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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2924-2933
Alterations in Protein-DNA Interactions in the  -Globin Gene
Promoter in Response to Butyrate Therapy
By
Tohru Ikuta,
Yuet Wai Kan,
Paul S. Swerdlow,
Douglas V. Faller, and
Susan P. Perrine
From the Hemoglobinopathy-Thalassemia Research Unit and Cancer
Research Center, Departments of Pharmacology and Experimental
Therapeutics, Pediatrics, and Medicine, Boston University School of
Medicine, Boston, MA; the Department of Medicine, Wayne State
University School of Medicine, Detroit, MI; and the Howard Hughes
Medical Institute, University of California, San Francisco, CA.
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ABSTRACT |
The mechanisms by which pharmacologic agents stimulate  -globin
gene expression in  -globin disorders has not been fully established at the molecular level. In studies described here, nucleated
erythroblasts were isolated from patients with -globin disorders
before and with butyrate therapy, and globin biosynthesis, mRNA, and
protein-DNA interactions were examined. Expression of -globin mRNA
increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in -globin protein synthetic levels above baseline globin ratios and a relative decrease in -globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in
erythroblasts of responsive patients in four regions of the -globin
gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1
sequences as a probe demonstrated that new binding of two
erythroid-specific proteins and one ubiquitous protein,  CP2,
occurred with treatment in the responsive patients and did not occur in
the nonresponder. The BRE-G1 sequence conferred butyrate inducibility
in reporter gene assays. These in vivo protein-DNA interactions in
human erythroblasts in which -globin gene expression is being
altered strongly suggest that nuclear protein binding, including
CP2, to the BRE-G1 region of the -globin gene promoter mediates
butyrate activity on -globin gene expression.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
EXTENSIVE INVESTIGATION has demonstrated
that the clinical severity of the -globin disorders can be
ameliorated by increased expression of the fetal globin
genes.1,2 Several pharmacologic agents stimulate fetal
globin production in patients with -globin disorders, including
chemotherapeutic agents and growth factors.3-8 Most of
these agents appear to act through altering erythroid growth kinetics.
The cytotoxic agents that produce a hypoproliferative marrow are
thought to induce -globin by selectively killing actively dividing
erythroid precursors, leading to the subsequent recruitment of more
primitive erythroid progenitors, which are programmed to produce higher
levels of hemoglobin F (HbF).7,8
Butyrate and other short-chain fatty acids have been shown to stimulate
fetal and embryonic globin gene expression in experimental models at
the level of transcription. These models include preferential -globin expression from the -globin locus transfected into
Xenopus oocytes, embryonic  -globin expression in chickens treated
with 5-azacytidine and butyrate, increased -globin expression in
human fetuses developing in the presence of elevated levels of
-amino-n-butyric acid and in butyric acid-treated fetal sheep,
nonhuman primates, and transgenic mice.9-13 Reporter gene
assays have demonstrated that several fatty acids and an amide
derivative, isobutyramide, stimulate -globin gene expression through
a proximal region of the -globin gene promoter.14-16 In
clinical studies, erythroid cells from patients have demonstrated
increased fetal globin expression at the mRNA, protein, or cellular
levels in patients treated with low-to-moderate, intermittent doses of
arginine butyrate and phenylbutyrate,17-20 although
prolonged high doses that inhibit cell growth are less effective.21 Neither arginine butyrate nor sodium
phenylbutyrate produces the myelosuppression that is associated with
administration of chemotherapeutic agents. Identification of the
specific mechanisms of action of butyrates may therefore be useful in
identifying potential combinations of therapeutic agents that have
different and complementary mechanisms of action.
To investigate the molecular effects of butyrate on fetal globin
expression, nucleated erythroblasts were purified from patients with
-globin disorders before and during butyrate treatment. In erythroid
cells from patients in whom -globin protein was increased by
butyrate therapy, increases in -globin mRNA and alterations in
protein-DNA binding to regulatory sequences in the -globin gene
promoter were observed. These changes were not detected in
erythroblasts isolated from the single patient studied here who did not
respond with an increase in -globin protein. These results suggest
that one of the mechanisms by which butyrate increases -globin is to
induce binding of transcription factors to specific cis-acting elements
of the -globin gene promoter.
