MOLECULAR AND CELLULAR BIOLOGY, JUlY 1992, p. 3183-3191 0270-7306/92/073183-09$02.00/0 Copyright ©D 1992, American Society for Microbiology
Vol. 12, No. 7
Transcriptional Regulation of the Apolipoprotein B100 Gene: Purification and Characterization of trans-Acting Factor BRF-2 HONGMING ZHUANG,' SAMUEL S. CHUANG,' AND HRIDAY K. DASl12* Department of Microbiology and Immunology, 1 and Department of Phannacology, 2* The Health Science Center, University of Tennessee-Memphis, Memphis, Tennessee 38163 Received 4 November 1991/Accepted 16 April 1992
Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions -128 and -70 (H. K. Das, T. Leff, and J. L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence - 128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.
distal positive element (-128 to -85) produces a fivefold increase in positive activity in HepG2 cells in a transient transfection assay (8). Recently, a rat liver nuclear factor, NF-BA1, with a molecular mass of 60 kDa, has been purified to apparent homogeneity; this factor binds to the apoB sequence from -79 to -63 within the proximal positive element and transactivates transcription in an in vitro transcription system (20). We have also purified another rat liver nuclear protein, BRF-1, which binds to the NF-BA1 cognate binding site (32a). The molecular mass of BRF-1 has been determined to be 68 kDa, and this protein appears to be different from NF-BA1. With a long-term objective to study the regulation of apoB gene transcription in detail, we undertook the task of purifying and characterizing protein factors which interact with both the distal (-128 to -85) and the proximal (-84 to -70) elements. In this paper, we report the purification and characterization of the apoB gene regulatory factor-2 (BRF2), which binds to the distal positive element (-128 to -85). The factor has been purified to apparent homogeneity from rat liver nuclei by using DNA affinity chromatography. BRF-2, whose molecular mass was determined to be 120 kDa, was found to interact with the apoB promoter sequence surrounding the hypersensitive site -95 as detected by copper-orthophenanthroline (Cu-OP) footprinting. A mutant double-stranded oligonucleotide, containing a trinucleotide (1TC) insertion between position -95 and -94 in the BRF-2 binding site, failed to interact with BRF-2, suggesting that the region surrounding the hypersensitive site -95 is involved in protein-DNA interaction. Using apoB promoter fragments containing various internal deletions as templates
Apolipoprotein B100 (apoB), the only protein component of low-density lipoprotein (LDL), is synthesised primarily in the liver and acts as a ligand for the LDL receptor to mediate the removal of LDL from plasma (11, 12). Elevated levels of plasma LDL cholesterol and apoB have been found to be associated with increased risk of coronary artery disease (18). On the other hand, moderately low levels of LDL cholesterol and apoB in plasma are associated with reduced risk of atherosclerosis (34). Mutations that lower the production of apoB decrease plasma levels of LDL cholesterol (5, 15, 36-38); mutations in the regulatory regions that result in decreased apoB gene transcription will diminish the plasma LDL cholesterol level. Thus, the apoB gene plays a crucial role in determining plasma LDL cholesterol levels. To better understand the regulation of apoB gene expression, a number of recent investigators have begun defining the cis-acting elements on the apoB promoter and the putative trans-acting factors involved in the transcriptional regulation of the apoB gene (2-4, 8, 19, 30). Hepatic cellspecific expression of the apoB gene is regulated by at least two positive cis-acting elements located from -128 to -70 (8); these elements are bound by several trans-acting protein factors (3, 4, 8, 19, 30). The DNA sequence from positions -84 to -70 has a 10-fold positive effect in HepG2 cells and is indispensable for liver-specific expression of the apoB gene (8). The proximal element and downstream sequences adjacent to it also bind to several rat liver nuclear proteins, including a heat-stable nuclear factor, C/EBP (19, 30). The *
Corresponding author. 3183
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TABLE 1. Purification of BRF-2 from rat liver'
BRF-2
I
Fraction
Hen -r--la G2
-261
-128
-84 -70 -60
-50
+1
-116
HeLa FIG. 1. Schematic diagram showing the regulatory regions of the human apoB gene active in HepG2 and HeLa cells. Open boxes delineate cis-acting sequences active in HepG2 cells; stippled boxes represent elements active in HeLa cells. + and -, positive and negative cis-acting elements, respectively, and their relative strength. Binding sites for apoB gene regulatory factors BRF-1, BRF-2, and C/EBP identified by gel mobility shift assay are marked.
for gel mobility shift assays, we found that the region -104 to -85 was important for interaction with BRF-2. This observation is supported by the earlier report which showed that mutations in the regions -105 to -100 and -96 to -91 reduced hepatic cell-specific expression of the apoB gene by fivefold in a transfection assay (19).
MATERLALS AND METHODS Materials. Heparin-agarose, phenylmethylsulfonyl fluoride, and dithiothreitol were purchased from Sigma. Cyanogen bromide-activated Sepharose 4B was purchased from Pharmacia. DEAE-cellulose was purchased from Collaborative Research. Restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were purchased from New England Biolabs. [.y-32P]ATP was obtained from DuPont. Centricon100 and silver stain kits were purchased from Amicon and BioRad, respectively. Purification of apoB gene regulatory factor-2 (BRF-2): nuclear extract preparation. Nuclear extracts from HeLa cells were prepared by the method of Dignam et al. (9). Nuclear extracts from rat livers were prepared by the procedure of Gorsky et al. (13) except that nuclei were lysed with nuclear lysis buffer containing 0.4 M KCI. Nuclear extract (260 ml; 940 mg of protein) was dialyzed against two changes of dialysis buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 7.9], 5 mM MgCl2, 100 mM KCI, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol). Step 1: DEAE-cellulose chromatography. The dialyzed nuclear extracts were applied to a DEAE-cellulose column (150 ml) equilibriated in buffer M containing 100 mM KCI. Buffer M contained 10 mM Tris (pH 7.9), 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 20% glycerol. After the nuclear extracts were absorbed in the column, the column was washed with three volumes of buffer M containing 100 mM KCI and was eluted with buffer M containing 0.5 M KCI. Fractions (10 ml) were collected, and the BRF-2 activity of each fraction was determined by DNA-binding gel mobility shift assay. Protein concentration of active fractions was measured by the colorometric method using a BioRad protein assay. Step 2: heparin-agarose chromatography. Active fractions (75 ml; total protein, 297 mg) from DEAE-cellulose chromatography were pooled and dialyzed against two changes of buffer M containing 50 mM KCI and loaded onto a column of
Protein
Vol
(mg)
(ml)
Activity (U)b
Sp act (U/mg)
Purifi(fold)
Re-
(%)
260 Nuclear extract 940.8 DEAE-cellulose 296.7 75 11,250 37.9 100 Heparin-agarose 41.6 30 10,500 252.4 6.66 93 Third oligo-affinity 0.0006 2.5 1,180 1,966,667 51,890 10.4 Centricon-100 0.0005 0.1 92 184,000 4,854 0.8 a One hundred rat livers (670 g) were used. b One unit is defined as the amount of protein bound to 0.1 ng of end-labeled F44 probe.
