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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1793-1801
Analysis of DNA Binding Proteins Associated With Hemin-Induced
Transcriptional Inhibition. The Hemin Response Element Binding
Protein Is a Heterogeneous Complex That Includes the Ku Protein
By
S.V. Reddy,
O. Alcantara, and
D.H. Boldt
From the Division of Hematology, Department of Medicine, University
of Texas Health Science Center, San Antonio, TX.
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ABSTRACT |
Hemin inhibits transcription of the tartrate resistant acid
phosphatase (TRAP) gene. Using deletion mutagenesis of the mouse TRAP
5 -flanking region, we previously identified a 27-bp DNA segment
containing a central GAGGC tandem repeat sequence (the hemin response
element [HRE]), which bound nuclear proteins (hemin response element
binding proteins [HREBPs]) from hemin-treated cells and appeared to
be responsible for mediating transcriptional inhibition in response to
hemin. We now have used affinity binding to
HRE-derivatized beads to identify four HREBP components with apparent
molecular masses of 133-, 90-, 80-, and 37-kD,
respectively. The 80- and 90-kD components correspond to the p70 and
p80/86 subunits of Ku antigen (KuAg) as documented by partial amino
acid microsequencing of tryptic digests and immunologic reactivity. Based on reactivity of the HREBP gel shift band with antibodies to the
redox factor protein (ref1) in shift Western experiments, it is shown
that the 37-kD component represents ref1. The 133-kD component appeared
to be a unique protein. KuAg participation in HREBP complexes was
specific as it was present in HREBPs bound to HRE microcircles. Results
of depletion/reconstitution experiments suggested that KuAg does not
bind alone or directly to HRE DNA, but does so only in conjunction with
the 133- and/or 37-kD proteins. We conclude that HREBP is a
heterogeneous complex composed of KuAg, ref1, and a unique 133-kD
protein. We speculate that the role of heme may be to promote
interactions among these components, thereby facilitating HRE binding
and downregulation of hemin responsive genes.
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INTRODUCTION |
RECENTLY WE HAVE described inhibition of
tartrate-resistant acid phosphatase (TRAP) gene transcription by hemin
(ferric protoporphyrin IX).1,2 Using deletion mutagenesis
of the mouse (m) TRAP gene 5 flanking region, we identified a
27-bp DNA segment containing a central GAGGC tandem repeat sequence
(the hemin response element [HRE]), which bound nuclear proteins
(hemin response element binding proteins [HREBPs]) from hemin-treated
cells and appeared to be responsible for mediating transcriptional
inhibition in response to hemin.2 A GENINFO data base
search showed several other mammalian genes with tandem GAGGC motifs in
noncoding regions, providing the possibility that additional genes may
be regulated in similar fashion by hemin at the level of
transcription.2 We now report that the HREBP is a complex
comprising up to four proteins and that both the Ku antigen (KuAg)
(p70/p86)3-5 and the redox factor (ref1)
protein6 are associated with this complex.
KuAg is a heterodimeric DNA binding protein consisting of p70 and
p80/86 subunits originally described as an autoantigen recognized by
antibodies from patients with systemic lupus erythematosus, overlap
syndrome, and other autoimmune disorders.3-5,7,8 KuAg plays
an important role in double-stranded break repair of DNA and V(D)J
recombination, both functions dependent on the targeting to DNA by KuAg
of the enzyme, DNA-dependent protein kinase (DNA-PK).9-14 KuAg appears to recognize and bind DNA structures containing double-to- single-stranded DNA transitions, as well as double-stranded DNA ends.15-18 A unique feature of the KuAg-DNA interaction is
that in vitro KuAg is able to translocate freely along DNA after its initial binding,19 although this translocation is blocked
in vivo by nucleosomes.20 It has been suggested that KuAg
may in this way serve as a general inhibitor of DNA-protein complex
formation and of transcription by displacing specific transcription
factors from DNA.21 In addition, Giffin et
al22,23 have described and characterized sequence-specific
KuAg binding to NRE1 (negative regulatory element 1), a DNA sequence
element in the long terminal repeat (LTR) of mouse mammary tumor virus
(MMTV). KuAg also has been described to possess DNA helicase
activity.24
An interaction between KuAg and the ref1 protein has been previously
described.25 Ref1 is a 37-kD protein that regulates the DNA
binding activity of the Fos-Jun heterodimer by mediating reduction of
conserved cysteine residues in the DNA-binding domains of these two
proteins.6
Recently, a specific interest of this laboratory has been hemin-induced
transcriptional inhibition of TRAP gene expression. We have shown that
this process is mediated by binding of nuclear proteins, the HREBP, to
a specific DNA element, the HRE. The aim of this study was to identify
and characterize the nuclear proteins comprising the HREBP.