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MATERIALS AND METHODS |
Isolation of nucleated erythroblasts.
Peripheral blood samples were collected from patients in whom there was
a high proportion of circulating nucleated erythroblasts, representing
between 70% and 90% of the mononuclear cells. Mononuclear cells were
isolated from the peripheral blood of 7 patients with -thalassemia
and from the bone marrow of 1 patient with sickle cell disease, before
and during therapy with butyrate, and from 1 normal adult control by
Ficoll-Paque (Pharmacia, Piscataway, NJ) density gradient
centrifugation. For mononuclear cell preparations that had 80% or more
nucleated erythroblasts, no further purification was performed and the
cell preparations were used as erythroblast-rich fractions. For samples
in which there were less than 80% erythroblasts, erythroblasts were
further purified using murine antihuman CD45 antibody and culture
flasks coated with goat antimurine IgG serum (Applied Immune Sciences,
Santa Clara, CA). Because CD45 is expressed on all hematopoietic cells
except erythroid cells,22 the CD45+ cells were
removed by treating erythroblast-rich preparations with murine
antihuman CD45 antibody according to the manufacturer's directions.
The purity of erythroblasts in the resulting nonadherent fractions was
confirmed by morphology after Wright's and benzidine staining.
Collection of samples for these studies was approved by the
Institutional Review Boards of the Boston Medical Center and the
University of California at San Francisco, and informed consent was
obtained from each patient.
For erythroid and nonerythroid controls, K562, Raji, and HeLa cells
were cultured in RPMI 1640 media (GIBCO, Grand Island, NY) containing
10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 100 IU/mL
penicillin, and 100 IU/mL streptomycin (GIBCO) and incubation with or
without butyric acid (Aldrich Chemical Co, St Louis, MO) at neutral
pH in humidified 5% CO2 and 95% air at 37°C.
Isolation and analysis of globin mRNA.
Total cytoplasmic RNA was extracted from erythroblasts by the method of
Chomoczynski and Sacchi.23 Globin mRNAs were analyzed by
primer extension as previously described.24,25
Oligonucleotides for -, -, and -globin mRNAs were 5
end-labeled with T4 polynucleotide kinase and [-32P]
ATP (New England Nuclear, Boston, MA) and were purified through Push
columns (Stratagene, La Jolla, CA) as described
previously.25 Five micrograms of total RNA was subjected to
primer extension analysis. The extension products were analyzed in 8%
urea-polyacrylamide gels. Autoradiography was performed by exposing
gels to Kodak X-AR films (Eastman Kodak, Rochester, NY)
overnight with an intensifying screen. The ratio of -globin mRNA to
-globin mRNA in patients with -thalassemia or -globin mRNA to
s-globin mRNA in 1 patient with sickle cell disease was
determined using PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
The DNA sequence of the oligonucleotide for -globin mRNA was as
follows: 5-TTACCCCAGGCGGCCTTGACGTTGGTCTTGTCGG-3 (34 mer);
the sequences for - and -globin mRNA were as described
previously.25
Globin biosynthesis.
Heparinized venous blood containing nucleated erythroblasts was
incubated in 20 mL of leucine-free minimal essential medium (GIBCO)
with 100 µCi [3H] leucine (New England Nuclear) at
37°C for 6 hours. Globin was purified and globin chain synthesis
was determined by column chromatography by the method of Clegg et
al.26 Multiple samples were obtained before and after
treatment and were assayed at least three times.
In vivo footprinting analyses.
In vivo methylation of cellular DNA was performed as described
previously.27,28 Dimethyl sulfate (DMS) treatment (0.1% [vol/vol]) was performed for 90 seconds at room temperature.
