Characterization of nuclear proteins that bind to the regulatory TGATTGGC motif in the human immunodeficiency virus type 1 long terminal repeat.

Abstract

We have recently elucidated the nature and function of transcription factors present in Jurkat, glial and neuronal cells that interact with modulatory region B, the nuclear receptor responsive element, in the long terminal repeat of human immunodeficiency virus type 1 (HIV-1). Considering the key role that the combination of host cell proteins plays in HIV-1 gene transcription, it appears essential to characterize proteins interacting with the adjacent region A. In vitro experiments revealed that the 5'-TGATTGGC-3'motif of region A is the target for at least three distinct proteins, one belonging to the nuclear factor I family, while two others are related to the cAMP response element binding (CREB) protein family. One of these proteins, present in DNA-protein complex C2, is formed by distinct polypeptides of relative molecular mass 43 000 and 50 000. We have purified the 43 kDa protein, which is distinct from CREB-43, and have shown that renatured p43 is able to specifically interact with site A. Transient expression experiments with vectors containing wild-type or mutant motif A revealed that basal HIV-1 gene transcription in Jurkat cells is regulated by antagonistic effects of the site A binding proteins.

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 1997 Oxford University Press
1177–1184
Nucleic Acids Research, 1997, Vol. 25, No. 6
Characterization of nuclear proteins that bind to the
regulatory TGATTGGC motif in the human
immunodeficiency virus type 1 long terminal repeat
Christian Schwartz, François Canonne-Hergaux, Dominique Aunis and
Evelyne Schaeffer*
Unité 338 INSERM, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France
Received December 4, 1996; Revised and Accepted January 30, 1997
ABSTRACT
We have recently elucidated the nature and function of
transcription factors present in Jurkat, glial and
neuronal cells that interact with modulatory region B,
the nuclear receptor responsive element, in the long
terminal repeat of human immunodeficiency virus type
1 (HIV-1). Considering the key role that the combination
of host cell proteins plays in HIV-1 gene transcription,
it appears essential to characterize proteins interacting
with the adjacent region A. In vitro experiments
revealed that the 5′-TGATTGGC-3′ motif of region A is
the target for at least three distinct proteins, one
belonging to the nuclear factor I family, while two
others are related to the cAMP response element
binding (CREB) protein family. One of these proteins,
present in DNA–protein complex C2, is formed by
distinct polypeptides of relative molecular mass 43 000
and 50 000. We have purified the 43 kDa protein, which
is distinct from CREB-43, and have shown that
renatured p43 is able to specifically interact with site A.
Transient expression experiments with vectors con-
taining wild-type or mutant motif A revealed that basal
HIV-1 gene transcription in Jurkat cells is regulated by
antagonistic effects of the site A binding proteins.
INTRODUCTION
Replication and gene expression of human immunodeficiency
virus type 1 (HIV-1) depend upon multiple sequences present in
the long terminal repeat (LTR). Transcription is regulated by an
interplay of viral and cellular host proteins with regulatory
elements of the LTR. Key elements such as the TATA, Sp1 and
NF-κB region, present in the proximal part of the LTR (+1 to
–112) have been extensively characterized (for reviews see 1,2).
The role of the NF-κB element especially has been investigated
in a variety of cell types, including lymphocytes, monocytes and
central nervous system-derived cells (3–7).
However, transcription control elements present in the –112 to
–459 modulatory region of the LTR have been poorly characterized.
This region includes regulatory elements that can mediate
transcription in many cell types, under a variety of growth and
differentiation conditions (8). Recently several reports have
described the nuclear receptor-responsive element (NRRE)
located within the –356/–320 region as representing a site for
complex regulatory interactions among hormone receptors and
orphan receptors, as well as the AP-1 transcription factor (9–12).