We now present data derived from affinity purification,
microsequencing, immunoblotting, and gel mobility assays, which
demonstrate complexing of KuAg, ref1, and a 133-kD protein with the HRE
of the mTRAP gene. These data provide evidence for additional roles for
KuAg and ref1 in transcriptional regulation of gene expression.
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MATERIALS AND METHODS |
Cell lines, antibodies, and oligonucleotides.
Human U937 cells,26 obtained from American Type Culture
Collection (Rockville, MD), are maintained in our laboratory.
Monoclonal antibodies recognizing KuAg components p70 (Ab-4, clone
N3H10), p80 (Ab-2, clone 11D), or both p70/p80 (Ab-3, clone 162) were obtained from NeoMarkers, Fremont, CA.27 Dr Tom Curran (St
Jude Children's Research Hospital, Memphis, TN) kindly provided
affinity-purified rabbit anti-ref1 antibody. Native, scrambled, and
biotinylated HRE 27-bp oligonucleotides were synthesized at GIBCO-BRL
(Gaithersburg, MD). The native 27-mer sequence is as follows:
5-ACCTTGGAGGCGAGGCGCAGGTAATGG-3.2 The tandem repeat HRE sequence is underlined. Scrambled
oligonucleotides used in some experiments had the following sequence:
5-GTTATAATGTGTACATCCATCACTGT-3.
Affinity isolation of HREBPs.
U937 cells were incubated with hemin (50 µmol/L) for 48 hours. Nuclear proteins were extracted as previously
described2 using the technique of Andrews and
Faller.28 We used a modification of the methods described
by Ren et al29 to isolate from the nuclear extracts
proteins binding to the 27-mer HRE DNA sequence. Briefly, biotinylated
authentic or scrambled oligonucleotides were coupled to
streptavidin-coated magnetic beads (Dynabeads M-280; Dynal, Lake
Success, NY). Beads were washed twice with 1% bovine serum albumin
(BSA) in phosphate-buffered saline (PBS), twice with 1 mol/L NaCl in
PBS, then twice with 10 mmol/L Tris-CL, 1 mmol/L EDTA, pH 8.0 (TE buffer). Oligonucleotides diluted in 0.5 mL TE buffer were added to
the washed beads and incubated with rotation for 30 minutes at room
temperature. Unbound oligonucleotides were removed by washing with TE
buffer. Derivatized beads contained 250 pmol of oligonucleotide per 400 µL of beads (6 to 7 × 108 beads/mL). To isolate
specific DNA binding proteins, nuclear extracts first were dialyzed
against 25 mmol/L HEPES, pH 7.6, 40 mmol/L KCl, 0.1 mmol/L EDTA, 10%
glycerol, 1 mmol/L dithiothreitol, 0.1 mmol/L phenylmethylsulfonyl
fluoride, 0.1 µg/mL aprotinin, 0.5 µg/mL N-tosyl-L-phenylalanine
chloromethyl ketone (TPCK) (dialysis buffer). Subsequent incubations
were scaled up many fold compared with those performed by Ren et
al,29 but buffers and proportions were the same. We used
400 µL of derivatized Dynabeads per 3 to 5 mL of nuclear proteins in
dialysis buffer. Binding reactions were performed for 30 minutes at
room temperature with rotation. For one part nuclear protein extract we
used 10 parts 5× gel shift buffer (1× gel shift buffer = 20 mmol/L Tris-HCl, pH 7.0, 100 mmol/L NaCl, 5 mmol/L EDTA, 0.1% Nonidet
P-40, 0.5 µg/mL BSA, 5% glycerol); 0.125 parts 1 mol/L
MgCl2; and 37.8 parts dialysis buffer. After incubation,
unbound proteins were removed by washing the beads with 1× gel
shift buffer containing 2.5 mmol/L MgCl2 and 0.1% Nonidet
P-40, but without BSA. Beads were collected using a Dynal magnetic
particle concentrator and proteins were recovered by boiling in Laemmli
sample buffer then separated by sodium dodecyl sulfate-polyacrylamide
gel electropheresis (SDS-PAGE) in 10% acrylamide gels.30
Proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes for immunoblotting and to obtain material for microsequencing. Microsequencing was performed in the Institutional Protein Core Laboratory, UTHSCSA, directed by Dr Lynda Bonewald.
Immunodetection of KuAg.