heparin-agarose (50 ml) equilibrated with buffer M containing 50 mM KCl. The column was washed with 3 volumes of buffer M containing 0.2 M KCl and eluted stepwise with buffer M containing 0.3, 0.6, and 1 M KCl. Fractions of 6 ml were collected and were analyzed for BRF-2 activity by gel mobility shift assay. BRF-2 activity was eluted at 0.3 M KCl. (Note: with brand-new heparin-agarose, BRF-2 binding may be stronger and thus BRF-2 activity can be eluted by using slightly higher salt concentrations). Active heparin-agarose fractions were pooled together and dialyzed against buffer M containing 50 mM KCl. Step 3: DNA sequence-specific affinity chromatography. Two synthetic complementary oligonucleotide strands corresponding to the apoB promoter sequence (-128 to -85) were annealed and polymerized to form oligomers (17): 5'-gatcGGCTCAAAGAGAAGCCAGTGTAGAAAAGCAAACAGGTCAGGCCC-3'
3'-OCGAGTTTCTCTTCGOTCACATCTTTTCGTTTOTCCAGTCCGGGoctag-5'
The oligomers (1 mg) were coupled to 3 g of cyanogen bromide-activated Sepharose 4B as described previously (17). Dialyzed heparin-agarose fractions (30 ml; 42 mg of protein) were incubated with 10 ,ug of sonicated calf thymus (CT) DNA per ml, 4 mM MgCl2, and 0.1% Nonidet P-40 in ice for 1 h. The incubated mixture was then loaded onto a 15-ml DNA-specific affinity column equilibrated in buffer M containing 50 mM of KCl, and the flowthrough fractions were recycled two more times through the affinity column. After the column was washed with 5 volumes of buffer M containing 125 mM KCl, 4 mM MgCl2, and 5 ,ug of CT DNA per ml, BRF-2 was eluted with a step gradient of 0.4 M and 1 M KCl in buffer M. Fractions of 1.5 ml were collected. BRF-2 activity was eluted at 0.4 M KCl. The BRF-2containing fractions were pooled, dialyzed to a final KCl concentration of 50 mM, and incubated with 5 ,g of CT DNA per ml and 4 mM MgCl2 for 1 h. The incubated mixture was then applied to a DNA-specific affinity column (7 ml). After the column was washed as described above, BRF-2 activity was eluted again at 0.4 M KCl. The BRF-2 factor was purified further by an additional cycle of DNA-specific affinity chromatography (3 ml). Fractions of 0.5 ml were collected. The purified protein was concentrated by centricon-100, frozen in small aliquots in liquid nitrogen, and stored at -80°C. Synthetic oligonucleotides. Oligonucleotides were synthesized by an ABI#380B DNA synthesizer and purified electrophoretically in a 12% acrylamide-7 M urea gel and lx TBE buffer (0.089 Tris borate, 0.089 M boric acid, and 0.002 M EDTA). Oligonucleotides were recovered from the gel by eluting with 0.1 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) followed by ethanol precipitation. Oligonucleotides were annealed (17). Double-stranded oligonucleo-
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TABLE 2. Oligonucleotides and apoB genomic fragments used in this study Fragment
Gene
HNF-1/LF-B1 HNF-2/LF-A1 HNF-3
Rat alpha 1 AT Human alpha 1 AT Mouse TTR Mouse TTR Simian virus 40 Human apoB Human apoB Human apoB Human Alb2
HNF-4 C/EBP BRF-1 BRF-2
BRF-21 NF1 AP1 F138 F128 F84 F44 F152C F152D F152E
Human apoB Human apoB Human apoB Human apoB Human apoB Human apoB Human apoB
Positiona
Reference
5'gatcGGCTCAAAGAGAAGCCAGTGTAGAAAAGCAAACAGGTCAGGCCC3' 5'GGCTCAAAGAGAAGCCAGTGTAGAAAAGCAAACAttcGGTCAGGCCC3' 5'gggAGTCAAACAATTTTTTGGCAAGAATATTATGAATCCC3'
-104 to -75 -125 to -96 -109 to -80 -151 to -130 +260 to +230 -84 to -60 -128 to -85 -128 to -85 -140 to -104
5'gatcGTGACTCAGCGCG3' EcoRI-XbaI SacI-XbaI SacI-XbaI EcoRI-SmaI (BRF-2 binding site) SacI-XbaI (containing -85 to -63 internal deletion) SacI-XbaI (containing -113 to -56 internal deletion) SacI-XbaI (containing -104 to -4 internal deletion)
-138 to +122 -128 to +122 -84 to +122 -128 to -85 -152 to +122 -152 to +112 -152 to +112
7 26 6 6 16 8 8 8 31 25 8 8 8 8 8
Sequence
5'gatcACCAAACTGTCAAATATTAACTAAAGGGAG3' 5'gatcATCCCAGCCAGTGGACTTAGCCCCTGTTTG3' 5'gatcTGACTAAGTCAATAATCAGAATCAGCAGGT3' 5'tcgaGGCAAGGTTCATATTTGTGTAG3' 5'gatcTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCT3' 5'gatcGGGAGGCGCCCTTTGGACCTTTTGC3'
a The location of the viral sequences is relative to the numbering system of the viral genome. Locations of the apoB, to the start of transcription.
tides and apoB promoter fragments were labeled at the 5' end by [y-_ P]ATP and polynucleotide kinase. Gel mobility shift assay. Nuclear protein-DNA binding reactions were carried out in a volume of 10 ,l containing 60 mM KCl, 20 mM HEPES (pH 7.9), 5% glycerol, and 4 mM MgCl2. Sonicated CT DNA (1,250 ng) was added to act as a nonspecific competitor. A typical reaction contained 10,000 to 20,000 cpm (approximately 0.5 ng) of 5'-labeled DNA and protein as indicated. After the addition of protein, samples were incubated at room temperature for 30 min and analyzed by electrophoresis on nondenaturing 4% polyacrylamide gels in 0.25 x TBE buffer as described before (8). For competition experiments, conditions were as described above except that the appropriate competitor DNA (as indicated) was added to the reaction mixture prior to addition of protein. Cu-OP footprint assay. The coding strand of the apoB promoter fragment F138 (-138 to + 122) was 5'-end labeled with [.y-32P]ATP and used as a template for footprinting. Fifty nanograms (approximately 100,000 cpm) of the fragment was incubated with 10 ,ul of affinity-purified BRF-2 in a total volume of 100 ,ul at room temperature for 30 min under the conditions used for the gel mobility shift assay. Free DNA fragment was separated from DNA-protein complex by gel electrophoresis as described before. After gel electrophoresis, the gel was soaked with 200 ml of 50 mM Tris (pH 8.0) and 20 ml of Cu-OP (0.45 mM CUSO4 2 mM OP), and 20 ml of 58 mM 3-mercaptopropionic acid was added. The digestion was carried out for 10 min at room temperature followed by quenching for 2 min with 20 ml of 28 mM 2,9 dimethyl OP solution as described previously (21). The gel was washed several times with double-deionized water. The wet gel was then exposed with a X-ray film overnight at 4°C. The autoradiogram was developed, and free and bound bands were excised from the gel. DNAs from the free and bound bands were extracted by incubating gel slices with 0.5 M ammonium acetate and 1 mM EDTA at 37°C overnight. Eluted DNAs were then precipitated with three volumes of ethanol, resuspended in sequencing dye, and analyzed on a 10% polyacrylamide-7 M urea gel. A G+A ladder, generated by sequencing (28) the same end-labeled fragment, was run in the same sequencing gel. Recovery of BRF-2 from native polyacrylamide gel. Affini-
alpha-i-AT, alb2, and TTR are relative
ty-purified BRF-2 was electrophoretically separated in a 8.5% native polyacrylamide gel at 4°C and visualized by silver stain. Protein corresponding to the position of the stained band was eluted from the parallel unstained gel slices by electroelution with gel running buffer in a dialysis tube at -20°C for 4 h. Eluted protein was concentrated by centricon-100. RESULTS We have previously shown that the DNA element (-128 to -85) produced fivefold-greater expression of the reporter CAT construct driven by the apoB promoter sequence (-84 to + 122) in HepG2 cells but showed negative activity in HeLa cells (8). Kardassis et al. showed that the element (-128 to -85) produced two DNase I footprints with rat liver nuclear extracts. Mutations in this region also reduced the level of apoB gene transcription in transfection experiments in HepG2 cells (19). Figure 1 shows the human apoB cis-acting regions in HepG2 and in HeLa cells and the binding sites of factors which have been identified by us to interact with these regions. To understand the function of the apoB gene regulatory factor BRF-2 in the regulation of apoB gene transcription, we have purified this factor from rat liver nuclear extracts. The purification steps are summarized in Table 1. The activity of BRF-2 was monitored throughout the purification process by gel mobility shift assay using a 44-bp apoB promoter fragment (F44) between nucleotides -128 and -85, generated by EcoRI and SmaI digestion of plasmid pKT-128B (8). Table 2 shows different apoB genomic fragments and double-stranded oligonucleotides used for the purification and characterization of the BRF-2 protein. Purification of BRF-2 protein from rat liver. Nuclear extracts were first applied to an anion-exchange DEAE-cellulose column at 100 mM KCI, and bound proteins were eluted with 0.5 M KCI in buffer M. The active fractions eluted from the DEAE-cellulose column were pooled and chromatographed on a heparin-agarose column. The activity was eluted from the heparin-agarose column at 0.3 M KCl (Fig. 2A), representing more than 93% of the total activity. The final purification step involved a sequence-specific