Extraction of genomic DNA from in vivo methylated cells was performed
as described.25 In vitro methylation of naked DNA was
performed as described previously.29
Methylated DNAs were cleaved with piperidine and DNAs were dissolved in
1× TE (10 mmol/L Tris-HCl, pH 8.0/1 mmol/L EDTA) at a final
concentration of 0.5 to 1 µg/µL.28 Ligation-mediated
polymerase chain reaction (PCR) was next performed by the method of
Mueller and Wold,27 with modifications.25 Briefly, 3 to 7 µg of DNAs was suspended in 15 µL of solution A (40 mmol/L Tris-HCl, pH 7.7/50 mmol/L NaCl) containing 0.6 pmol of primer 1 (see below). First-strand synthesis was performed by adding 7.5 µL of
solution B (20 mmol/L MgCl2/ 20 mmol/L dithiothreitol [DTT]/60 µmol/L dNTP) and 1.5 µL of 1:4 diluted
Sequenase version 2.0 (US Biochemical, Cleveland, OH) in 1× TE at
50°C for 10 minutes. The reaction was quenched by heating at
68°C for 10 minutes, adding 6 µL of 310 mmol/L Tris-HCl (pH 7.7)
and subsequently extracting with phenol/chloroform/isoamyl alcohol
(25:24:1) and with ether. Ligation of the PCR common linker was
performed as described by Mueller and Wold.27 DNA sequences
were amplified by performing 16 to 18 cycles of PCR using 10 pmol each
of primer 2 (see below) and the longer oligomer of the common
linker.27 The amplified products were then digested with
mung bean nuclease (New England Biolabs, Beverly, MA), as described
previously.28 Primers 1, 2, and 3 were used for
first-strand synthesis, PCR, and primer extension, respectively.
Footprint ladders were visualized by performing a primer extension
using 5 end-labeled primer and subsequent analysis on 8%
urea-polyacrylamide gels. Autoradiography was performed by exposing
gels to Kodak X-AR films with an intensifying screen. The
reproducibility of in vivo footprints was confirmed by scanning with
PhosphorImager. The percentage of protection on each G residue and
identification of hyperreactive G residues were defined as described
previously.25-30
Electrophoretic mobility shift assays (EMSA).
Nuclear extracts used for gel mobility shift assays were prepared from
purified nucleated erythroblasts as previously described.31 Oligonucleotides were annealed to form double-stranded DNA, 5 labeled with T4 polynucleotide kinase, and purified through Push columns (Stratagene, La Jolla, CA).24 A double-stranded
oligonucleotide containing the TFIID consensus sequence (Promega,
Madison, WI) was used as a control to verify the integrity of the
nuclear extracts. Ten micrograms of nuclear extracts was mixed with 500 ng of poly[(dA-dT)] or poly[(dI-dC)] in 20 µL of binding buffer
(25 mmol/L HEPES, pH 7.5/50 mmol/L KCl/12.5 mmol/L MgCl2/1
mmol/L DTT/10 µmol/L ZnSO4/5% [vol/vol] Glycerol/0.1%
[vol/vol] NP-40) at room temperature for 30 minutes. After the
addition of probe labeled with 5 × 104 cpm, the
mixture was incubated for 30 minutes at room temperature. Competition
experiments were performed by adding a cold competitor at a 100-fold
molar excess before the addition of a labeled probe. In these
experiments, an oligonucleotide with sequences corresponding to CP2
was used. Protein-DNA complexes were separated on 5% nondenaturing polyacrylamide gels in 0.5× TBE (1× TBE is 9 mmol/L
Tris-borate/2 mmol/L EDTA) at 200 V for 90 minutes at room temperature.
Gels were dried and exposed to Kodak X-AR films overnight. The DNA sequences of oligonucleotide probes used for these assays were as
follows: BRE-G1, 5-GAGTATCCAGTGAGGCCAAGGGGCCG
GCGGCTGGCTAGGG-3; mutant BRE-G1,
5-GAGTATCCATTTAGGCCAAGGGGCCGGCGGCTGGCTAGGG-3; CP2,
5-CCCTAACAAGTTTTACTGGGTAGAGCAAGCACAAACCAGCCAATGAG-332;
and TFIID,
5-GCAGAGCATATAAGGTGAGGTAGGA-3.33
Plasmid constructions and reporter gene assays.