Immunoblotting was performed as previously described31 with
KuAg subunit specific monoclonal antibodies used at a dilution of
1:500. Secondary antibody was goat antimouse IgG
F(ab)2 conjugated to horseradish peroxidase (Pierce
Chemical Co, Rockport, IL) used at a dilution of 1:4,000 followed by
treatment with chemiluminescence solution (Pierce Supersignal
Chemiluminescent, Rockford, IL) and visualization by autoradiography.
Gel mobility assays.
Gel mobility assays were performed as previously described2
using nuclear extracts prepared according to Andrews and
Faller.28 HRE oligonucleotide probes (10 pmol/L) were
radiolabeled with 10 mCi/mL  -32P (specific activity,
3,000 Ci/mmol) by reaction with T4 polynucleotide kinase
for 30 minutes at 37°C. The reaction was stopped with 0.5 mol/L
EDTA, and probes were purified by the Qia quick nucleotide removal kit
(Qiagen, Valencia, CA) used according to the manufacturer's instructions. Probes were suspended in TE buffer and used in binding reactions (10,000 to 20,000 cpm) with 2 to 10 µg nuclear extract protein incubated at 37°C for 30 minutes with BSA and poly-(dI-dC). After incubation, reaction mixtures were analyzed by electrophoresis in
a 4% low ionic strength native polyacrylamide gel with an
acrylamide:bisacrylamide ratio of 80:1, followed by autoradiography.
For supershift experiments, incubations with oligonucleotide probes
were performed as described, then followed by an additional incubation
for 30 minutes with antibody, 10 µg per reaction mixture.
Construction of HRE microcircles and gel mobility assays with
microcircles.
Tandem repeats of HRE containing complementary oligomers were
synthesized (GIBCO-BRL) with BamHI restriction enzyme sites flanking the ends. Both strands were annealed and subcloned into pBluescript II SK+ vector at the corresponding site. Direct sequencing confirmed incorporation of the correct 27-bp HRE sequence. A 310-bp fragment containing the HRE sequences was excised from this plasmid construct (pHRE#5) by PVU II/Xho I restriction
enzyme digestion and subjected to fill-in reaction in the presence of
32P labeled deoxycytidine triphosphate (dCTP) using
Klenow-enzyme. Alternatively, a 1.69-kb DNA fragment containing the HRE
sequences was also excised from the pHRE#5 plasmid by Bgl I and
end labeled in the presence of 32P-labeled ATP using T4
polynucleotide kinase. The radiolabeled fragments were self-ligated
using T4 DNA ligase and the products were digested with 1 U each of
Bal 31 and S 1 nucleases. The resulting HRE-containing
microcircles resistant to these exonucleases were gel purified and used
in gel mobility shift assays. Gel mobility assays with microcircles
were performed using 1% agarose gels rather than the standard 4%
polyacrylamide used for the same assays with double-stranded
oligonucleotides because microcircles did not enter the polyacrylamide
when incubated with nuclear extract.
Immunodepletion experiments.
Immunodepletion was used to remove KuAg from nuclear extracts before
some gel mobility assays. In brief, KuAg specific monoclonal antibody,
Ab-3, was bound to tosylactivated magnetic beads (Dynabeads M-280
Tosylactivated; Dynal) according to the manufacturer's instructions. Derivatized beads were incubated with nuclear extracts prepared as
described above for 30 minutes at room temperature, then collected magnetically, and washed with 1× gel shift buffer. The captured proteins were eluted from the beads by incubation for 30 minutes at
4°C in high salt extraction buffer, 20 mmol/L HEPES-KOH, pH 7.9, 24% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 0.2 mmol/L phenylmethylsulfonyl fluoride at 4°C.
Shift-Western blotting.
The method of Demczuk et al32 was used to identify the
protein components of gel mobility assays in combination with
immunoblotting. After completion of gel mobility assays performed as
described above, electrophoretic transfer was performed at room
temperature in 48 mmol/L Tris/39 mmol/L glycine/20% methanol, pH 8.5. Stacked membranes were used with the first membrane below the gel being nitrocellulose (Millipore, Bedford, MA) to bind proteins, but not
double-stranded DNA probes, followed by a polyvinylidene difluoride (PVDF) membrane (Millipore) to retain radiolabeled probe. The membrane
components were separated with Whatman 3MM paper (Whatman, Hillsboro,
OR) during transfer and all were preequilibrated in the transfer
buffer. Subsequent to transfer, the radiolabeled probe was identified
by autoradiography, and protein bands retained on the nitrocellulose
membrane were analyzed by Western blotting as described above.
Ultraviolet (UV) cross-linking experiments.
