver RNA, 20 µg of total RNA from the human cell lines HEL, K562, HeLa, and HL60, or 20 µg of tRNA. Hybrids were digested with a mixture of the nucleases RNase A and RNase T1 (0.125 and 5 µ, respectively, per assay) at 37°C for 30 minutes. After digestion, protected fragments were detected by autoradiography after electrophoresis in 6% polyacrylamide 7 mol/L urea gels. Further
increases in nuclease concentration or length of incubation did not
alter the pattern of the protected fragment (not shown).
5' rapid amplification of cDNA ends The 1 µg of total human fetal liver RNA was reverse transcribed using primer A (Table 1) and avian myeloblastosis virus (AMV) reverse transcriptase (Promega Corp). Single-stranded oligonucleotide ligation and polymerase chain reaction (PCR) amplification were carried out as described using primers B+D and C+D.23,24 Amplification products were subcloned and sequenced.Cell culture The tissue culture cell lines K562 and HEL (erythroid), SH-SY5Y (neural), and HeLa (nonerythroid) were used to study expression of the putative promoter of the ankyrin-1 gene. K562, HEL, and SH-Sy5Y cells were maintained in RPMI 1640 medium, containing 10% fetal calf serum. HeLa cells were maintained in Eagle's minimal essential media, supplemented with 10% fetal calf serum.Preparation of nuclear extracts Nuclear extracts were prepared from K562, HEL, MEL (murine erythroleukemia, NIGMS GM00086E), and HeLa cells by hypotonic lysis, followed by high salt extraction of nuclei as described by Andrews and Faller.25DNase I footprinting in vitro Probes for DNAse I footprinting were by produced by PCR amplification of plasmid p656 (see below) as template and 1 of 2 pairs of oligonucleotide primers, E+F and G+H. One oligonucleotide in each reaction was 5' end labeled with 32P-ATP using polynucleotide kinase before use in PCR. Footprinting reaction mixes contained 1 to 20 µg MEL cell nuclear extracts, 20 000 cpm of labeled probe, and 1 µg of poly (dI-dC). After digestion with DNase I, samples were electrophoresed in 6% polyacrylamide gels, the gels were dried and subjected to autoradiography.Preparation of promoter-reporter plasmids for transfection assays Test plasmids were prepared by inserting an approximately 2270-bp fragment of the 5' flanking ankyrin-1 genomic DNA upstream of the firefly luciferase reporter gene in the plasmid pGL2B (Promega Corp). Serial truncations of this 2270-bp fragment in the pGL2B plasmid were constructed using convenient restriction enzyme sites or PCR amplification. One promoter fragment, p656, was inserted into pGL2B in both orientations. All test plasmids were sequenced to exclude cloning or PCR-generated artifacts.Transient transfection analyses All plasmids tested were purified using Qiagen columns (Qiagen, Inc, Chatsworth, CA) or cesium chloride plasmid purification and at least 2 preparations of each plasmid were tested. The 107 K562, HEL, and SH-SY5Y cells were transfected by electroporation with a single pulse of 300 V at 960 microfarad (µF) with 20 µg of test plasmid and 0.5 µg of pCMV , a mammalian reporter plasmid expressing -galactosidase driven by the human cytomegalovirus immediate early gene promoter (Clontech). The 105 HeLa or
C2C12 (murine myoblast, ATCC 1772-CRL) cells were transfected with 2.0 µg test plasmid and 0.25 µg of the pCMV plasmid by lipofection using 4 µL Lipofectamine (Gibco BRL Life Technologies, Inc,
Gaithersburg, MD). Twenty-four hours after transfection, cells were
harvested, lysed, and the activity of both luciferase and
-galactosidase activity determined in cell extracts. All assays were
performed in triplicate. Differences in transfection
efficiency were determined by cotransfection with the pCMV
plasmid. For transactivation assays, HeLa cells were
transfected using 1 µg of reporter plasmid and varying amounts of
GATA-1, BKLF (basic Kruppel-like factor), or EKLF (erythroid
Kruppel-like factor) cDNA expression plasmids26-28 (see
below) and the reporter gene activity assayed.