To construct the wild-type plasmid containing the human growth hormone
gene driven by a -globin gene promoter, a 335-bp Alu fragment of the
A-globin gene promoter extending from  299 to +36
with respect to the transcription initiation site was subcloned into
the HindIII site of the plasmid p0GH (Nichols Institute, San
Juan Capistrano, CA). A mutant plasmid was constructed with primer 5 MUT-1T, 5-GAGTATCCATTTAGGCCAGGGGC CGGCGGCTGGCTAGGG-3, which contains two mutations (G  T) introduced in the BRE-G1 sequence that eliminate the binding of
proteins in nuclear extracts of patients' erythroblasts observed
during butyrate therapy, using a transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA). The sequence of mutations were verified
by dideoxy sequencing using the DNA polymerase sequencing kit
(Clontech). All plasmid DNAs were prepared using Qiagen columns (Qiagen, Santa Clara, CA). For each transient transfection, 1 × 107 K562 cells were electroporated with 50 µg of reporter
construct in duplicate. The two duplicate sets of K562 cells
transfected with each plasmid were pooled and then divided into two
fractions. Sodium butyrate was added to one of the fractions at a final
concentration of 1 mmol/L, and the cells were incubated for 72 hours at
37°C in 5% CO2 and air. Human growth hormone was
assayed in the media 72 hours after transfection using the HGH-TGES
Gene Expression kit (Nichols Institute) according to directions from
the manufacturer. The assays were performed at least three times.
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RESULTS |
Increases in -globin mRNA and protein biosynthesis with butyrate
treatment.
Characterization of the patients studied here and their responses to
butyrate administration are shown in Table
1. In 6 of the 7 -thalassemia patients studied before and during or
after butyrate therapy, the ratios of -globin mRNA to -globin
mRNA in erythroblasts increased above pretreatment levels in the same subjects by 2.8-to 6-fold (Fig 1). In
erythroid cells from the patient with sickle cell anemia, -globin
mRNA relative to s-globin mRNA increased fivefold above
the baseline ratio of :s-globin (Fig 1). Increases in
-globin mRNA were consistently associated with, on average, twofold
increases above baseline in -globin protein synthesis in the
thalassemic subjects, as shown in Fig 2.
For example, in a pretreatment sample from patient no. 4, the ratio of
non--globin ( + ) to -globin was 0.4 (Fig 2A). With a
moderate dose of arginine butyrate (1,200 mg/kg/d), -globin
increased, and the overall non-:-globin chain ratio was corrected
to 0.84, with relative -globin synthesis diminished compared with
baseline (Fig 2B). Similar increases in -globin synthesis and
decreases in globin in + thalassemia subjects
were observed in the other patients in whom -globin mRNA increased
over baseline (data not shown). The modest doses studied (250 to 800 mg/kg for 3 to 4 days of 14 days total) have increased the hemoglobin F
protein in two thirds of another group of adult sickle cell subjects to
a mean of 22% hemoglobin F.20
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| Fig 1.
Analysis of globin mRNAs by primer extension in 7 patients with -thalassemia (patients no. 1 through 7), 1 patient
with sickle cell anemia (patient no. 8), and a normal adult subject
(NS). Samples obtained before therapy and during therapy are designated
with and + above the lane, respectively. Patient no. 5 was
analyzed before therapy was completed (), 24 hours after therapy was
completed (+), and 44 hours after therapy ended (*). Fold
increases of -globin over the subject's baseline are shown at the
bottom of the lanes. Patient no. 4 was the single subject studied in
whom an increase in -globin mRNA was not observed.
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| Fig 2.
Globin chain synthesis in a representative patient with
+ thalassemia (patient no. 5) who received
butyrate therapy. (A) Baseline profile (pretreatment); (B) with
butyrate (moderate dose, 1,200 mg/kg/d) therapy. The elution positions
of the , , , and acetylated (AC) are
indicated. Non-: globin chain balance improved by approximately
twofold with butyrate therapy due to an increase in acetylated
-globin and -globin synthesis and despite a relative decrease in
-globin synthesis.
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New binding of transcription factors is observed in vivo during
butyrate treatment.