To estimate sizes of DNA-binding proteins or protein complexes detected
by gel mobility assays, UV cross-linking was performed as described by
Chodosh.33 For these experiments, nuclear extracts and
radiolabeled nucleotide probes were incubated under conditions used for
gel mobility assays, then subjected to an additional 60-minute
incubation with UV illumination (324 nm at 5 cm). After incubation,
reaction mixtures were analyzed by PAGE and autoradiography.
| |
RESULTS |
Affinity isolation of HREBP.
Figure 1 shows gel mobility assays
performed with nuclear extracts from hemin-treated U937 cells before
and after affinity depletion by beads derivatized with the 27-bp HRE in
wild-type or scrambled configurations. Extracts subjected to multiple
rounds of specific affinity depletion no longer produced a band shift, whereas those treated with the scrambled oligomer produced a pattern indistinguishable from that given by unmanipulated extracts. Therefore, specific depletion of HREBPs was accomplished. Protein components bound
to HRE-derivatized beads were analyzed by SDS-PAGE
(Fig 2A). Protein components of 133-, 90-, and 80-kD were regularly obtained. As illustrated by the results in Fig
2, a component of 37 kD also was isolated from some, but not all,
preparations. It is unknown why variable recovery of this component
occurred despite identical affinity isolation conditions. Nuclear
extracts derived from 2.5 × 108 cells contained
approximately 5 mg of total protein from which was derived 500 ng of
the 90-kD component.
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| Fig 1.
Analyses of HREBPs in nuclear extracts by gel mobility
assays. Gel mobility assays were performed as described in Materials and Methods using nuclear extracts from hemin-treated U937 cells and
radiolabeled 27-mer HRE probes. For the experiments in lanes 2 and 4, extracts were preincubated x 5 with beads derivatized with irrelevant (scrambled) or wild-type 27-mer HREs, respectively. Lane 1, free probe. Lane 2, U937 extract preincubated with scrambled 27-mer beads. Lane 3, unmanipulated extract. Lane 4, extract
preincubated with beads derivatized with wild-type 27-mer HRE
sequence.
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| Fig 2.
Analyses of protein bound to HRE-derivatized magnetic
beads. (A) SDS-PAGE analysis. Nuclear extracts from hemin-treated U937 cells were incubated with magnetic beads derivatized with the 27-mer
HRE DNA sequence as described in Materials and Methods. Bound proteins
were released by boiling in Laemmli buffer, then separated by SDS-PAGE
in 10% acrylamide gels. Protein bonds were visualized by silver or
Coomassie blue staining. Lanes 1 and 2 depict results of two separate
isolations. (B) Microsequence analysis of two peptides from the 90-kD
HREBP band. Digests were sequenced on an Applied Biosystems 477A
sequencer with an ABI 120 HPLC. KuAg p86 sequences are from the BLAST
data base. Swiss-Prot accession:P13010; NCBI Seq ID:125731. (C)
Immunoblot analyses of isolates in (A) with anti-KuAg monoclonal
antibodies. Lane 1, Ab-3, clone 162 recognizing p70/p80; lane 2, Ab-4,
clone N3H10 recognizing p70; lane 3, Ab-2, clone 11D recognizing p80.
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Identification of KuAg as a component of the HREBP complex.
Sequencing of the 90-kD HREBP yielded two amino acid sequences
homologous to ATP-dependent DNA helicase II, the KuAg p80/86 subunit3 (Fig 2B). There was complete identity between a
10-amino acid sequence of the 90-kD HREBP and the p86 KuAg, amino acids 264 to 273; and another sequence of the HREBP was 83% homologous to
the p86 KuAg sequence at amino acids 581 to 586.3 To
confirm that KuAg was present in the HREBPs affinity-purified from
hemin-treated U937 nuclear extracts, immunoblotting was performed with
KuAg subunit specific monoclonal antibodies.27 Results are
given in Fig 2C and indicated that both p80/86 and p70 KuAg components were recovered as HREBPs with the 90-kD band representing KuAg p80/86
and the 80-kD band representing KuAg p70.
Identification of KuAg and ref1 in gel mobility assays.