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l-
b
10
20
30
Fraction Number
B
op
W
FIG. 2. Purification of BRF-2 by heparin-agarose and DNAspecific affinity chromatography. (A) Elution profile of proteins fractionated on heparin-agarose column. Conditions used for fractionation are described in Materials and Methods. Fractions were analyzed for BRF-2 activity by DNA binding and gel mobility shift assays. One microliter of each fraction was used for the assay. An autoradiogram corresponding to the active fractions is shown in the inset. (B) Sequence-specific affinity chromatography. The active fractions from the heparin-agarose column were pooled and incubated with the DNA affinity resins as described in Materials and Methods. The apoB genomic fragment containing sequences from -128 to -85 (F44) was used for the DNA binding assay. A 1.5-pl portion of each fraction eluted was assayed for BRF-2 binding activity. The top band labeled as BRF-2 indicates the protein-DNA complex, and the bottom band indicated by F44 represents the free probe. The first lane contains the pooled BRF-2 active fractions from the second cycle of DNA-specific affinity chromatography. FT., flowthrough from the third oligo affinity column. W, wash fraction of the third oligo affinity column with buffer M containing 0.125 M KCl. Lanes 1 to 14, fraction numbers eluted from the third oligo affinity column with buffer M containing 0.4 M KCl.
affinity column. A double-stranded oligonucleotide containing apoB promoter sequence -128 to -85 was ligated to produce multimers which were covalently ligated to cyanogen bromide-activated Sepharose 4B beads. The 0.3 M KCl fractions from heparin-agarose were dialyzed with buffer M containing 50 mM KCl and chromatographed on the oligonucleotide affinity column. The BRF-2 activity was eluted at 0.4 M KCl. The BRF-2 factor was purified further by two additional cycles of DNA-specific affinity chromatography. Figure 2B shows the protein elution profile from the third DNA-specific affinity column as monitored by gel mobility shift assay. Table 1 summarizes the purification of BRF-1 from rat liver nuclear extracts. The overall purification and
the yield of the specific DNA binding activity were approximately 52,000 fold and 10.4%, respectively. The BRF-2 binding activity is expressed in units, where one unit is defined as the amount of protein bound to 0.1 ng of F44 probe. The affinity-purified BRF-2 was concentrated by centricon-100 and stored at -80°C. During concentration for several hours, the recovery of BRF-2 activity was found to have diminished by 10- to 12-fold. The concentrated protein is active for approximately a week if stored in ice at -20°C. Identification of the BRF-2 factor as a 120-kDa protein. A sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis of the pools of BRF-2 binding activity obtained by heparinagarose and third oligo affinity column chromatography is shown in Fig. 3A. Significant degrees of purification were achieved by three cycles of DNA-specific affinity chromatography. Only one major protein band corresponding to a molecular mass of 120 kDa was detected by silver stain after three cycles of DNA-specific affinity chromatography (lane 2). To verify that the 120-kDa band is indeed BRF-2, we performed a preparative native polyacrylamide gel electrophoresis (PAGE) of affinity-purified BRF-2 and visualized it with a silver stain; a single polypeptide band was detected (Fig. 3B). Protein corresponding to the position of the stained band was eluted from the parallel unstained gel slices by electroelution at -20°C and concentrated by centricon100. The concentrated protein was then subjected to SDSPAGE, and a single band corresponding to a molecular mass of 120 kDa was visualized with a silver stain (Fig. 3C). This molecular mass is in agreement with gel filtration data obtained with affinity-purified BRF-2 (data not shown). Protein eluted from the native polyacrylamide gel was assayed with F44 and F128 probes (Table 2) in a gel mobility shift experiment. As shown in Fig. 3D, eluted 120-kDa protein produced a mobility shift at the position corresponding to affinity-purified BRF-2 (lanes 2, 3, 4, and 5). Cu-OP footprint analysis with affinity-purified BRF-2 protein. We have reported previously (8) that we could not detect DNase I footprints with HeLa or HepG2 cells or rat liver nuclear extracts in the regulatory region (-128 to -85) of the apoB gene. We also failed to detect a DNase I footprint with affinity-purified BRF-2 protein, suggesting that BRF-2 binds with low affinity with the element (- 128 to -85). To circumvent the problem associated with DNase I footprinting, we used a chemical footprinting technique with Cu-OP to define BRF-2 protein-DNA interaction. The DNA fragment from - 138 to + 122 (F138) was 5'-end labeled at the coding strand and used as a template for footprint analysis. After digestion of DNA in the gel matrix by Cu-OP, DNA corresponding to free and bound complex was eluted from the gel. DNA was then subjected to electrophoresis in a sequencing gel, and an autoradiogram was prepared. As seen in Fig. 4A, purified BRF-2 failed to produce a detectable footprint. However, we repeatedly observed a strong hypersensitive site compared with naked DNA at position -95, indicating that the region surrounding the -95 nucleotide position could be the binding site for BRF-2. Because of the high dissociation constant of DNA-BRF-2 complex, we failed to detect the footprint in this region. To ascertain that the region surrounding the nucleotide position -95 was the bona fide protein binding site for BRF-2, we introduced a trinucleotide (TTC) in the position between -95 and -94 of the element (-128 to -85). We used the double-stranded oligonucleotide as a template in a gel mobility shift assay. As shown in Fig. 4B, the wild-type oligonucleotide (-128 to -85) produced a strong protein-DNA complex with purified
PURIFICATION AND CHARACTERIZATION OF BRF-2
VOL. 12, 1992 M
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FIG. 3. Analysis of purification of BRF-2 by SDS and native PAGE. (A) SDS-8.5% PAGE of BRF-2 active fractions obtained by heparin-agarose and DNA-specific affinity chromatography. Lane M indicates protein molecular mass markers as follows: ovalbumin, 46 kDa; bovine serum albumin, 68 kDa; ,B-galactosidase, 116 kDa; and myosin, 205 kDa. Lanes 1 and 2 show the profile of proteins present in pooled active heparin-agarose fractions and pooled fractions eluted from the third oligo affinity column with buffer M containing 0.4 M KCI and concentrated by centricon-100, respectively. (B) Native 8.5% PAGE of BRF-2 fractions obtained by third oligo affinity column chromatography and concentrated by centricon-100. (C) SDS-8.5% PAGE of BRF-2 recovered from native polyacrylamide gel matrix by electroelution. Lanes indicated by M represent protein molecular mass markers as follows: ovalbumin, 46 kDa; bovine serum albumin, 68 kDa; phosphorylase b, 97 kDa; p-galactosidase, 116 kDa. Lane 1 shows the profile of protein present in BRF-2 recovered from the native polyacrylamide gel matrix. (D) Gel mobility shift assays of BRF-2 recovered from the native polyacrylamide gel matrix. apoB genomic fragments F44 and F128 were used as templates in this assay. BRF-2 indicates the position of protein-DNA complex. F44 and F128 indicate the positions of the two free templates used in this assay. Lane 1 contains 1 pl of BRF-2 purified on the second oligo affinity column. Lanes 2 and 4 contain 1 p,l of BRF-2 recovered from the native polyacrylamide gel; lanes 3 and 5 contain 2 ,ul of BRF-2 recovered from the native polyacrylamide gel matrix.