Electrophoretic mobility shift analyses Binding reactions were carried out as described.26 Competitor oligonucleotides were added at molar excesses of 10- or 100-fold. Resulting complexes were separated by electrophoresis through 6% polyacrylamide gels in 0.5X tris-borate-EDTA at 21°C at 200W for 2 hours. Gels were dried and subjected to autoradiography.Computer analyses Computer-assisted analyses were performed using the sequence analysis software package of the University of Wisconsin Genetics Computer Group (UW GCG; Madison, WI) and the BLAST algorithm, National Center for Biotechnology Information (Bethesda, MD).29,30
Cloning of chromosomal gene: isolation and analysis of recombinant clones Primary screening of a human genomic DNA library with the ankyrin-1 cDNA probe ANK58 (Figure 1A) yielded 5 hybridization-positive plaques. Selected recombinants were analyzed and 1 clone identified, AN261, that spanned about 16-kilobase (kb) of
DNA containing the ankyrin-1 gene. A limited restriction map of this
region is shown in Figure 1B.
The 5'-flanking genomic DNA sequence of the human
ankyrin-1 gene exhibits features of a housekeeping gene promoter.
The nucleotide sequence of the 5' flanking genomic DNA of the
human ankyrin-1 gene is shown in Figure 2.
Inspection of the sequence reveals features characteristic of a
housekeeping gene promoter, including lack of consensus TATA, InR, or
CCAAT sequences.31,32 In addition, this region appears to
be a HpaII tiny fragment (HTF) island, based on a high G+C
content (77% between positions
Mapping the human ankyrin-1 erythroid messenger RNA transcription initiation sites and identification of 5' cDNA sequences To identify the 5' end of the human ankyrin-1 cDNA, primer extension analyses and RNase mapping with RNase A and RNase T1 nucleases were performed. These experiments identified 6 transcription initiation sites. The longest fragment obtained by RNase protection (Figure 3) and primer extension (not shown) predicted the presence of an additional 23-bp in the messenger RNA (mRNA) upstream of the 5' end of the sequence obtained from cDNA cloning. These additional 23-bp of upstream 5' untranslated sequence were obtained by 5' rapid amplification of cDNA ends (RACE) (Figure 2) and verified by comparison to corresponding genomic DNA sequences. No additional ATGs were present in the 5' untranslated sequences. Taken together, these data suggest that these sequences are at or very near the 5' end of the human ankyrin-1 erythroid cDNA.
An ankyrin-1 gene promoter fragment is active in erythroid
cells.
To investigate whether the region from
The ankyrin-1 erythroid promoter contains binding sites for GATA-1,
Sp1, and CACCC-related binding proteins.
The 286-bp minimal promoter fragment, p296, contains consensus binding
sequences for GATA, CACCC, and CGCCC-binding proteins. To identify
binding sites for transcription factors within the ankyrin-1 promoter,
in vitro DNase I footprinting analysis with nuclear extracts from K562
cells was performed in 2 steps. Footprints at 2 protected sites were
observed. Site 1, GCCGATAAG, contained consensus binding
sequences for GATA-1 (Figure 5). GATA-1 is
a transcription factor that plays a critical role in erythropoiesis via
its binding to the sequence GATA of the promoters and/or enhancers of
nearly all erythroid-expressed genes. The second site,
GCCACCCCTCCGCCC, consists of sequences
recognized by members of the Kruppel-like family of transcription
factors and other CACCC-related proteins (not shown). These include
Sp1, EKLF, and BKLF. EKLF, erythroid Kruppel-like factor, is a
transcription factor that is important in
GATA-1 binds the ankyrin-1 gene promoter site in vitro.
To determine whether nuclear proteins could bind this GATA-1 site in
vitro, double-stranded (DS) oligonucleotides containing the
corresponding ankyrin-1 promoter GATA-1 sequences (I+J, Table 1) or
control sequences (K+L; Table 1)35 were prepared and used
in gel shift analyses. When DS oligonucleotides containing the
footprinted GATA-1 sequences were used in gel shift analyses, a single
retarded species was observed in K562 extracts (Figure 6A), but not in HeLa extracts (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. The inclusion of GATA-1 antisera abolished most or all
of the DNA binding (Figure 6B). These data indicate that GATA-1 binds
to this site in the ankyrin-1 gene promoter in vitro.