Gel mobility assays performed with nuclear extracts of hemin-treated
cells and radiolabeled 27-mer HRE oligonucleotides produced a
characteristic pattern2 (Fig 1). To determine if KuAg were involved in this gel mobility shift observed after hemin treatment, two
types of additional experiments were performed: (1) supershift experiments with anti-Ku monoclonal antibodies, and (2) shift-Western blotting.31 Results of a representative supershift
experiment using the monoclonal anti-Ku antibody, Ab-3 (reactive with
both KuAg components), are illustrated in
Fig 3A. The appearance of a gel mobility
supershift in the presence of antibody provides evidence for presence
of KuAg in the HREBP complex. For the shift-Western experiments,
various KuAg subunit specific monoclonal antibodies27 were
used to probe proteins retained on the nitrocellulose filters after
electrophoretic transfer to stacked membranes was performed as
described in Materials and Methods. Results in Fig 3B indicate that
both the p80/86 and p70 KuAg components were present in the band shifts
of gel mobility assays performed under standard conditions. To provide
additional confirmation that KuAg contributed to the gel mobility shift
results, nuclear extracts were prepared as usual, then immunodepleted
of KuAg, as described in Materials and Methods. Immunoblotting was
performed to determine the extent of KuAg depletion (Fig 3C). Using
KuAg-depleted nuclear extracts in the standard gel mobility assay
resulted in abrogation of the usual gel shift pattern compared with
that given by undepleted extracts (Fig 3C). Therefore, we conclude that
KuAg is a component of the HREBP involved in the gel mobility shift
observed with nuclear extracts after hemin treatment of the cells.
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| Fig 3.
KuAg is bound to HREs in gel mobility assays with nuclear
extracts from hemin-treated U937 cells. (A) Supershift experiment. Gel
mobility assays with radiolabeled 27-bp HRE probes were performed as
described in Materials and Methods. Supershift experiments included an
additional 30-minute incubation with Ab-3. Lane 1, standard gel
mobility assay; lane 2, parallel supershift experiment with Ab-3. The
arrow indicates the supershifted band. (B) Shift-Western experiments.
Gel mobility assays with HRE probes were performed, followed by
electrophoretic transfer of proteins and probes to stacked
nitrocellulose and PVDF membranes as described in Materials and
Methods. Panel 1, autoradiography of PVDF membrane. Remaining panels
are immunoblots of nitrocellulose membrane with the anti-KuAg monoclonals Ab-4 (anti-P70), panel 2; and Ab-2 (anti-p80), panel 3; or
with the anti-ref1 antibody, panel 4. Each panel depicts duplicate
lanes. The arrow indicates the location of the shift band. The band at
the top of each lane corresponds to the position of the sample wells.
(C) Gel mobility assays with Ku depleted extracts. Nuclear extracts
were prepared from hemin-treated U937 cells, then depleted of KuAg by
serial incubations with Ab-3-derivatized magnetic beads as described
in Materials and Methods. Panel I depicts standard gel mobility assays
with radiolabeled 27-bp HRE probe. Lane 1, free probe; lane 2, undepleted extract; lanes 3 to 6 extract after 1, 2, 3, or 4 incubations, respectively, with anti-KuAg beads. Panel II: immunoblot
of KuAg depleted extracts using antibody Ab-3. Lanes correspond to
those of panel I. The arrow indicates the position of the largest gel
shift band.
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In other experiments, shift Western analyses using affinity purified
rabbit ref1 antiserum34 demonstrated presence of ref1 in
the HREBP complex (Fig 3B). This antiserum did not bind to the complex
in the presence of specific ref1 blocking peptide (Santa Cruz
Biotechnology, Santa Cruz, CA; data not shown).
Gel mobility assays with HRE microcircles.
The potential for KuAg to represent a confounding variable for the
interpretation of gel mobility assays has been
recognized.35 To avoid the potential problem of nonspecific
KuAg binding to the free ends of radiolabeled oligonucleotide probes,
Giffin et al22,23 constructed DNA
microcircles containing NRE1 to demonstrate sequence-specific KuAg
binding. We used a similar technique to prepare HRE microcircles, then
used 32P-labeled microcircles in agarose gel mobility and
immunoblot assays. Microcircles were resistant to S1 or Bal 31 nuclease
digestion (Fig 4A). Gel shift patterns with
nuclear extracts are illustrated in Fig 4B and C. Incubation of
radiolabeled microcircles with nuclear extracts from hemin-treated U937
cells resulted in a gel mobility shift (Fig 4B). Concentrations of
bovine serum albumin more than sixfold the concentration of nuclear
extract used did not produce a shift band (Fig 4B). Cold competition
experiments with authentic 27-bp HRE in linear form demonstrated
specific dose-dependent inhibition by authentic HRE sequences (Fig 4C), thus confirming specificity of the gel shift pattern observed with
microcircles. Parallel competition experiments with scrambled 27-bp HRE
(Fig 4C) or with 27-bp HRE in which the tandem GAGGC repeat was
replaced with a different 10-bp nucleotide sequence ( CCGGCTTCCC) gave no inhibition (data not shown).
Immunoblotting with anti-KuAg showed KuAg bound to microcircles in the
shift band (Fig 4D). Persistence of a mobility shift pattern with the microcircles and identification of KuAg by immunoblotting establishes the specificity of this component for the HREBP complex.