BRF-2, but the mutant template failed to exhibit proteinDNA interaction. apoB promoter region -104 to -85 is important for BRF-2 binding. To localize the binding site of BRF-2, a series of labeled DNA fragments missing specific regions between -128 and + 122 were used in a gel mobility shift assay. The radiolabeled fragments -128 to -85 (F44) and -128 to + 122 (F128) and a radiolabeled fragment prepared from F152C (containing a -85 to -63 internal deletion) bound to BRF-2. When radiolabeled fragments prepared from F152D (containing a -113 to -56 internal deletion) and from F152E (containing a -104 to -4 internal deletion) were used in a gel mobility shift assay, no protein-DNA complex appeared (Fig. SA). These data suggest that the binding site of BRF-2 is not located between -128 and -105. Therefore, the region spanning nucleotides -104 to -85 is important for BRF-2 binding. The binding of BRF-2 with different apoB promoter mutants is summarized in Fig. SB. Competition gel mobility shift assays with other DNA binding sites. Several liver-enriched factors (e.g., HNF-1, HNF-2, HNF-3, HNF-4, C/EBP, NF-1, and Ap-1) interact with cis-acting elements of a number of liver-specific genes and regulate their expression (1, 6, 7, 10, 14, 16, 23, 24, 26, 27, 31-33, 35). Double-stranded oligonucleotides containing cognate binding sites of several such factors were used as
competitors in a gel mobility shift assay with affinity-purified BRF-2. As shown in Fig. 6, bound complex could only be competed for efficiently by the BRF-2 binding site, indicating that the BRF-2 transcription factor is unique. BRF-2 was found to be heat sensitive and lost DNA binding when heated at 65°C for 10 min (data not shown). Presence of BRF-2-related activity in HeLa cells. We have reported previously that the distal element (-128 to -85) of the apoB promoter acts as a positive element in hepatic cells and a negative element in HeLa cells (8). Therefore, it was of interest to test whether an F44 template could detect the presence of trans-acting factor(s) in HeLa cell extracts. As shown in Fig. 7, the retarded bands had different migrations. It is conceivable that the putative factor(s) is either a posttranslationally modified homolog of BRF-2 or a completely different factor. DISCUSSION We showed previously that hepatic cell-specific expression of the human apoB gene is determined by two cis-acting elements located between -128 and -70 (8). The proximal element (-84 to -70) acts as a strong positive element in HepG2 cells and as a mildly positive element in HeLa cells; this element appears to be absolutely necessary for liver-
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FIG. 4. Cu-OP footprint analysis of BRF-2 binding to the apoB promoter. (A) A fragment of the apoB promoter (50 ng; 100,000 cpm) extending from -138 to + 122 was radiolabeled at the 5' end of the coding strand and was incubated in the presence and absence of third oligo affinity column-purified BRF-2 and subjected to electrophoresis followed by the treatment with Cu-OP reagent described in Materials and Methods. DNAs corresponding to the bound and free bands were eluted from the gel matrix, precipitated with ethanol, and subjected to gel electrophoresis on a 10% sequencing gel. A sequencing reaction (G+A) of the same labeled fragment was run as a marker. Numbering begins at the transcription initiation site (+ 1). Lane 1 contains free DNA fragments recovered from the mobility shift gel. Lane 2 contains DNA fragments recovered from the mobility shift gel corresponding to the protein-DNA complex. Arrow indicates the hypersensitive site at nucleotide position -95 present in lane 2. (B) Gel mobility shift assay of third oligo affinity column-purified BRF-2 with wild-type and mutant apoB oligo templates. Labeled apoB templates (1 ng) were either wild-type BRF-2 oligo (-128 to -85) in lane 1 or mutant oligo containing a trinucleotide (TTC) insertion at position between -95 and -94 (BRF-21) in lane 2. These apoB oligos have blunt ends and were labeled only at the 5' end of the coding strand. One microliter of third oligo affinity column-purified BRF-2 was used in each lane. F, free labeled oligo; BRF-2, position of the protein-DNA complex.
specific expression of the apoB gene (8). Addition of the distal element (-128 to -85) produced fivefold-greater activity over the level of CAT construct containing the proximal positive element (-84 to -70) in HepG2 cells but had a negative effect in HeLa cells (8). We also showed that a protein factor(s) from HepG2 cell and mouse liver nuclear extracts binds to the proximal element (8). The findings described above were confirmed recently by two groups of investigators (3, 4, 19). Metzger et al. have shown that two transcription factors, AF-1 and C/EBP, bind to overlapping sites (-86 to -61 and -69 to -52, respectively) of the apoB promoter (30). Kardassis et al. have purified a rat liver nuclear factor, NF-BA1, which interacts with the apoB promoter sequence (-79 to -63) as well as with other
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FIG. 5. Gel mobility shift assay using wild-type and mutated apoB promoter fragments. (A) Radiolabeled apoB promoter fragments F44 (-128 to -85), F128 (-128 to +122), F152C, F152D, and F152E (1 ng= 12,000 cpm) were used as templates to detect the binding of BRF-2. One microliter of third oligo affinity columnpurified BRF-2 was used in each binding assay. F, free labeled templates; BRF-2, position of protein-DNA complex. (B) Schematic diagram showing the different apoB promoter fragments used in Fig. 5A and a summary of BRF-2 binding with different apoB promoter mutants. Mutations were produced by deletion of sequences from -85 to -63 (F152C), from -113 to -56 (F152D), and from -104 to -4 (F152E). The positions of nucleotides at the endpoints of each fragment are also marked. + and - symbols indicate the presence and absence of BRF-2-DNA complex, respectively.
related sequences present in apoAI, apoAII, and apoCIII genes, indicating the involvement of NF-BA1 in other apolipoprotein gene regulation (20). These NF-BA1- and AF-1like activities are also found to be present in HeLa and CaCo2 cells (20, 30). Genes encoding hepatocyte nuclear factors HNF-1, HNF-2, HNF-3, and HNF-4, which regulate the transcription of other liver-specific genes, have been cloned for structure-function analysis (10, 23, 24, 29, 35). HNF-2 and HNF-4 are identical proteins having a molecular mass of 54 kDa and also bind to the apoB sequence (-86 to -61) (35). Ladias and Karathanasis have cloned a gene encoding an apoAI gene regulatory factor, ARP-1, which also binds to the apoB proximal element (-84 to -60) but negatively regulates apoAI and apoCIII sites (22). We have also purified a protein factor, BRF-1, from rat liver nuclei which binds to the apoB proximal element and appears to have a molecular mass different from those of NF-BA1, ARP-1, and HNF-2 and HNF-4 (32a). Therefore, a clear picture is emerging from these studies that a group of closely related proteins can bind to the apoB proximal element (-84 to -60) and related sequences present in other apolipoprotein and liver-specific genes. Selective gene expression may be governed by multiple promoter-dependent protein-protein inter-
PURIFICATION AND CHARACTERIZATION OF BRF-2
VOL. 12, 1992 N4
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Vol. 12, No. 7
Transcriptional Regulation of the Apolipoprotein B100 Gene: Purification and Characterization of trans-Acting Factor BRF-2 HONGMING ZHUANG,' SAMUEL S. CHUANG,' AND HRIDAY K. DASl12* Department of Microbiology and Immunology, 1 and Department of Phannacology, 2* The Health Science Center, University of Tennessee-Memphis, Memphis, Tennessee 38163 Received 4 November 1991/Accepted 16 April 1992
Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions -128 and -70 (H. K. Das, T. Leff, and J. L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence - 128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.