Sp1 and BKLF bind to the ankyrin-1 gene promoter CACCC site in
vitro.
Site 2 identified by DNase I footprinting contains CACCC and CGCCC,
consensus binding sites for CACCC-related binding proteins and members
of the Kruppel family of transcription factors. Although CACCC-binding
proteins and Kruppel-like proteins both bind CACCC and CGCCC sequences,
they show distinct binding preferences. To determine whether the
nuclear proteins Sp1, BKLF, or EKLF bind these sites in vitro,
electrophoretic mobility shift assays were performed. When DS
oligonucleotides containing the corresponding ankyrin-1 promoter site
Sp1 (M+N; Table 1) or CACCC (E+O, Table 1) sequences or control
sequences (Sp1: P+Q;36,37 CACCC: R+S,28 Table
1) were used in gel shift analyses with K562 extracts, 1 larger,
slower-migrating species and 2 smaller, faster-migrating species were
detected. These species migrated at the same location as those obtained
using control oligonucleotides containing either Sp1 (Figure
7A) or CACCC consensus binding sequences
(Figure 7B). All 3 species were effectively competed by an
excess of unlabeled homologous oligonucleotide and an excess of
unlabeled Sp1 or CACCC control oligonucleotides. The inclusion of Sp1
antisera supershifted the larger, slower migrating species in the gel
when either the ankyrin Sp1 oligonucleotide (Figure 7C) or the CACCC
oligonucleotide (not shown) was used.
GATA-1 and CACCC-related proteins are both major activators of
the human erythroid ankyrin-1 gene promoter.
To assess the relative importance of these
transcription factor binding sites in promoter function,
mutations were introduced into each of the 3 consensus
binding sites protected in DNase I footprinting experiments and the
affect assayed in mutant promoter/reporter plasmids on
expression in transient transfections (Figure 4B). Mutation of the
GATA site to GTTA, a mutation shown to
disrupt GATA-1 binding,39 reduced promoter activity nearly
to background, indicating that this site is of major importance in the
ankyrin-1 gene promoter. Mutation of the CACCC site to
CACGC, a mutation of the human Transactivation of the ankyrin-1 gene erythroid promoter in
heterologous cells
These studies show that the human ankyrin-1 erythroid promoter is
compact, ie, a very short fragment of DNA directs high-level expression
in erythroid cells. Like other erythroid gene promoters, the
combination of GATA-1 and CACCC-binding proteins appears to be
essential for high-level expression of the ankyrin-1
gene.41-43 The consensus binding sites for GATA-1 and
CACCC-binding proteins are present in very close proximity in the
ankyrin-1 promoter. This combination may lead to cooperation between
GATA-1 and CACCC-binding proteins to enhance transcription, as has been
shown in several reports.44-48 CACCC-box binding proteins
and members of the Kruppel-family of transcription factors both bind
CACCC and CGCCC sequences; however, they show distinct binding
preferences. Although Sp1 and BKLF were found to bind to the CACCC site
of the ankyrin gene erythroid promoter in vitro, it is unknown what
transcription factors bind this site in vivo. The interactions of Sp1
and/or BKLF with a broad spectrum of erythroid gene promoters make them likely candidates for binding to this site.
We thank Drs Crossley, Orkin, and Bieker for sharing reagents with
us and we thank C. Wong for skilled technical assistance.
Submitted February 7, 2000; accepted April 3, 2000.
Supported in part by grants from the National Institutes of
Health, the March of Dimes Birth Defects Foundation, and the American Heart Association-Connecticut Affiliate.
Reprints: Patrick G. Gallagher, Department of
Pediatrics, Yale University School of Medicine, 333 Cedar St, PO Box 208064, New Haven, CT 06520-8064; email: patrick.gallagher{at}yale.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
1 to 