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| Fig 4.
Experiments with microcircles. (A) Nuclease digestion
experiments. Radiolabeled linear 27-bp HRE linear probes or
microcircles containing HRE sequences were incubated with S1 nuclease
(1 U, 15 minutes, 37°C) or Bal 31 nuclease (1 U, 15 minutes,
37°C), then analyzed by PAGE and autoradiography. Lane 1, untreated
linear probe. Lane 2, untreated microcircles after ligation reaction and before gel purification. The arrow indicates unincorporated residual linear probe. Lanes 3 and 4, linear probe after digestion with
S1 nuclease or Bal 31 nuclease, respectively. Lanes 5 and 6, microcircles after digestion with S1 nuclease or Bal 31 nuclease, respectively. (B) Mobility shift assays with nuclear extracts. Radiolabeled HRE microcircles were incubated with nuclear extracts from
hemin-treated U937 cells (12 µg) or with bovine serum albumin, 4 to
80 µg, then analyzed by agarose gel electrophoresis and
autoradiography. Lane 1, free microcircle probe. Lanes 2 and 3, probe
plus nuclear extracts. Lanes 4, 5, 6, and 7, probe plus BSA, 4, 8, 40, or 80 µg, respectively. (C) Cold competition experiments. Gel
mobility shift assays were performed with radiolabeled HRE microcircle probes. Competition experiments include a 15-minute preincubation with
increasing concentrations of unlabeled linear oligonucleotides, either
authentic 27-bp HRE, or scrambled HRE sequences. Panel I: Competition
study with authentic probe. Lane 1, free microcircle probe. Lanes 2 and
3, probe plus nuclear extracts. Lanes 4, 5, and 6, probe, nuclear
extract plus 50-fold, 100-fold, or 250-fold excess of linear 27-bp HRE.
Panel II: Competition study with irrelevant probe. Lane 1, free
microcircle probe; Lane 2, probe plus nuclear extract. Lanes 3, 4, and
5, probe, nuclear extract, plus 50-fold, 100-fold, or 200-fold excess
of linear scrambled HRE. (D) Immunoblot analyses of gel shift bonds.
Radiolabeled HRE microcircles were incubated with 12, 40, or 80 µg of
nuclear extract from hemin-treated U937 cells, then analyzed by agarose
gel electrophoresis. Panel I: Analysis by autoradiography (48-hour
exposure). Lane 1, free microcircle probe. Lanes 2, 3, and 4, probe
plus 12, 40, or 80 µg of nuclear extract. Panel II: Immunoblot
analysis with anti-KuAg (Ab-3) (chemiluminescence exposure, 2 minutes).
Lanes 1 to 4, same as panel I. Panel III: Immunoblot analysis with
mouse anti-c-myc IgG (chemiluminescence exposure, 2 minutes). Lanes 1 to 4, same as panels I and II.
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UV cross-linking experiments.
UV cross-linking experiments were performed to estimate molecular mass
of protein complexes bound to the 27-mer HRE oligonucleotides during
gel mobility assays and to serve as an indication as to whether KuAg
was present alone or complexed with other proteins. UV cross-linking
was performed as described in Materials and Methods34 and
products were analyzed on denaturing gels. Results are presented in
Fig 5. Two species of approximately 300 and
174 kD were present, with the former predominant in unmanipulated
nuclear extracts. Conversely, in extracts first depleted of KuAg by
incubation with antibody-coated beads, the predominant species was the
174-kD species. These data suggest that the KuAg may not itself
interact directly with the HRE, but may bind only in association with
other protein components, which in turn, may provide the DNA binding specificity. Based on estimated molecular weight (MW) of the HRE binding complex from Ku-depleted extract, DNA recognition and binding
specificity appears to be provided by the 133-kD protein (p133)
and/or the ref1 protein present in the affinity isolates.
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| Fig 5.
UV cross-linking experiments. UV cross-linking
experiments with hemin-treated U937 nuclear extracts and radiolabeled
HRE probes were performed as described in Materials and Methods.
Autoradiographs of the PAGE analyses are illustrated. Lane A,
undepleted extract; lane B, KuAg-depleted (5 rounds of depletion)
extract.
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Depletion/reconstitution experiments.