distal positive element (-128 to -85) produces a fivefold increase in positive activity in HepG2 cells in a transient transfection assay (8). Recently, a rat liver nuclear factor, NF-BA1, with a molecular mass of 60 kDa, has been purified to apparent homogeneity; this factor binds to the apoB sequence from -79 to -63 within the proximal positive element and transactivates transcription in an in vitro transcription system (20). We have also purified another rat liver nuclear protein, BRF-1, which binds to the NF-BA1 cognate binding site (32a). The molecular mass of BRF-1 has been determined to be 68 kDa, and this protein appears to be different from NF-BA1. With a long-term objective to study the regulation of apoB gene transcription in detail, we undertook the task of purifying and characterizing protein factors which interact with both the distal (-128 to -85) and the proximal (-84 to -70) elements. In this paper, we report the purification and characterization of the apoB gene regulatory factor-2 (BRF2), which binds to the distal positive element (-128 to -85). The factor has been purified to apparent homogeneity from rat liver nuclei by using DNA affinity chromatography. BRF-2, whose molecular mass was determined to be 120 kDa, was found to interact with the apoB promoter sequence surrounding the hypersensitive site -95 as detected by copper-orthophenanthroline (Cu-OP) footprinting. A mutant double-stranded oligonucleotide, containing a trinucleotide (1TC) insertion between position -95 and -94 in the BRF-2 binding site, failed to interact with BRF-2, suggesting that the region surrounding the hypersensitive site -95 is involved in protein-DNA interaction. Using apoB promoter fragments containing various internal deletions as templates
Apolipoprotein B100 (apoB), the only protein component of low-density lipoprotein (LDL), is synthesised primarily in the liver and acts as a ligand for the LDL receptor to mediate the removal of LDL from plasma (11, 12). Elevated levels of plasma LDL cholesterol and apoB have been found to be associated with increased risk of coronary artery disease (18). On the other hand, moderately low levels of LDL cholesterol and apoB in plasma are associated with reduced risk of atherosclerosis (34). Mutations that lower the production of apoB decrease plasma levels of LDL cholesterol (5, 15, 36-38); mutations in the regulatory regions that result in decreased apoB gene transcription will diminish the plasma LDL cholesterol level. Thus, the apoB gene plays a crucial role in determining plasma LDL cholesterol levels. To better understand the regulation of apoB gene expression, a number of recent investigators have begun defining the cis-acting elements on the apoB promoter and the putative trans-acting factors involved in the transcriptional regulation of the apoB gene (2-4, 8, 19, 30). Hepatic cellspecific expression of the apoB gene is regulated by at least two positive cis-acting elements located from -128 to -70 (8); these elements are bound by several trans-acting protein factors (3, 4, 8, 19, 30). The DNA sequence from positions -84 to -70 has a 10-fold positive effect in HepG2 cells and is indispensable for liver-specific expression of the apoB gene (8). The proximal element and downstream sequences adjacent to it also bind to several rat liver nuclear proteins, including a heat-stable nuclear factor, C/EBP (19, 30). The *
Corresponding author. 3183
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TABLE 1. Purification of BRF-2 from rat liver'
BRF-2
I
Fraction
Hen -r--la G2
-261
-128
-84 -70 -60
-50
+1
-116
HeLa FIG. 1. Schematic diagram showing the regulatory regions of the human apoB gene active in HepG2 and HeLa cells. Open boxes delineate cis-acting sequences active in HepG2 cells; stippled boxes represent elements active in HeLa cells. + and -, positive and negative cis-acting elements, respectively, and their relative strength. Binding sites for apoB gene regulatory factors BRF-1, BRF-2, and C/EBP identified by gel mobility shift assay are marked.
for gel mobility shift assays, we found that the region -104 to -85 was important for interaction with BRF-2. This observation is supported by the earlier report which showed that mutations in the regions -105 to -100 and -96 to -91 reduced hepatic cell-specific expression of the apoB gene by fivefold in a transfection assay (19).
MATERLALS AND METHODS Materials. Heparin-agarose, phenylmethylsulfonyl fluoride, and dithiothreitol were purchased from Sigma. Cyanogen bromide-activated Sepharose 4B was purchased from Pharmacia. DEAE-cellulose was purchased from Collaborative Research. Restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were purchased from New England Biolabs. [.y-32P]ATP was obtained from DuPont. Centricon100 and silver stain kits were purchased from Amicon and BioRad, respectively. Purification of apoB gene regulatory factor-2 (BRF-2): nuclear extract preparation. Nuclear extracts from HeLa cells were prepared by the method of Dignam et al. (9). Nuclear extracts from rat livers were prepared by the procedure of Gorsky et al. (13) except that nuclei were lysed with nuclear lysis buffer containing 0.4 M KCI. Nuclear extract (260 ml; 940 mg of protein) was dialyzed against two changes of dialysis buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 7.9], 5 mM MgCl2, 100 mM KCI, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol). Step 1: DEAE-cellulose chromatography. The dialyzed nuclear extracts were applied to a DEAE-cellulose column (150 ml) equilibriated in buffer M containing 100 mM KCI. Buffer M contained 10 mM Tris (pH 7.9), 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 20% glycerol. After the nuclear extracts were absorbed in the column, the column was washed with three volumes of buffer M containing 100 mM KCI and was eluted with buffer M containing 0.5 M KCI. Fractions (10 ml) were collected, and the BRF-2 activity of each fraction was determined by DNA-binding gel mobility shift assay. Protein concentration of active fractions was measured by the colorometric method using a BioRad protein assay. Step 2: heparin-agarose chromatography. Active fractions (75 ml; total protein, 297 mg) from DEAE-cellulose chromatography were pooled and dialyzed against two changes of buffer M containing 50 mM KCI and loaded onto a column of
Protein
Vol
(mg)
(ml)
Activity (U)b
Sp act (U/mg)
Purifi(fold)
Re-
(%)
260 Nuclear extract 940.8 DEAE-cellulose 296.7 75 11,250 37.9 100 Heparin-agarose 41.6 30 10,500 252.4 6.66 93 Third oligo-affinity 0.0006 2.5 1,180 1,966,667 51,890 10.4 Centricon-100 0.0005 0.1 92 184,000 4,854 0.8 a One hundred rat livers (670 g) were used. b One unit is defined as the amount of protein bound to 0.1 ng of end-labeled F44 probe.