To determine whether the increases in -globin mRNA observed with
butyrate treatment are associated with changes in binding of
transcription factors to the -globin gene promoter, in vivo footprinting analysis of established regulatory sequences for -globin gene expression was performed. Although in vitro DNA binding
assays do not necessarily provide direct evidence for tissue-specific
binding of transcription factors, in vivo footprinting has demonstrated
protein-DNA interactions that are functionally relevant to
tissue-specific gene expression.25,28,34 Furthermore, previous studies have shown that, in the -globin gene promoter, a
DNA fragment extending up to 200 bp relative to the
transcription start site is sufficient to direct efficient
transcription of the -globin genes,14-16,35,36 although
several potentially important regulatory sequences have also been
identified at further upstream regions.37,38 We focused in
vivo footprinting analysis for the -globin promoter on a region
extending to 200 bp upstream of the mRNA cap site, which has
demonstrated functional importance for butyrate-inducibility in
previous reporter assays in three different
laboratories.14-16 As shown in
Fig 3, four promoter regions were newly and heavily footprinted, demonstrating protection on G
residues with characteristic hyperreactive G residues adjacent to the
protected bands in erythroblasts obtained from patients during butyrate
treatment. These heavily footprinted sequences were demonstrated in at
least four different subjects and were designated butyrate response
elements G1-G4 (BRE-G1 to BRE-G4). These new footprints were not
detected in the patient who did not respond to butyrate therapy.
Erythroblasts isolated from -thalassemic patients who responded to
butyrate treatment demonstrated new detectable footprints consistently
over BRE-G1 and BRE-G2, and sporadically over BRE-G3 and BRE-G4, during
therapy. Several sites of point mutations that result in hereditary
persistence of fetal hemoglobin (HPFH) were noted to be in close
proximity of the BRE sequences.41 It should be noted that
three G residues immediately 3 of the CACCC sequence, which were
footprinted in K562 cells as well as in human-murine erythroleukemia
hybrid cell lines that express human -globin,25,28 were
not footprinted in erythroblasts isolated from patients before or
during butyrate therapy, indicating that the protein-DNA interactions
of the -globin promoter are not identical between erythroleukemia
cells and -hemoglobinopathy patients' erythroblasts. These in vivo
footprinting analyses on erythroblasts isolated from multiple patients
have clearly demonstrated that new nuclear protein binding to multiple
regulatory sites of the -globin gene promoter develop during
butyrate therapy in erythroblasts that respond by increasing -globin
mRNA and protein.
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| Fig 3.
In vivo footprinting analyses of the -globin gene
promoter. (A) A sickle cell anemia patient (patient no. 8). Lane 1, hemin-induced K562; lane 2, before therapy; lane 3, during therapy;
lane 4, naked DNA. (B) A -thalassemia patient (patient no. 7).
Lane 1, before therapy; lane 2, during therapy; lane 3, naked DNA. The
location of the canonical elements is shown at the right of figures.
( ) Footprinted G residues; ( ) G residues that showed
hyperreactivity to DMS. The arrow indicates the location of three G
residues that were footprinted in K562 cells but were not footprinted
in patients' samples.
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EMSAs with BRE-G1.
To further characterize transcription factors binding to BREs, gel
mobility shift assays using nuclear extracts prepared from patients'
erythroblasts before and during therapy were performed. We specifically
focused on transcription factors binding to BRE-G1 of the -globin
promoter, for two reasons. First, reporter gene assays in our
laboratory and others have previously demonstrated that a small region
of the -globin promoter, extending to 60 bp from the
transcription start site, was adequate to respond to butyrate with
upregulation of -globin gene transcription, although other upstream
regions may augment this effect.11,14-16 Second, the stage
selector element, which has been implicated in competitively silencing
the -globin gene in the fetal stage,39,40 has been
mapped to a region 50 bp upstream of the -globin promoter cap site.
Because the BRE-G1 region defined in this study spans 68 to
43 bp of the -globin promoter, it is possible that the BRE-G1
region includes these two regions of potential interest (Fig 4).
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| Fig 4.
Summary of in vivo footprints of the -globin gene
promoter observed with butyrate treatment. Canonical elements are
boxed. Closed and stippled symbols are for sickle cell anemia and
-thalassemia, respectively. Symbols: circles, footprinted G
residues; triangles, G residues hyperreactive to DMS. Asterisks
indicate sites of mutations that result in HPFH. The transcriptional
start site of the -globin gene is denoted by the arrow.