To explore further the relationships among the components of the HREBP
complex, we prepared nuclear extracts depleted of HREBP by extensive
sequential absorptions with HRE-derivatized, then anti-KuAg-derivatized beads. As expected, the usual gel shift pattern
was abolished in assays using the depleted extract
(Fig 6). The emergence of a lower
molecular weight gel shift band in assays with depleted extracts most
likely represents a low-affinity HRE binding component masked in the
presence of HREBP. We next prepared nuclear extract fractions A and B. Fraction A was the 0.42 mol/L NaCl eluate from anti-
KuAg-derivatized beads and contained predominantly KuAg
components. Fraction B was the 0.42 mol/L NaCl eluate from
HRE-derivatized beads to which first had been applied a KuAg-depleted
nuclear extract. Fraction B contained predominantly non-KuAg HRE
components. A series of reconstitution experiments were then performed
in which depleted nuclear extract was mixed with Fraction A, Fraction
B, or Fractions A+B. Results are shown in Fig 6. Under the experimental
conditions, reconstitution with Fraction A containing predominantly
KuAg did not produce the usual gel mobility shift pattern. However,
either Fraction B alone or together with Fraction A added to depleted
extract did partially reconstitute the pattern. Reconstitution with
Fraction B alone produced a major lower MW and minor high MW bands
consistent with some contamination of Fraction B with KuAg. These data
are additional evidence that KuAg requires interactions with the other
components of the HREBP complex to interact with the HRE DNA sequence.
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| Fig 6.
Depletion/reconstitution experiments. Nuclear extracts
were prepared from hemin-treated U937 cells, then depleted of KuAg by
affinity absorption to KuAg-derivatized beads followed by serial incubations with HRE-derivatized beads to remove non-KuAg HREBPs. Fraction A (primarily KuAg) was the eluate from KuAg-derivatized beads.
KuAg-depleted extract was incubated with HRE-derivatized beads to
obtain Fraction B, the eluate from these beads comprising predominantly
non-KuAg HREBPs. Gel mobility assays were then performed with
radiolabeled HRE probes and these various extracts alone, or
reconstituted with fraction A, B, or A+B. Lane 1, free probe; lane 2, unmanipulated extract; lane 3, depleted extract; lane 4, depleted
extract + Fraction A; lane 5, depleted extract + Fraction B; lane
6, depleted extract + Fractions A + B. The arrow indicates the
position of the major gel shift band.
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DISCUSSION |
Previously we have identified a novel mechanism of transcriptional
inhibition mediated by hemin.2 Induction of TRAP mRNA in
cultured peripheral mononuclear cells was inhibited by hemin. This
inhibition was dependent on nucleotide sequences in the mTRAP 5-flanking region localized by deletion mutagenesis experiments to a 27-bp sequence at  1815 to 1789 bp relative to
ATG.2 A tandem repeat sequence, GAGGC; GAGGC, contained
within this DNA segment was shown to be involved in specific binding of
HREBP in response to hemin. Highly homologous sequences have been
identified in the 5-flanking region of the hTRAP
gene.36 A 607-bp segment of the mTRAP 5-flanking
region containing the HRE and adjacent sequences conferred hemin
regulation on the viral SV40 promoter.2 Southwestern
blotting experiments probing nuclear extracts of hemin-treated U937
cells with the 27-mer HRE sequence identified two protein bands at 37 and 133 kD representing candidate HREBPs.2
The present studies begin to elucidate the composition of HREBPs. Using
affinity isolation techniques, we have identified four protein
components binding specifically to immobilized HRE DNA sequences (Fig
2). The 80- and 90-kD components correspond to the p70 and p80/86
subunits of KuAg, respectively, as documented by partial amino acid
microsequence data and by immunologic reactivity with KuAg specific
mouse monoclonal antibodies (Fig 2). The two components of 133 and 37 kD may correspond to the two components previously identified by
Southwestern blotting.2 The demonstration by shift Western
blotting that HREBP complexes contain the ref1 protein (Fig 3B) makes
it likely that the 37-kD component represents ref1. Limited amino acid
sequence data indicate that p133 may be a unique protein (O. Alcantara
and D.H. Boldt, unpublished results, October 1997).
Because KuAg may be capable of nonspecific binding to double-stranded
DNA ends,21,35 it is important to exclude this type of
nonspecific interaction as an explanation for the findings. Several
lines of evidence argue against this. First, KuAg was not removed from
nuclear extracts by a scrambled double-stranded 27-mer HRE
oligonucleotide used as a control for the affinity isolation procedure
(Fig 1). Second, the shift Western blotting experiments demonstrated
that KuAg was present in specific HREBP complexes identified by gel
mobility assays (Fig 3). The specificity of these complexes for binding
to the HRE tandem repeat GAGGC sequence has been established
previously.2 Third, the Southwestern blotting experiments
previously reported demonstrated no interaction of double-stranded
27-mer HRE probes with either p70 or p80/86 KuAg
components.2 Fourth, KuAg alone was unable to reproduce the
characteristic gel shift pattern when added to nuclear extracts first
depleted of specific HRE-binding components (Fig 6). Finally, experiments with HRE microcircles confirmed the presence of KuAg in
HREBP complexes (Fig 4). The use of microcircles to overcome potential
problems of nonspecific KuAg-DNA interactions has been previously
documented.22,23,37
Although the results of our experiments establish the specificity of
the KuAg participation in HREBP binding to DNA, they do not establish
the determinants of that specificity. Which component or components of
HREBP binds directly to DNA and the sequence and manner of complex
assembly remain to be determined, as does also the role of hemin
induction. However, the UV cross-linking experiments (Fig 5) and the
depletion/reconstitution experiments (Fig 6) strongly suggest that KuAg
does not directly bind to HRE, but interacts only in the context of the
HREBP complex.
Recent reports have highlighted the capability of KuAg to mediate
sequence specific DNA binding.22,23 In those reports, KuAg
was identified as a transcription factor capable of binding specifically to NRE1. When bound, KuAg recruited DNA-PK and the resulting complex inhibited glucocorticoid-induced gene transcription via phosphorylation and inactivation of glucocorticoid
receptors.22 Although a consensus DNA sequence required for
KuAg recognition has not been determined rigorously, limited data
suggest that a polypurine/polypyrimidine core represents a minimal
requirement.23 In the study of Giffin et al,23
this characteristic plus substantial sequence identity to NRE1 were
common features of DNA sequences interacting with KuAg. Neither are
features of HRE.2 Therefore, this is additional evidence
that binding of the HREBP complex to DNA is not mediated directly by
KuAg. Additional experiments will be required to determine this point
directly.
A number of other potential sequence-specific KuAg DNA binding sites
have been proposed, but in general, these lack clear homology to NRE1
and potential confounding variables have not been rigorously
excluded.38-42 It has been suggested that accumulation of
KuAg at these sites may result from translocation after initial binding
of KuAg to free ends, nicks, or other DNA structural
features.23
In at least two instances, some data suggest that the interaction of
KuAg with additional proteins may confer unique DNA binding specificities.25,41 Of particular interest to the findings in this report is the suggestion that interaction of KuAg with ref1
facilitates protein complex interaction with the DNA sequence comprising the negative calcium-responsive element (nCaRE) resulting in
regulation of gene expression.25 Further experimentation will be required to determine whether such an interaction is
responsible for the hemin-induced transcriptional inhibition we have
observed. Although shift-Western experiments demonstrated ref1 in HREBP complexes, we were unable to confirm the presence of ref1 by
immunoreactivity in complexes bound to HRE microcircles (data not
shown). Although this result was observed repeatedly, these experiments
were all performed with one U937 cell nuclear extract. We observed
earlier that the 37-kD component could not be isolated from every
nuclear extract examined (Fig 2A). The data provide the possibility
that ref1 may participate in HRE binding, but its precise role remains equivocal.
The observations summarized above and the data in this report emphasize
the variety of potential KuAg-dependent transcriptional regulatory
mechanisms and also the potentially great flexibility inherent in a
system in which DNA binding specificity might vary according to the
specific protein partner complexing with KuAg. In this context, it is
intriguing to speculate that hemin may function by promoting
interaction(s) between KuAg and p133 and/or ref1 to produce the
HREBP complex and lead to downregulation of hemin responsive genes.
Such a role has recently been proposed for heme in regulating
transcription of the CYP2B1/B2 gene in rat liver.43
In summary, we have demonstrated that the HREBP complex consists of the
Ku antigen, a unique 133-kD protein, and the ref1 protein. Because
binding of this complex to the HRE mediates transcriptional inhibition
of TRAP gene expression in response to hemin treatment, future studies
will be directed to understanding the potential role of hemin in
complex assembly and/or DNA interaction and how binding of the
complex functions to block transcription. Additional studies are in
progress to clone and characterize the 133-kD protein.
| |
FOOTNOTES |
Submitted October 1, 1997;
accepted October 28, 1997.
Supported by Grant No. DK 50425 from the National Institute of
Diabetes and Digestive and Kidney Diseases and Grant No. CA 40035 from the National Cancer Institute, National Institutes of Health,
Bethesda, MD.
Address reprint requests to D.H. Boldt, MD, Medicine/Hematology,
University of Texas Health Science Center, 7703 Floyd Curl Dr, San
Antonio, TX 78284-7880.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Jue Yang for technical assistance and Cheryl Muzzi
Adams for secretarial support. We are grateful to Dr Tom Curran for
providing anti-ref1 antibody.
| |
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Abstract
Introduction
Abstract