heparin-agarose (50 ml) equilibrated with buffer M containing 50 mM KCl. The column was washed with 3 volumes of buffer M containing 0.2 M KCl and eluted stepwise with buffer M containing 0.3, 0.6, and 1 M KCl. Fractions of 6 ml were collected and were analyzed for BRF-2 activity by gel mobility shift assay. BRF-2 activity was eluted at 0.3 M KCl. (Note: with brand-new heparin-agarose, BRF-2 binding may be stronger and thus BRF-2 activity can be eluted by using slightly higher salt concentrations). Active heparin-agarose fractions were pooled together and dialyzed against buffer M containing 50 mM KCl. Step 3: DNA sequence-specific affinity chromatography. Two synthetic complementary oligonucleotide strands corresponding to the apoB promoter sequence (-128 to -85) were annealed and polymerized to form oligomers (17): 5'-gatcGGCTCAAAGAGAAGCCAGTGTAGAAAAGCAAACAGGTCAGGCCC-3'
3'-OCGAGTTTCTCTTCGOTCACATCTTTTCGTTTOTCCAGTCCGGGoctag-5'
The oligomers (1 mg) were coupled to 3 g of cyanogen bromide-activated Sepharose 4B as described previously (17). Dialyzed heparin-agarose fractions (30 ml; 42 mg of protein) were incubated with 10 ,ug of sonicated calf thymus (CT) DNA per ml, 4 mM MgCl2, and 0.1% Nonidet P-40 in ice for 1 h. The incubated mixture was then loaded onto a 15-ml DNA-specific affinity column equilibrated in buffer M containing 50 mM of KCl, and the flowthrough fractions were recycled two more times through the affinity column. After the column was washed with 5 volumes of buffer M containing 125 mM KCl, 4 mM MgCl2, and 5 ,ug of CT DNA per ml, BRF-2 was eluted with a step gradient of 0.4 M and 1 M KCl in buffer M. Fractions of 1.5 ml were collected. BRF-2 activity was eluted at 0.4 M KCl. The BRF-2containing fractions were pooled, dialyzed to a final KCl concentration of 50 mM, and incubated with 5 ,g of CT DNA per ml and 4 mM MgCl2 for 1 h. The incubated mixture was then applied to a DNA-specific affinity column (7 ml). After the column was washed as described above, BRF-2 activity was eluted again at 0.4 M KCl. The BRF-2 factor was purified further by an additional cycle of DNA-specific affinity chromatography (3 ml). Fractions of 0.5 ml were collected. The purified protein was concentrated by centricon-100, frozen in small aliquots in liquid nitrogen, and stored at -80°C. Synthetic oligonucleotides. Oligonucleotides were synthesized by an ABI#380B DNA synthesizer and purified electrophoretically in a 12% acrylamide-7 M urea gel and lx TBE buffer (0.089 Tris borate, 0.089 M boric acid, and 0.002 M EDTA). Oligonucleotides were recovered from the gel by eluting with 0.1 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) followed by ethanol precipitation. Oligonucleotides were annealed (17). Double-stranded oligonucleo-
VOL. 12, 1992
PURIFICATION AND CHARACTERIZATION OF BRF-2
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TABLE 2. Oligonucleotides and apoB genomic fragments used in this study Fragment
Gene
HNF-1/LF-B1 HNF-2/LF-A1 HNF-3
Rat alpha 1 AT Human alpha 1 AT Mouse TTR Mouse TTR Simian virus 40 Human apoB Human apoB Human apoB Human Alb2
HNF-4 C/EBP BRF-1 BRF-2
BRF-21 NF1 AP1 F138 F128 F84 F44 F152C F152D F152E
Human apoB Human apoB Human apoB Human apoB Human apoB Human apoB Human apoB
Positiona
Reference
5'gatcGGCTCAAAGAGAAGCCAGTGTAGAAAAGCAAACAGGTCAGGCCC3' 5'GGCTCAAAGAGAAGCCAGTGTAGAAAAGCAAACAttcGGTCAGGCCC3' 5'gggAGTCAAACAATTTTTTGGCAAGAATATTATGAATCCC3'
-104 to -75 -125 to -96 -109 to -80 -151 to -130 +260 to +230 -84 to -60 -128 to -85 -128 to -85 -140 to -104
5'gatcGTGACTCAGCGCG3' EcoRI-XbaI SacI-XbaI SacI-XbaI EcoRI-SmaI (BRF-2 binding site) SacI-XbaI (containing -85 to -63 internal deletion) SacI-XbaI (containing -113 to -56 internal deletion) SacI-XbaI (containing -104 to -4 internal deletion)
-138 to +122 -128 to +122 -84 to +122 -128 to -85 -152 to +122 -152 to +112 -152 to +112
7 26 6 6 16 8 8 8 31 25 8 8 8 8 8
Sequence
5'gatcACCAAACTGTCAAATATTAACTAAAGGGAG3' 5'gatcATCCCAGCCAGTGGACTTAGCCCCTGTTTG3' 5'gatcTGACTAAGTCAATAATCAGAATCAGCAGGT3' 5'tcgaGGCAAGGTTCATATTTGTGTAG3' 5'gatcTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCT3' 5'gatcGGGAGGCGCCCTTTGGACCTTTTGC3'
a The location of the viral sequences is relative to the numbering system of the viral genome. Locations of the apoB, to the start of transcription.
tides and apoB promoter fragments were labeled at the 5' end by [y-_ P]ATP and polynucleotide kinase. Gel mobility shift assay. Nuclear protein-DNA binding reactions were carried out in a volume of 10 ,l containing 60 mM KCl, 20 mM HEPES (pH 7.9), 5% glycerol, and 4 mM MgCl2. Sonicated CT DNA (1,250 ng) was added to act as a nonspecific competitor. A typical reaction contained 10,000 to 20,000 cpm (approximately 0.5 ng) of 5'-labeled DNA and protein as indicated. After the addition of protein, samples were incubated at room temperature for 30 min and analyzed by electrophoresis on nondenaturing 4% polyacrylamide gels in 0.25 x TBE buffer as described before (8). For competition experiments, conditions were as described above except that the appropriate competitor DNA (as indicated) was added to the reaction mixture prior to addition of protein. Cu-OP footprint assay. The coding strand of the apoB promoter fragment F138 (-138 to + 122) was 5'-end labeled with [.y-32P]ATP and used as a template for footprinting. Fifty nanograms (approximately 100,000 cpm) of the fragment was incubated with 10 ,ul of affinity-purified BRF-2 in a total volume of 100 ,ul at room temperature for 30 min under the conditions used for the gel mobility shift assay. Free DNA fragment was separated from DNA-protein complex by gel electrophoresis as described before. After gel electrophoresis, the gel was soaked with 200 ml of 50 mM Tris (pH 8.0) and 20 ml of Cu-OP (0.45 mM CUSO4 2 mM OP), and 20 ml of 58 mM 3-mercaptopropionic acid was added. The digestion was carried out for 10 min at room temperature followed by quenching for 2 min with 20 ml of 28 mM 2,9 dimethyl OP solution as described previously (21). The gel was washed several times with double-deionized water. The wet gel was then exposed with a X-ray film overnight at 4°C. The autoradiogram was developed, and free and bound bands were excised from the gel. DNAs from the free and bound bands were extracted by incubating gel slices with 0.5 M ammonium acetate and 1 mM EDTA at 37°C overnight. Eluted DNAs were then precipitated with three volumes of ethanol, resuspended in sequencing dye, and analyzed on a 10% polyacrylamide-7 M urea gel. A G+A ladder, generated by sequencing (28) the same end-labeled fragment, was run in the same sequencing gel. Recovery of BRF-2 from native polyacrylamide gel. Affini-
alpha-i-AT, alb2, and TTR are relative
ty-purified BRF-2 was electrophoretically separated in a 8.5% native polyacrylamide gel at 4°C and visualized by silver stain. Protein corresponding to the position of the stained band was eluted from the parallel unstained gel slices by electroelution with gel running buffer in a dialysis tube at -20°C for 4 h. Eluted protein was concentrated by centricon-100. RESULTS We have previously shown that the DNA element (-128 to -85) produced fivefold-greater expression of the reporter CAT construct driven by the apoB promoter sequence (-84 to + 122) in HepG2 cells but showed negative activity in HeLa cells (8). Kardassis et al. showed that the element (-128 to -85) produced two DNase I footprints with rat liver nuclear extracts. Mutations in this region also reduced the level of apoB gene transcription in transfection experiments in HepG2 cells (19). Figure 1 shows the human apoB cis-acting regions in HepG2 and in HeLa cells and the binding sites of factors which have been identified by us to interact with these regions. To understand the function of the apoB gene regulatory factor BRF-2 in the regulation of apoB gene transcription, we have purified this factor from rat liver nuclear extracts. The purification steps are summarized in Table 1. The activity of BRF-2 was monitored throughout the purification process by gel mobility shift assay using a 44-bp apoB promoter fragment (F44) between nucleotides -128 and -85, generated by EcoRI and SmaI digestion of plasmid pKT-128B (8). Table 2 shows different apoB genomic fragments and double-stranded oligonucleotides used for the purification and characterization of the BRF-2 protein. Purification of BRF-2 protein from rat liver. Nuclear extracts were first applied to an anion-exchange DEAE-cellulose column at 100 mM KCI, and bound proteins were eluted with 0.5 M KCI in buffer M. The active fractions eluted from the DEAE-cellulose column were pooled and chromatographed on a heparin-agarose column. The activity was eluted from the heparin-agarose column at 0.3 M KCl (Fig. 2A), representing more than 93% of the total activity. The final purification step involved a sequence-specific