We have recently reported that AP-1 is unable to directly bind to
the NRRE sequence but interacts indirectly via nuclear receptors
belonging to the steroid–thyroid–retinoid nuclear receptor super-
family (13–14). Moreover, we have recently reported the role of
retinoic acid receptors and the orphan receptor COUP-TF on
HIV-1 gene transcription in human central nervous system-derived
cell lines (14). Since recent data have highlighted the importance
of the U3 region of the LTR in determining the pathogenicity of
HIV-1 (15), studies concerning the nature and role of cellular
transcription factors that interact with various elements of the
LTR appear essential.
In this study we have characterized the regulatory element
spanning the –385 to –362 region of the LTR, which is directly
adjacent to the NRRE. This region has previously been footprinted
with nuclear extracts from Jurkat T cells and named site A
(16–17). Our previous studies have revealed that this sequence is
the binding site for nuclear proteins present in a great variety of
cell lines, such as Jurkat, HeLa, astrocytoma, oligodendroglioma
and neuronal cells (13). However, the nature and function of the
nuclear proteins interacting with site A remain elusive. Here we
demonstrate that the TGATTGGC core sequence is the binding
site for at least three distinct proteins present in Jurkat and in HeLa
cells. By in vitro experiments we have analyzed the nature of the
proteins forming the different DNA–protein complexes. We have
shown that one nuclear protein belongs to the nuclear factor I
(NFI) family (18), while other factors are related to the CREB
protein family (for a review see 19). We report here the
purification of a nuclear factor of relative molecular mass 43 000,
distinct from CREB-43, which specifically binds to site A.
Moreover, we provide evidence, by transient expression experi-
ments, that site A binding proteins play antagonistic roles in the
control of basal transcription of the HIV-1 genome in Jurkat cells.
MATERIALS AND METHODS
Plasmid constructs
To construct the 1N/tk-CAT, 1L/tk-CAT, 1Lm/tk-CAT, 2×1L/tk-
CAT and 2×1Lm/tk-CAT vectors, one copy of the 1N, 1L and
*To whom correspondence should be addressed. Tel: +33 3 88 45 67 18; Fax: +33 3 88 60 08 06; Email: schaeffer@neurochem.u-strasbg.fr

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1Lm oligonucleotides and two copies of the 1L and 1Lm
oligonucleotides respectively were subcloned in sense orientation
in the blunt-ended SalI site of pBLCAT2, containing the herpes
simplex virus thymidine kinase promoter in front of the CAT gene
(20; a gift of C.Kedinger, Unit INSERM 184, Strasbourg,
France). The 1L-LTR-CAT and 1Lm-LTR-CAT vectors were
constructed by site-directed mutagenesis with oligonucleotides
1L and 1Lm (Transformer TM Site-directed Mutagenesis Kit;
Clontech). Mutations were controlled by DNA sequencing.
Cell culture and nuclear extract preparation
Jurkat and HeLa cells were respectively grown in RPMI 1640 and
DMEM medium supplemented with 10% fetal calf serum and
10 mM HEPES in the presence of penicillin and streptomycin
(100 U/ml). Nuclear extracts were prepared as previously described
(21). A phosphatase inhibitor, 0.5 mM β-glycerophosphate, was
added during the preparation of nuclear extracts from Jurkat cells.
Transfections and CAT assays
Jurkat cells (5 × 106–107 cells/transfection) were transfected by
the DEAE–dextran technique with 3 pmol plasmid DNA. After
48 h, cells were collected and chloramphenicol acetyltransferase
(CAT) assays were performed as described previously (21) with
20 µg protein extract and a 2 h incubation at 37?C.
Electrophoretic mobility shift assays
Protein–DNA binding reactions were performed with 5 or 10 µg
crude nuclear extract as previously described (13). For competition
assays, unlabeled oligonucleotides were added at a 50- or
200-fold molar excess at the same time as the probe. When
purification columns were analyzed, 10 µl of the fractions were
used. The sequences of the oligonucleotides used as probes or
competitors are shown in Figure 1. In supershift assays performed
as previously described (13), we used antibodies directed against
CREB-1 and ATF-1 and reactive with ATF-1 p35, CREB-1 p43
and CREM-1 (Santa Cruz Biotechnology). Binding reactions
were carried out with the protein CREB bZIP (Santa Cruz
Biotechnology).