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Binding activities of nuclear extracts prepared from cultured erythroid
and nonerythroid cells were first examined. Nuclear extracts from
uninduced K562 cells revealed several major protein-DNA complexes,
which differed slightly from the binding protein pattern observed in
extracts from butyrate-treated K562 cells
(Fig 5A, compare lanes 1 and 2). With exposure to butyrate, the fastest migrating band decreased in intensity and two additional protein-DNA complexes (shown by the arrows) appeared. In contrast, nuclear extracts
from nonerythroid cells, Raji and HeLa cells, produced patterns of
shifted bands similar to extracts from untreated K562, and butyrate
treatment of these nonerythroid cells did not change the patterns of
shifted bands (data not shown). Because no in vivo footprints were
detected in this region in these nonerythroid cell lines, these data
suggest that nuclear factors forming some of the prominent complexes in
vitro are derived from ubiquitous, not erythroid-specific,
transcription factors.
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| Fig 5.
EMSAs using the BRE-G1 sequence of the -globin gene
promoter or TFIID as probe. (A) Nuclear extracts from uninduced K562 or
a patient's erythroblasts (patient no. 5) without treatment are shown
by the ; butyrate-induced K562 cells or patient's erythroblasts on
treatment (patient no. 5) are designated by the +. Nuclear extracts
prepared from erythroblasts of a normal subject are designated NS. Lane
designations are as follows: lane 1, K562 (untreated); lane 2, K562
(butyrate-treated); lane 3, patient no. 5 (before treatment); lane 4, patient no. 5 (on butyrate treatment); lane 5, patient no. 5 (28 hours
after treatment); lane 6, patient no. 5 (44 hours after treatment);
lane 7, K562 (untreated); lane 8, patient no. 5 (on treatment); lane 9, normal subject (N.S.). Four major protein-DNA complexes, designated a
through d, were detected. Arrows denote protein-DNA complexes that
appeared in K562 extracts during butyrate treatment. The asterisk
denotes a band that is diminished with exposure to butyrate. Nuclear
extracts from patient no. 5 are shown before (), during (+), and
28 and 44 hours after therapy (28hr and 44hr) are shown. Three
new shifted bands are demonstrated during butyrate therapy and persist
for at least 44 hours, at which time globin protein synthesis is still
altered with an increase in -globin and decrease in -globin
compared with baseline. (B) EMSA using a TFIID probe. The origins of
nuclear extracts and addition of butyrate are shown on the top of
figure. Lane designations are as follows: lane 1, patient no. 5 (before
treatment); lane 2, patient no. 5 (on treatment); lane 3, patient no. 5 (28 hours after treatment); lane 4, patient no. 5 (44 hours after
treatment); lane 5, K562 (untreated); lane 6, patient no. 5 (on
treatment); lane 7, normal subject (N.S.). The arrow indicates the
major protein-DNA complex observed with the probe. No new or shifted
complexes are observed with butyrate treatment. (C) The wild-type -globin promoter probe (W) and a mutant probe (M) were used in EMSA. Probes and the cellular
origin of nuclear extracts are shown above the lanes. The positions of
the four major protein-DNA complexes, a through d, are shown on the
right of figure. A decrease in binding of bands b and c was observed
with the BRE-G1 sequence in which mutations were introduced, compared
with binding of the normal sequence. (D) Competition assays with an
CP2 sequence oligonucleotide. Assays were performed in the absence
(lanes 1 and 3) or presence (lanes 2 and 4) of 100-fold molar excess of
a cold competitor. Probes and the cellular origin of the nuclear
extracts assayed are shown above the lanes. Binding of the top band was
abolished in the presence of excess cold CP2.