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l-
b
10
20
30
Fraction Number
B
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W
FIG. 2. Purification of BRF-2 by heparin-agarose and DNAspecific affinity chromatography. (A) Elution profile of proteins fractionated on heparin-agarose column. Conditions used for fractionation are described in Materials and Methods. Fractions were analyzed for BRF-2 activity by DNA binding and gel mobility shift assays. One microliter of each fraction was used for the assay. An autoradiogram corresponding to the active fractions is shown in the inset. (B) Sequence-specific affinity chromatography. The active fractions from the heparin-agarose column were pooled and incubated with the DNA affinity resins as described in Materials and Methods. The apoB genomic fragment containing sequences from -128 to -85 (F44) was used for the DNA binding assay. A 1.5-pl portion of each fraction eluted was assayed for BRF-2 binding activity. The top band labeled as BRF-2 indicates the protein-DNA complex, and the bottom band indicated by F44 represents the free probe. The first lane contains the pooled BRF-2 active fractions from the second cycle of DNA-specific affinity chromatography. FT., flowthrough from the third oligo affinity column. W, wash fraction of the third oligo affinity column with buffer M containing 0.125 M KCl. Lanes 1 to 14, fraction numbers eluted from the third oligo affinity column with buffer M containing 0.4 M KCl.
affinity column. A double-stranded oligonucleotide containing apoB promoter sequence -128 to -85 was ligated to produce multimers which were covalently ligated to cyanogen bromide-activated Sepharose 4B beads. The 0.3 M KCl fractions from heparin-agarose were dialyzed with buffer M containing 50 mM KCl and chromatographed on the oligonucleotide affinity column. The BRF-2 activity was eluted at 0.4 M KCl. The BRF-2 factor was purified further by two additional cycles of DNA-specific affinity chromatography. Figure 2B shows the protein elution profile from the third DNA-specific affinity column as monitored by gel mobility shift assay. Table 1 summarizes the purification of BRF-1 from rat liver nuclear extracts. The overall purification and
the yield of the specific DNA binding activity were approximately 52,000 fold and 10.4%, respectively. The BRF-2 binding activity is expressed in units, where one unit is defined as the amount of protein bound to 0.1 ng of F44 probe. The affinity-purified BRF-2 was concentrated by centricon-100 and stored at -80°C. During concentration for several hours, the recovery of BRF-2 activity was found to have diminished by 10- to 12-fold. The concentrated protein is active for approximately a week if stored in ice at -20°C. Identification of the BRF-2 factor as a 120-kDa protein. A sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis of the pools of BRF-2 binding activity obtained by heparinagarose and third oligo affinity column chromatography is shown in Fig. 3A. Significant degrees of purification were achieved by three cycles of DNA-specific affinity chromatography. Only one major protein band corresponding to a molecular mass of 120 kDa was detected by silver stain after three cycles of DNA-specific affinity chromatography (lane 2). To verify that the 120-kDa band is indeed BRF-2, we performed a preparative native polyacrylamide gel electrophoresis (PAGE) of affinity-purified BRF-2 and visualized it with a silver stain; a single polypeptide band was detected (Fig. 3B). Protein corresponding to the position of the stained band was eluted from the parallel unstained gel slices by electroelution at -20°C and concentrated by centricon100. The concentrated protein was then subjected to SDSPAGE, and a single band corresponding to a molecular mass of 120 kDa was visualized with a silver stain (Fig. 3C). This molecular mass is in agreement with gel filtration data obtained with affinity-purified BRF-2 (data not shown). Protein eluted from the native polyacrylamide gel was assayed with F44 and F128 probes (Table 2) in a gel mobility shift experiment. As shown in Fig. 3D, eluted 120-kDa protein produced a mobility shift at the position corresponding to affinity-purified BRF-2 (lanes 2, 3, 4, and 5). Cu-OP footprint analysis with affinity-purified BRF-2 protein. We have reported previously (8) that we could not detect DNase I footprints with HeLa or HepG2 cells or rat liver nuclear extracts in the regulatory region (-128 to -85) of the apoB gene. We also failed to detect a DNase I footprint with affinity-purified BRF-2 protein, suggesting that BRF-2 binds with low affinity with the element (- 128 to -85). To circumvent the problem associated with DNase I footprinting, we used a chemical footprinting technique with Cu-OP to define BRF-2 protein-DNA interaction. The DNA fragment from - 138 to + 122 (F138) was 5'-end labeled at the coding strand and used as a template for footprint analysis. After digestion of DNA in the gel matrix by Cu-OP, DNA corresponding to free and bound complex was eluted from the gel. DNA was then subjected to electrophoresis in a sequencing gel, and an autoradiogram was prepared. As seen in Fig. 4A, purified BRF-2 failed to produce a detectable footprint. However, we repeatedly observed a strong hypersensitive site compared with naked DNA at position -95, indicating that the region surrounding the -95 nucleotide position could be the binding site for BRF-2. Because of the high dissociation constant of DNA-BRF-2 complex, we failed to detect the footprint in this region. To ascertain that the region surrounding the nucleotide position -95 was the bona fide protein binding site for BRF-2, we introduced a trinucleotide (TTC) in the position between -95 and -94 of the element (-128 to -85). We used the double-stranded oligonucleotide as a template in a gel mobility shift assay. As shown in Fig. 4B, the wild-type oligonucleotide (-128 to -85) produced a strong protein-DNA complex with purified
PURIFICATION AND CHARACTERIZATION OF BRF-2
VOL. 12, 1992 M
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FIG. 3. Analysis of purification of BRF-2 by SDS and native PAGE. (A) SDS-8.5% PAGE of BRF-2 active fractions obtained by heparin-agarose and DNA-specific affinity chromatography. Lane M indicates protein molecular mass markers as follows: ovalbumin, 46 kDa; bovine serum albumin, 68 kDa; ,B-galactosidase, 116 kDa; and myosin, 205 kDa. Lanes 1 and 2 show the profile of proteins present in pooled active heparin-agarose fractions and pooled fractions eluted from the third oligo affinity column with buffer M containing 0.4 M KCI and concentrated by centricon-100, respectively. (B) Native 8.5% PAGE of BRF-2 fractions obtained by third oligo affinity column chromatography and concentrated by centricon-100. (C) SDS-8.5% PAGE of BRF-2 recovered from native polyacrylamide gel matrix by electroelution. Lanes indicated by M represent protein molecular mass markers as follows: ovalbumin, 46 kDa; bovine serum albumin, 68 kDa; phosphorylase b, 97 kDa; p-galactosidase, 116 kDa. Lane 1 shows the profile of protein present in BRF-2 recovered from the native polyacrylamide gel matrix. (D) Gel mobility shift assays of BRF-2 recovered from the native polyacrylamide gel matrix. apoB genomic fragments F44 and F128 were used as templates in this assay. BRF-2 indicates the position of protein-DNA complex. F44 and F128 indicate the positions of the two free templates used in this assay. Lane 1 contains 1 pl of BRF-2 purified on the second oligo affinity column. Lanes 2 and 4 contain 1 p,l of BRF-2 recovered from the native polyacrylamide gel; lanes 3 and 5 contain 2 ,ul of BRF-2 recovered from the native polyacrylamide gel matrix.