Methylation interference assays
Methylation interference assays were performed with oligonucleo-
tide 1L as described previously (13).
UV cross-linking analysis
Crude nuclear extract (10 µg) from Jurkat cells and partially
purified proteins (10 µg) of a 0.4 M KCl fraction from the
heparin–Sepharose column were incubated for 15 min on ice with
the 5′-end-labeled 1L probe substituted with bromodeoxyuridine
under standard mobility shift assay conditions. Following
electrophoresis, the gel was irradiated for 12 min using a UV
transilluminator (254 nm; Bioblock Scientific). Radioactive
bands corresponding to complexed and free DNA identified by
autoradiography were excised and incubated in 40 µl denaturing
buffer (62 mM Tris–HCl, pH 6.7, 2% SDS, 10% glycerol, 50 mM
dithiothreitol) and heated at 100?C for 5 min. The gel slices and
the denaturing buffer were loaded on a 10% SDS–polyacrylamide
gel. The gel was fixed, dried under vacuum and the proteins
directly involved in the DNA interaction were identified by
autoradiography. We used prestained molecular weight markers
(BioRad).
Southwestern blotting
Nuclear proteins partially purified by heparin–Sepharose (20 µg)
or by DNA affinity chromatography (5 µg) were resolved on a
10% SDS–PAGE gel and electrotransferred onto nitrocellulose
membranes. The membranes were washed three times for 1 h with
renaturation buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 10 mM
dithiothreitol, 2.5% Nonidet P-40, 10% glycerol, 3% BSA) (22).
The membranes were then placed in a heat-sealable pouch in
binding buffer (10 mM Tris–HCl, pH 7.5, 40 mM NaCl, 1 mM
DTT, 1 mM EDTA, 8% v/v glycerol, 3% BSA, 60 µg poly(dI·dC),
5 mM MgCl2, 5 µg/ml 32P-labeled oligonucleotide 1L or 1Lm).
After 16 h constant agitation, membranes were washed for 2 h at
room temperature with 10 mM Tris–HCl, 50 mM NaCl with
several changes and autoradiographed on Kodak X-OMAT film.
Purification of a 43 kDa protein
Aliquots of 25 mg nuclear protein extract, obtained from 5 × 109
Jurkat cells were applied to a 5 ml heparin–Sepharose column
equilibrated with buffer Z (20 mM HEPES, pH 7.9, 0.2 mM
EDTA, 20% glycerol, 1 mM dithiothreitol) containing 50 mM KCl.
Proteins were separated using a step gradient from 0.1 to 1 M KCl
in buffer Z. Activity was detected in fractions eluted between 0.3
and 0.4 M KCl by gel mobility shift assay. These fractions were
pooled and dialyzed against buffer Z containing 0.1 M KCl. The
pool was mixed with salmon sperm DNA (25 µg/ml), incubated
on ice for 15 min and loaded onto a 2 ml oligonucleotide 1Lm
affinity column equilibrated in buffer Z containing 0.1 M KCl.
The column was washed with the same solution. The flow-through
was loaded onto a 2 ml oligonucleotide 1L affinity column
equilibrated in buffer Z containing 100 mM KCl. The column was
washed with the same solution and eluted with a step gradient
from 0.1 to 1 M KCl in buffer Z. Oligonucleotide 1L binding
activity was detected in fractions at 0.5 M KCl. These fractions
were pooled, dialyzed against buffer Z containing 0.1 M KCl and
recycled on the same column. An aliquot (10 µl) of each fraction
was assayed by gel retardation assay using the oligonucleotide 1L
as probe. The active fractions were also analyzed by 10%
SDS–PAGE. The sequence-specific DNA affinity columns were
prepared according to Kadonaga and Tjian (23) using CNBr-
activated Sepharose 4B (Pharmacia).
Denaturation and renaturation experiments
Fractions (20 µg) containing peak activity after heparin–Sepharose
chromatography were subjected to a preparative SDS–10%
PAGE gel. After electrophoresis, the gel was cut horizontally into
1 mm slices between 40 and 70 kDa. The proteins were eluted
from these slices as described by Hager and Burgess (24). After
elution, proteins were precipitated with acetone and resuspended
in buffer H (50 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.05%
Nonidet P-40, 20% glycerol, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 0.1 M NaCl and 5 µg/ml leupeptin
and pepstatin) containing 6 M guanidine–HCl for 20 min. The
denaturing agent was diluted 50-fold with buffer H and renaturation
was allowed for 4 h at 4?C. Activity of renatured proteins was

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Figure 1. Gel mobility shift analysis of nuclear proteins from HeLa and Jurkat
cells binding to region A of the HIV-1 LTR. (A) Sequences of wild-type and
mutant oligonucleotides 1N, 1L and 1Lm, corresponding to the LTR region A,
and of oligonucleotides CRE, CRI and CR. The bold type represents mutant
oligonucleotides. (B) Gel retardation assays were carried out by incubating 10
µg nuclear extract from HeLa or Jurkat cells with probes 1N, 1L and 1Lm.
Binding reactions were performed either in the absence of competitor (lanes –)
or in the presence of a 50-fold molar excess of unlabeled 1N, 1L or CR
oligonucleotide or a 200-fold molar excess of unlabeled CRE or CRI
oligonucleotide, as indicated on top. DNA–protein complexes C1–C4 and the
free probe (F) are indicated.
tested by gel mobility shift assay in the absence of non-specific
competitors.
RESULTS
Multiple host nuclear factors bind to the site A of the
HIV-1 LTR
The sequence spanning the –385/–362 distal region of the HIV-1
LTR has previously been footprinted with nuclear extracts from
different cell types and named site A (13,16,17). A number of
studies have shown that the adjacent region B is the binding site
of proteins belonging to the nuclear receptor superfamily
(11–13). In order to analyze the nuclear proteins interacting with
site A, we performed DNA mobility shift assays using oligo-
nucleotides 1N, 1L and 1Lm (Fig. 1A). The 1N sequence, which
corresponds to site A, gave rise to four DNA–protein complexes,
C1–C4, with extracts from HeLa cells (Fig. 1B, lane 1) and to
three complexes, C1, C2 and C4, with extracts from Jurkat cells
(lane 3). Specificity of binding was ascertained by competition
experiments using a 50-fold molar excess of unlabeled homologous
1N oligonucleotide. The formation of complexes C1, C2 and C3
was specifically competed by oligonucleotide 1N (lanes 2 and 6).
Under similar conditions the formation of complex C4 appeared
less specific, since half of the complex was still detected (lanes 2
and 6).
Analysis of complex C1
Identification of the proteins binding to site A was first achieved
using competition gel shift experiments. A close inspection of the
1N oligonucleotide revealed the sequence TGGCA, which
corresponds to the consensus binding site of proteins of the
CTF/NFI family (18). We therefore used as a probe the mutant
oligonucleotide 1L containing the TGACA 1 bp mutation;
interestingly this mutation abolished formation of complex C1
(lane 4). When oligonucleotide 1L was used in a 50-fold molar
excess as a competitor of the 1N probe, formation of complex C1
was unaffected, while formation of complexes C2–C4 was
abolished (lane 7). To further assess that the protein forming
complex C1 belongs to the NFI family, we used as a competitor
oligonucleotide CR, previously shown to be the binding site of the
NFI protein in the promoter of the human transferrin gene (25).
As expected, in the presence of a 50-fold molar excess of CR
competitor, formation of complex C1 was specifically prevented
(lane 11). As a control, the mutant oligonucleotide 1Lm, which
contains 4 bp mutations, was unable to give any specific complex
when used as a probe (lane 5) or to compete for formation of any
complex in competition experiments (lane 8). A recent report
described that transcription factor YB-1, a member of the NFI
family, is able to interact with site A of the HIV-1 LTR (26).
However, supershift experiments with antibodies directed against
YB-1 did not alter formation of complex C1 (results not shown).
Taken together, these results suggest that complex C1 is formed
by a member of the NFI protein family, distinct from YB-1.
Analysis of complexes C2–C4
Oligonucleotide 1L contains the TGAC half-site consensus
sequence of the cAMP response element (CRE), in direct repeat
with the sequence TGAT, which varies by one base from the CRE
consensus (19). We therefore performed gel shift assays, using as
a competitor in 200-fold excess a CRE oligonucleotide containing
the consensus palindromic sequence TGACGTCA. Formation of
complexes C2 and C4 was totally abolished (lane 9). As a control,
a mutant CRI oligonucleotide containing mutations in the CRE
site did not affect formation of complexes C1 and C2 (lane 10).
Similarly, when oligonucleotide TRE containing the TGACTCA
binding site for proteins of the AP-1 family was used as a
competitor, the pattern was unaffected (results not shown). These
results suggest that the protein forming complex C2 is related to
the ATF/CRE binding protein family. We therefore performed
shift assays in the presence of antibodies directed against CREB-1
(27) or ATF-1 (28). All the complexes were unaffected (results
not shown), indicating that the CRE-binding protein(s) in
complex C2 is (are) different from CREB-1 and ATF-1.
We next tested whether a truncated CREB-1 protein, containing
the binding and dimerization domain, was able to bind to
oligonucleotides 1L and 1N (Fig. 2). Interestingly, with the 1N
probe CREB-1 was able to form a weak retarded complex. With
the 1L probe, containing a TGAC site, and with the CRE probe,
containing the palindromic TGACGTCA site, CREB-1 gave a
more intense retarded complex. This result demonstrates that
CREB proteins are able to interact with the 1L sequence and with
a weaker affinity with the 1N sequence. Taken together, these
results indicate that complex C2 is formed by a protein distinct
from CREB-1 and ATF-1 and related to proteins of the
CREB/ATF family. In HeLa cells, the additional complex C3 had
a behavior similar to that of complex C2 (results not shown),

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Figure 2. Binding activity of CREB-1 bZIP. Gel retardation assays were
performed by incubating different amounts of protein CREB-1 bZIP (from
Santa Cruz Biotechnology) with probes 1N, 1L and CRE. Lanes a, b and c
correspond to 100, 500 and 10 ng protein respectively.
suggesting that proteins forming complex C3 are also related to
the CREB/ATF family.
In order to precisely identify the binding site of proteins forming
complex C2, compared with proteins forming complexes C3 and
C4, we performed methylation interference experiments using
the 1L probe and nuclear extracts from HeLa cells. Figure 3
shows that methylation at the guanine residues at positions –372
and –376 on the coding strand and at position –370 on the
non-coding strand resulted in a strong interference with methylation.
In addition to a strong interference with methylation of these
guanine residues, methylation of the G residues at –378, –379,
–380 and –383 of the non-coding strand slightly decreased
binding in complex C3. In complex C4, only weak interference
with methylation was observed. These results indicate that all the
G residues in the TGATTGAC motif on both strands are critical
in formation of complex C2. Similar results were obtained for
complexes C2 and C4 formed with nuclear extracts from Jurkat
cells (results not shown). The importance of these G residues is
further confirmed by the results of the gel shift assays presented
in Figure 1 (lane 5): with the 1Lm probe, which contains point
mutations of the critical G residues, no complex C2 was detected.
Functional importance of the site A binding proteins in
Jurkat cells
To evaluate the functional effect of the different proteins interacting
with site A of the LTR, we constructed a series of pBLCAT2
reporter plasmids containing one copy of either oligonucleotide
1N, 1L or 1Lm or two copies of 1L or 1Lm in front of the herpes
simplex virus thymidine kinase promoter upstream of the CAT
gene. These vectors were transfected into Jurkat cells and CAT
activities were determined (Fig. 4). Interestingly, the activity of
all the vectors was higher than that of the control pBLCAT2
vector. Transfection of the 1Lm/tk-CAT and 2×1Lm/tk-CAT
vectors resulted in 1.8- and 4.0-fold stimulation of CAT activity
respectively, reflecting the positive action of the protein(s)
forming complex C4. Transfection of the 1L/tk-CAT and
2×1L/tk-CAT constructs led to 3.2- and 6.2-fold transcriptional
activation respectively, indicating that proteins forming complexes
C2 and C4 are able to optimally stimulate transcription.
Figure 3. Methylation interference pattern of DNA–protein complexes C2–C4
formed with the 1L probe. Oligonucleotide 1L was labeled at the 5′-end of one
strand. Guanine residues were partially methylated with dimethylsulfate. The
oligonucleotide was used in a gel shift assay with nuclear proteins from HeLa
cells. The DNA–protein complexes C2–C4 and free DNA were eluted from a
band shift gel and cleaved at methylated guanine residues with piperidine. The
protein-bound DNA was compared with free DNA on a sequencing gel.
(Bottom) Summary of the pattern obtained with complexes C2–C4. Closed and
open circles indicate strong and weak interference of binding respectively.
Interestingly, the CAT activity of the 1N/tk-CAT construct was
reduced, when compared with that of 1L/tk-CAT, which could
suggest mutual inhibition between proteins forming complexes
C1 and C2. Another explanation could be that complex C1
contains a protein(s) that acts as an inhibitor of site A promoter
activity and that is overwhelmed by the positive activity of
proteins forming complexes C2 and C4.
To examine the effect of mutagenesis of site A in the context of
the intact LTR, we have constructed 1L-LTR-CAT and 1Lm-LTR-
CAT vectors, by site-directed mutagenesis with the 1L and the
1Lm oligonucleotides respectively (Fig. 5). With the 1L-LTR-CAT
vector, where binding of the protein forming complex C1 is
prevented, CAT activity was increased 1.4-fold, suggesting that
this protein acts as a weak inhibitor of LTR-driven transcription.
CAT activity of the 1Lm-LTR-CAT vector was stimulated 2-fold,
suggesting that binding of proteins forming complexes C1 and C2
inhibits LTR transcription. This observation indicates that in the
context of both the tk and LTR promoters the protein forming

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Figure 4. Functional effect of site A in the tk-CAT reporter vectors. Jurkat cells
were transfected with pBLCAT2 reporter plasmids containing one or two copies
of oligonucleotides 1N, 1L or 1Lm (shown in the right panel). After 48 h, CAT
activities were determined as described in Materials and Methods. Values on the
right are the mean ± SE for at least three independent experiments performed
in duplicate and are expressed relative to the CAT activity of pBLCAT2, set at
1. An autoradiogram of one typical CAT assay is shown on the right.
Figure 5. Functional significance of site A within the HIV-1 LTR. Jurkat cells
were transfected with the wild-type LTR-CAT and mutant 1L-LTR-CAT and
1Lm-LTR-CAT vectors. CAT activities were determined as in Figure 4 and are
expressed relative to that of LTR-CAT.
complex C4 acts as an activator, while the protein forming
complex C1 acts as an inhibitor. Proteins forming complex C2 act
as activators in the context of the tk promoter and function as
inhibitors in the context of the LTR promoter. These data stress
both the promoter-dependent activity and the antagonistic effects
of the site A binding proteins.
Analysis and purification of a protein present in complex C2
To estimate the apparent molecular mass of the protein(s) present
in the major complex C2, we performed UV cross-linking
experiments. Gel shift assays were carried out with bromodeoxy-
uridine-substituted 32P-labeled 1L probe in the presence of either
crude nuclear extract from Jurkat cells or with partially purified
proteins eluted from a heparin–Sepharose column. After electro-
phoresis, the native gel was UV irradiated and the band
corresponding to complex C2 was excised and subjected to
SDS–10% PAGE. As shown in Figure 6, two oligonucleotide–
protein complexes were detected with apparent molecular masses
of 67 and 60 kDa. As a control, similar experiments performed
with the C4 complex revealed a unique band migrating in the 80 kDa
range (results not shown). These results suggest that the C2
complex is composed of two proteins, with apparent molecular
masses of ∼43 (± 5) and 50 (± 5) kDa, while the C4 complex is
formed of one protein of ∼60–66 kDa.
The molecular mass of the protein(s) able to specifically bind
to the 1L sequence was further confirmed with the Southwestern
technique. This experiment was carried out with crude nuclear
extracts and with partially purified proteins eluted at 0.4 M KCl
from a heparin–Sepharose column (Fig. 7). When the renatured
Figure 6. UV cross-linking analysis of Jurkat nuclear proteins forming
complex C2. 10 µg Jurkat crude nuclear extract (CE) and 8 µg nuclear proteins
eluted at 0.4 M KCl from a heparin–Sepharose column (HS) were incubated
with a 32P-labeled bromodeoxyuridine-substituted 1L probe. After separation
on a 6% polyacrylamide gel and UV cross-linking, complex C2 was excised
and analyzed on a SDS–10% polyacrylamide gel. Arrows indicate the two
oligonucleotide–protein complexes of 67 and 60 kDa. Since the molecular mass
of oligonucleotide 1L alone is 17 kDa, the molecular mass of the two proteins
forming complex C2 is estimated as 50 (± 5) and 43 (± 5) kDa.
Figure 7. Southwestern blots of partially purified Jurkat proteins binding to site
A of the LTR. Aliquots (20 µg) of Jurkat proteins eluted from a heparin–Sepharose
column were analyzed by Southwestern blotting as described in Materials and
Methods. The blot was probed with 32P-labeled oligonucleotides 1L or 1Lm,
indicated on top. The labeled proteins were visualized by autoradiography.
Molecular weight markers are shown on the right. The left arrow indicates the
43 kDa protein.
proteins were probed with radioactively labeled oligonucleotide
1L, three major protein bands of 43, 62 and 66 kDa were detected.
In contrast, when we used the 1Lm probe, the 43 kDa protein was
poorly detected, while the 62 and 66 kDa proteins were labeled
with a similar intensity as with the 1L probe. A control TRE probe
failed to bind to any of the three major species (results not shown).
These data confirm that the 43 kDa protein is likely to correspond
to one of the proteins forming complex C2. They also suggest that
the 50 kDa protein is not easily renatured. Since complex C4 is
detected with both the 1L and 1Lm probes in gel shift assays
(Fig. 1), the 62 or the 66 kDa species may correspond to the
protein forming complex C4.
In order to further characterize the proteins forming complex
C2, we developed the following purification strategy. Throughout
the purification procedure, 1L binding activity was monitored by
gel shift assay (Fig. 8A). Crude nuclear extract from Jurkat cells
was first loaded onto a heparin–Sepharose column and proteins
were eluted with a step gradient of 0.1 to 1.0 M KCl in buffer Z.
Complexes C2 and C4 were detected in fractions eluted at 0.4 M
KCl. The pooled fractions were loaded onto a DNA affinity
column containing multimers of oligonucleotide 1Lm, to remove
most of the proteins forming complex C4. The flow-through
fraction, which contained the proteins involved in formation of