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Significant changes in existing patterns and new DNA-protein binding
were detected comparing erythroid nuclear extracts from patients
pretreated and during treatment. As shown in Fig 5A, erythroid nuclear
extracts prepared from a representative butyrate-responsive thalassemic
patient (patient no. 5) demonstrated two weak DNA-binding activities to
the BRE-G1 element before therapy. Consistently, nuclear extracts
prepared from erythroblasts of a normal adult who has not received
butyrate therapy and does not express -globin exhibited a similar
pattern of retarded bands to that seen with the patient off treatment
(Fig 5A, lane 9). However, during and after butyrate therapy, three
new, strong protein-DNA complexes were generated with the patients'
nuclear extracts. The integrity of the nuclear extracts was confirmed
by performing an EMSA with a TFIID probe (Fig 5B). Although one new
band (band a in Fig 5A) migrated in parallel with a protein-DNA complex
detected in K562 nuclear extracts, two other new shifted bands (bands b
and c in Fig 5A) from treated patients' nuclear extracts migrated
slightly, but consistently, more slowly than two similar bands formed
with K562 nuclear extracts. These two distinct shifted bands were not detected in nuclear extracts from Raji and HeLa cells, even when treated with butyrate (data not shown). These results suggest that the
nuclear factors forming two of the prominent complexes (bands b and c)
during reactivation of -globin gene expression with butyrate therapy
in vivo may be erythroid-specific, whereas the other complex is likely
formed by a ubiquitous factor(s). To determine which band(s) are
responsible for forming the in vivo footprints spanning a 60 to
70 region, we performed an EMSA using a probe in which the
footprinted G residues of the region were mutated (G T). As
seen in Fig 5C, with the mutant probe, the top bands are intact but the
intensities of the middle two bands are significantly decreased in both
K562 and patients' nuclear extracts, indicating that both bands b and
c result from transcription factors binding to the 60 to
70 region. The binding activities for bands b and c were not
competed away with oligonucleotides corresponding to AP1, AP2, SP1, or
CREB (data not shown).
Because the wild-type oligonucleotide probe used for these EMSAs for
BRE-G1 extended from 77 to 39 bp of the -globin
promoter, it is possible that the stage selector protein complex, a
heteromeric complex between CP2 and a nuclear protein of 45 kD,40 binds to this oligonucleotide probe. To determine if
any of the newly shifted bands result from a protein-DNA complex due to
part of the stage selector protein complex, a competition assay using a
cold CP2 probe was performed. The top band (slowest-migrating complex) was completely abolished by the cold competitor, whereas three
other bands were not affected (Fig 5D). This band was not detected in
nuclear extracts prepared form a normal adult subject who did not
express globin (see Fig 5A). This result strongly suggests that the
slowest migrating complex induced during butyrate therapy results from
the binding of at least one part of the stage selector protein complex,
the transcription factor CP2.
Reporter gene assays with BRE-G1.
To evaluate a potential functional role of BRE-G1 in induction of
-globin gene expression by butyrate, transient transfection assays
were performed using DNA constructs containing a human growth hormone
gene (HGH) as a reporter driven by a 335-bp fragment of the -globin
gene promoter, which was previously found to be sufficient and
necessary for induction by butyrate or isobutyramide.15 Induction of HGH expression by butyrate was compared between constructs containing the wild-type promoter and a promoter in which mutations abolishing DNA-protein complex formation to BRE-G1 in EMSAs were introduced. The results are summarized in
Table 2. Transfection of the wild-type
construct into K562 cells and subsequent treatment of the transfected
cells with 1 mmol/L sodium butyrate resulted in a sixfold increase in
the reporter gene expression over the level of HGH expressed by
untreated control cells. No change in HGH expression was observed when
the mutant construct of BRE-G1 was treated with butyrate compared with
the untreated mutant construct, indicating loss of butyrate
responsiveness with the mutation in the BRE-G1 sequence.
| |
DISCUSSION |
Augmentation of -globin chain synthesis, Hb F, or
F-reticulocytes and total hemoglobin and hematocrit has been
demonstrated in some patients with -globin disorders treated with
sodium phenylbutyrate and with arginine butyrate
intermittently.18-20 Substantial increases in -globin
mRNA were also observed in 7 of 8 patients studied here who received
variable doses of butyrate therapy. The increases in -globin mRNA
and protein were found to be associated with alterations of
transcription factors binding to regulatory sequences of the -globin
gene promoter that differed from the DNA-binding protein patterns
observed before treatment in the responsive patients.