BRF-2, but the mutant template failed to exhibit proteinDNA interaction. apoB promoter region -104 to -85 is important for BRF-2 binding. To localize the binding site of BRF-2, a series of labeled DNA fragments missing specific regions between -128 and + 122 were used in a gel mobility shift assay. The radiolabeled fragments -128 to -85 (F44) and -128 to + 122 (F128) and a radiolabeled fragment prepared from F152C (containing a -85 to -63 internal deletion) bound to BRF-2. When radiolabeled fragments prepared from F152D (containing a -113 to -56 internal deletion) and from F152E (containing a -104 to -4 internal deletion) were used in a gel mobility shift assay, no protein-DNA complex appeared (Fig. SA). These data suggest that the binding site of BRF-2 is not located between -128 and -105. Therefore, the region spanning nucleotides -104 to -85 is important for BRF-2 binding. The binding of BRF-2 with different apoB promoter mutants is summarized in Fig. SB. Competition gel mobility shift assays with other DNA binding sites. Several liver-enriched factors (e.g., HNF-1, HNF-2, HNF-3, HNF-4, C/EBP, NF-1, and Ap-1) interact with cis-acting elements of a number of liver-specific genes and regulate their expression (1, 6, 7, 10, 14, 16, 23, 24, 26, 27, 31-33, 35). Double-stranded oligonucleotides containing cognate binding sites of several such factors were used as
competitors in a gel mobility shift assay with affinity-purified BRF-2. As shown in Fig. 6, bound complex could only be competed for efficiently by the BRF-2 binding site, indicating that the BRF-2 transcription factor is unique. BRF-2 was found to be heat sensitive and lost DNA binding when heated at 65°C for 10 min (data not shown). Presence of BRF-2-related activity in HeLa cells. We have reported previously that the distal element (-128 to -85) of the apoB promoter acts as a positive element in hepatic cells and a negative element in HeLa cells (8). Therefore, it was of interest to test whether an F44 template could detect the presence of trans-acting factor(s) in HeLa cell extracts. As shown in Fig. 7, the retarded bands had different migrations. It is conceivable that the putative factor(s) is either a posttranslationally modified homolog of BRF-2 or a completely different factor. DISCUSSION We showed previously that hepatic cell-specific expression of the human apoB gene is determined by two cis-acting elements located between -128 and -70 (8). The proximal element (-84 to -70) acts as a strong positive element in HepG2 cells and as a mildly positive element in HeLa cells; this element appears to be absolutely necessary for liver-
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FIG. 4. Cu-OP footprint analysis of BRF-2 binding to the apoB promoter. (A) A fragment of the apoB promoter (50 ng; 100,000 cpm) extending from -138 to + 122 was radiolabeled at the 5' end of the coding strand and was incubated in the presence and absence of third oligo affinity column-purified BRF-2 and subjected to electrophoresis followed by the treatment with Cu-OP reagent described in Materials and Methods. DNAs corresponding to the bound and free bands were eluted from the gel matrix, precipitated with ethanol, and subjected to gel electrophoresis on a 10% sequencing gel. A sequencing reaction (G+A) of the same labeled fragment was run as a marker. Numbering begins at the transcription initiation site (+ 1). Lane 1 contains free DNA fragments recovered from the mobility shift gel. Lane 2 contains DNA fragments recovered from the mobility shift gel corresponding to the protein-DNA complex. Arrow indicates the hypersensitive site at nucleotide position -95 present in lane 2. (B) Gel mobility shift assay of third oligo affinity column-purified BRF-2 with wild-type and mutant apoB oligo templates. Labeled apoB templates (1 ng) were either wild-type BRF-2 oligo (-128 to -85) in lane 1 or mutant oligo containing a trinucleotide (TTC) insertion at position between -95 and -94 (BRF-21) in lane 2. These apoB oligos have blunt ends and were labeled only at the 5' end of the coding strand. One microliter of third oligo affinity column-purified BRF-2 was used in each lane. F, free labeled oligo; BRF-2, position of the protein-DNA complex.
specific expression of the apoB gene (8). Addition of the distal element (-128 to -85) produced fivefold-greater activity over the level of CAT construct containing the proximal positive element (-84 to -70) in HepG2 cells but had a negative effect in HeLa cells (8). We also showed that a protein factor(s) from HepG2 cell and mouse liver nuclear extracts binds to the proximal element (8). The findings described above were confirmed recently by two groups of investigators (3, 4, 19). Metzger et al. have shown that two transcription factors, AF-1 and C/EBP, bind to overlapping sites (-86 to -61 and -69 to -52, respectively) of the apoB promoter (30). Kardassis et al. have purified a rat liver nuclear factor, NF-BA1, which interacts with the apoB promoter sequence (-79 to -63) as well as with other
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FIG. 5. Gel mobility shift assay using wild-type and mutated apoB promoter fragments. (A) Radiolabeled apoB promoter fragments F44 (-128 to -85), F128 (-128 to +122), F152C, F152D, and F152E (1 ng= 12,000 cpm) were used as templates to detect the binding of BRF-2. One microliter of third oligo affinity columnpurified BRF-2 was used in each binding assay. F, free labeled templates; BRF-2, position of protein-DNA complex. (B) Schematic diagram showing the different apoB promoter fragments used in Fig. 5A and a summary of BRF-2 binding with different apoB promoter mutants. Mutations were produced by deletion of sequences from -85 to -63 (F152C), from -113 to -56 (F152D), and from -104 to -4 (F152E). The positions of nucleotides at the endpoints of each fragment are also marked. + and - symbols indicate the presence and absence of BRF-2-DNA complex, respectively.
related sequences present in apoAI, apoAII, and apoCIII genes, indicating the involvement of NF-BA1 in other apolipoprotein gene regulation (20). These NF-BA1- and AF-1like activities are also found to be present in HeLa and CaCo2 cells (20, 30). Genes encoding hepatocyte nuclear factors HNF-1, HNF-2, HNF-3, and HNF-4, which regulate the transcription of other liver-specific genes, have been cloned for structure-function analysis (10, 23, 24, 29, 35). HNF-2 and HNF-4 are identical proteins having a molecular mass of 54 kDa and also bind to the apoB sequence (-86 to -61) (35). Ladias and Karathanasis have cloned a gene encoding an apoAI gene regulatory factor, ARP-1, which also binds to the apoB proximal element (-84 to -60) but negatively regulates apoAI and apoCIII sites (22). We have also purified a protein factor, BRF-1, from rat liver nuclei which binds to the apoB proximal element and appears to have a molecular mass different from those of NF-BA1, ARP-1, and HNF-2 and HNF-4 (32a). Therefore, a clear picture is emerging from these studies that a group of closely related proteins can bind to the apoB proximal element (-84 to -60) and related sequences present in other apolipoprotein and liver-specific genes. Selective gene expression may be governed by multiple promoter-dependent protein-protein inter-
PURIFICATION AND CHARACTERIZATION OF BRF-2
VOL. 12, 1992 N4
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