This study has focused primarily on whether increases in -globin
mRNA and protein of patients who received butyrate were associated with
molecular changes that have been reported to affect transcription of
the -globin gene in other experimental
systems.14-17,25,37,39,42 Several changes in protein-DNA
interactions were observed during and after treatment with butyrate in
vivo, observed as prominent new footprints in the proximal -globin
promoter, at sites that several laboratories have found to be
functionally related to globin
transcription.14-16,34-44 Of note, these changes in
protein-DNA binding occurred only in patients who responded to butyrate
therapy with an increase in -globin mRNA and protein and were not
observed in cells of the one patient who did not demonstrate any
changes in globin gene expression with therapy. This correlation
strongly suggests that butyrate-responsiveness is mediated, at least
partially, through the DNA-binding protein changes observed.
It is also noteworthy that nucleotide mutations that have been found in
HPFH syndromes are located close to, or within, the DNA sequences of
the BREs in the -globin promoter.41-44 Because the
molecular mechanisms underlying high-level expression of the -globin
gene in HPFH may represent a physiologic response, butyrate may result
in an alteration of the developmental expression of globin genes in a
manner similar to the physiologic response in HPFH. It is of interest
that the G residues immediately 3 to the CACCC sequence, which
are footprinted in the human erythroleukemia cell line
K562,25,28 are not footprinted in erythroblasts of patients
who received butyrate therapy. Furthermore, BRE-G3 and BRE-G4 are not
footprinted in K562, but were footprinted in patients' erythroblasts
during butyrate treatment. These findings suggest that the molecular
mechanisms by which butyrate stimulates expression of -globin in
patients appear somewhat distinct from those operative in K562 cells.
Binding of these additional protein factors may contribute to the high
level of -globin expression induced in these patients, compared with
the low-level expression typical of K562 cells.
How the nuclear protein binding to these DNA sequences that is induced
with butyrate treatment results in increased transcription of the
-globin gene remains to be determined. Although BRE-G1 was found to
be essential for butyrate responsiveness of the -globin gene in
previous and current functional assays, additional promoter elements
also increase basal transcription of the -globin
gene.36-38 BRE-G2 was also footprinted in erythroblasts
isolated from both -thalassemic and sickle cell patients who
received butyrate, as well as in K562 cells. Interestingly, BRE-G2 is
composed of perfect direct-inverted repeats of 6-bp long
(GCCTTGACAAGGC), suggesting that BRE-G2 is composed of two binding
units. Other regulatory sequences of the -globin locus may also
contain elements which contribute to
butyrate-responsiveness.16,38 Because a relative decrease
in -globin protein synthesis is frequently observed before increases
in -globin protein synthesis during butyrate treatment, footprints
of these other regions, particularly the locus control region that may
amplify the effects of butyrate, should be examined to completely
elucidate all the DNA-protein interactions that mediate globin gene
switching in vivo in response to butyrate therapy.
These analyses demonstrate that a pharmacologic therapy that results in
increased -globin expression in vivo is associated with changes in
DNA-protein binding interactions between specific globin gene
regulatory sequences and multiple DNA-binding proteins in erythroid
cells from patients undergoing this therapy. Furthermore, these changes
were observed solely in the nucleated erythroid cells of patients who
responded with increased -globin expression and were not observed in
a nonresponding patient. Changes in other specific binding proteins
were not detected in these nuclear extracts. These findings may
eventually contribute to a better understanding of the molecular
mechanisms that govern the variable responsiveness of patients to
pharmacologic therapy that alters globin gene expression. These regions
may also provide molecular targets for development of additional fetal
globin inducers or effective combinations of inducing agents with
different mechanisms of action.
| |
FOOTNOTES |
Submitted May 28, 1997;
accepted June 2, 1998.
Supported by National Institutes of Health Grants No. HL-37118 and
HL-15157, American Cancer Society Institutional Grant No. IN97-R, the
American Heart Association Grant-in-Aid program, Cooley's Anemia
International, Inc, and the Cooley's Anemia Foundation.
Address reprint requests to Tohru Ikuta, MD, PhD,
Hemoglobinopathy-Thalassemia Research Unit and Cancer Research Center,
Boston University School of Medicine, 80 E Concord St, L-911, Boston, MA 02118-2394.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract