Vol. 147, No. 2, 1987

September 15, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 824-830

A NUCLEAR FACTOR WHICH INTERACTS WITH AN AT-CLUSTER IN THE FIRST INTRON OF RAT a2-MACROGLOBULIN GENE

Takashi Ito and Yoshiyuki Sakaki

Research Laboratory for Genetic Information, Kyushu University, 18, Fukuoka 812, Japan

Received July 22, 1987

We have identified a nuclear protein which binds specifically to the first intron of rat e2-macroglobulin gene. The protein became insoluble at low salt concentration retaining the binding specificity. Its molecular weight was estimated to be 22 kilodalton by a protein blotting procedure. The binding site of the protein determined by DNase I footprinting was an AT-strech which shared 80% homology with the cleavage consensus of Drosophila topoisomerase II. 0 1987 Academic Press, Inc.

Rat CY~ -macroglobulin (ct2M) is an acute phase reactant, concentration of which in serum increases more than up to 100- fold during the course of inflammation (1). Hybridization experiments using or2M cDNA have shown that the induction is due to drastic increase of cr2M mRNA in liver (2, 3, 4). No significant amount of c12M mRNA was detected in other organs. Thus, rat ct2M gene may provide a good system for studying the molecular mechanism of tissue specificity and induction of gene expression. We have recently isolated genomic DNA clones of rat a2-M gene containing the promoter region, and identified several possible regulatory signals (5).

It is generally thought that trans-acting factors, which interact with such regulatory signals, have crucial roles in the regulation of gene expression. As an approach to identify such trans-acting reguratory factors, we have tried to detect sequence-specific DNA binding proteins which bind specifically to ci2M gene. In the course of such trials, we have found a nuclear protein which binds specifically to the first intron of e2M gene.

MATERIALS AND METHODS Preparation of nuclear extract: Rat livers (lOQl5g) were homogenized in 150ml of 2.2M sucrose, and nuclei were recovered by centrifugation at 30,OOOg for 60-90 min at 4OC. After

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Vol. 147, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

washing with lOm1 of a solution containing 1OmM Tris-HCl (PH7.6), 1OmM MgC12, 1OmM 2-mercaptoethanol, 0.2% (v/v) Triton X-100 and 0.25M sucrose, pelleted nuclei were stored at -80°C until use. Approximately lo8 nuclei were resuspended in EB (5mM Tris-HCl (pH7.6), 0.9mM MgC12, 0.12mM spermidine, 10% (v/v) glycerol) and the volume of the suspension was adjusted to 22O)Jl. The NaCl concentration of the suspension was then brought to 420mM by adding 30~1 of EB/3.5M NaCl. After standing on ice for 30 min, nuclei were pelleted by centrifugation (15,000g for 20 min). The supernatant was filtered through a DE-81 (Whatman) minicolumn. Insoluble materials in the filtrate were removed by centrifugation, and the supernatant was stored at -8O'c. A protease inhibitor, (p-Amidinophenyl) methanesulfonyl fluoride, was added to all solutions described above to a final concentration of 1uM.

For DNase I footprinting, nuclear extract prepared from lo8 nuclei was boiled for 10 min at 95'C and centrifuged to remove aggregated materials. The supernatant was dialyzed against 1OmM Tris-HCl (pH7.6), 50mM NaCl, O.lmM EDTA and 20% (v/v) glycerol. Since the binding protein precipitated during the dialysis as described below, precipitates were collected by centrifugation and dissolved in 25~1 of a solution containing 20mM Tris-HCl (PH7.6) I 840mM NaCl, and 0.2mM EDTA. After addition of isovolume of 40% (v/v) glycerol, the solution was centrifuged to remove insoluble material (15,OOOg for 20 min) and the supernatant was stored in small aliquots at -80°C.

Binding Assays: Plasmids containing various fragments around the promoter region were digested w'th appropriate restriction enzymes, terminally labeled with 3i2 P and used as probes for binding assays. Standard reaction mixture (100~1) contained 1OmM Tris-HCl (pH7.6), 50mM NaCl, 1OmM MgC12, O.lmM EDTA, 50uglml BSA, 5%lOpg of sonicated E. extract. After the mixture was-kept at 4°C for 45 min,

coli DNA and 5~1 of nucjq;,

labeled probe (lo-20ng) was added to the mixture and incubated for 30 min at room temperature. The mixture was subjected to filter binding assay, essentially as described by Siebenlist et al (6).

- - Alternatively, the mixture was centrifuged at 15,000g for 10 min at 4'C to precipitate the protein-DNA complex. DNA fragments in the complex were extracted and analyzed by agarose gel electrophoresis and autoradiography. This assay was based on the observation that the protein becomes insoluble at the salt concentration of 50mM retaining its binding specificity (see below).

Protein blotting and DNase I footprinting were performed essentially as described by Miskimins et al (7) and Galas and Schmitz (S), respectively.

--

RESULTS To identify sequence-specific DNA binding proteins for rat

or2-M gene, we used plasmids containing various fragments around

the promoter region (covering from -l.Okb to +4.3kb) as probes for filter binding assays (data not shown). In the course of such experiments, we have detected a DNA binding activity specific for the XI l.lkb fragment derived from the first

intron (Fig. 1). The 31 l.lkb fragment was subcloned into

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Vol. 147, No. 2. 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a b M 12 3 4

\ \ I \ I \ 1 \ \

Fig. 1. Specific binding activity to the first intron of rat ax-M gene. (a) Genomic organization of rat cr2-M gene promoter region (see ref. 5 for details) and plasmid used in this experiment. (E: EcoRI, P: =I, T: TaqI, X: XbaI). 7 The TaqI l.lkb frasment derived from the first intron was subcloned into AccI site-of pUC13. XI digestion of the plasmid genera four fragments (A to D), which were terminally labeled with and used as a probe for binding assays. The fragment B was the -1 l.lkb fragment derived from the first intron, and the fragments A, C and D were derived from pUC13. (b) Filter binding assay. Lanes l-4 contained 0, 2.5, 5 and 1Oug of E. coli DNA as competitors, respectively. M: Marker. (c) Solubility of the bindinq activity. After the reaction, DNAs were recovered from the supernatant and the pellet and subjected to gel electrophoresis. Lane 1: DNA from the supernatant. Lane 2: DNA from the pellet. Lane 3: as lane 2, but boiled nuclear extract was used.

AccI site of pUC13, and four fragments (A to D) generated by -1 digestion of the plasmid were terminally labeled with 32P, and used as a probe for the filter binding assay. As shown in Fig. lb, the fragment B, corresponding to the XI l.lkb fragment, was retained on the filter even in the presence of enough competitor E.coli DNA to abolish the non-specific binding -- of other vector-derived fragments (A, C and D). The binding activity was sensitive to proteases, indicating its protein nature. We call this protein nuclear factor-or (NF-or) hereinafter.

Some properties of NF-c( were examined. After the nuclear extract was incubated with the 32P-labeled probe in the standard reaction mixture (see MATERIALS AND METHODS), the mixture was

centrifuged at 15,000g for 10 min. DNAs were then recovered from the supernatant and the pellet fractions, respectively. Interestingly, the TaqI l.lkb fragment, which contains the NF-or binding site, was recovered from the pellet fraction, but not

Vol. 147, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

PROBE -l--2-

RY 1 1 I I I II

b

1 2

Fig. 2. Protein Blotting. (a) Probes used in this experiment. Probe 1 was the EcoRV-TaqI promoter and the first exon.

1.2kb fragment containing the Probe 2 was the TaqI l.lkb

fragment containing the binding site of NF-cr (RV: EcoRV, T: TaqI). (b) Protein blotting experiments were carrixout essentially as described by Miskimins et al (7). Probes 1 and 2 -- were used in lanes 1 and 2, respectively.

from the supernatant fraction. (Fig. lc, lanes 1 and 2). Thus, NF-a became insoluble at low salt concentration while retaining

the binding specificity. NF-c! seemed to be a heat-stable

protein, because heating of the nuclear extract at 95OC for 10

min did not abolish the binding activity (Fig. lc, lane 3). NF-

OL required Mg2+ for binding (data not shown).

Molecular weight of NF-cr was estimated by protein blotting

experiments. Two probes were used: Probe 1, to which NF-cr did

not bind, and probe 2 containing the binding site (Fig. 2a). As

shown in Fig. 2b, two signals were detected. The proteins in

the upper bands seemed to bind to DNA non-specifically. On the

other hand, the lower one bound only to probe 2. These results

suggested that the lower band having the molecular weight of

22KD was NF-cr.

To map the binding site more precisely, the binding assay

was carried out using three fragments generated by HaeIII

digestion of the TaqI l.lkb fragment. The 3'-fragment (HaeIII-

XI 0.37kb) was preferentially recovered in the protein-DNA

complex (Fig. 3), suggesting that it contained the binding site.

Using the HaeIII-aI 0.37kb fragment, DNase I footprinting

analysis was performed to determine the binding site. An AT-

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Vol. 147, No. 2. 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a b 1 2 lkb

I 1 E

.$$$q :, &?,>f

P T P I II

T X E

I

7 ‘\\

I V

II llIN VVI ‘\

I ‘\

I ‘1

‘\

I ‘\

‘1 A

T’ ‘\ B

H H ‘\ T Ir ‘l C

A C B

Fig. 3. Fine mapping of the binding site (a) Probes used in the experiments. HaeIII (H) digestion of the -1 l.lkb fragment generated three subfragments (A to C) as shown in the Figure. (b) Binding assay using three fragments described above. Lane 1, input DNA. Lane 2, bound DNA. Arrow shows the fragment B, the HaeIII-XI 0.37kb fragment, to which NF-(r bound preferentially. 7: vector (pUC13).

a Ml 23

b ATAAAAMAATGAGAATTGTTCATGAGTTATTTTGCAGCATAAAGAAAT

CATAGTATAAACTCTGGATAATAGTAAGTGGCACAAAAATTAGAATTTTT

. . *.*. TGAGCATTACAAAAAGAACCTATACA~~~~~ATTATTTTCTCT

l * t t t t * t * * t t

Top011 site GTNTATATTNATNNA AC G

CGTTGTTCATTTGTTTGTTTTGAGACAGGATTTTGCTAAGGGTGCCTGAC

Fig..l. DNase I footprinting. (a) DNase I footprinting was carried out essentially as described by Galas and Schmitt (8). M: size marker (pUC18/=11) lane 1: without nuclear extract. Lanes 2, 3: with partially purified nuclear extracts (Lane 2: 2.5~1, Lane 3: 5.0~1). Protected region was indicated by the vertical line. (b) Nucleotide sequence around the binding site. Protected region was shown by the horizontal line. Homology of the binding site with the topo II site proposed by Sander and Hsieh (14) was also shown.

Vol. 147, No. 2, 1987 8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cluster was protected from digestion in the presence of nuclear extract (Fig. 4). The precise boundaries of the protected region were obscure, because for unknown reasons the segments flanking the region were relatively resistant to DNase I digestion even in the absence of nuclear extract (see Fig. 4, lane 1). Since no other significantly protected regions were observed, the site shown in Fig. 4 was considered to be the binding site of NF-a, which contains characteristic TTTA

repeats.

DISCUSSION

We have identified a nuclear protein (NF-a) which binds specifically to an AT-cluster in the first intron of rat a2-M

gene. NF-or seemed to differ from other proteins which bind preferentially to AT-rich sequences, such as a-protein (9, 10) BA (11) and Dl (12, 13), in its molecular weight, solubility, Mg2+ -requirement and more strict binding specificity. We have also noticed that the binding site of NF-a shares 80% homology with topoisomerase II cleavage consensus (topo II site) (14). However, NF-ci is distinct from topoisomerase II, judging from its molecular weight, solubility and lack of topoisomerase II activity (data not shown). We think that NF-a is a novel DNA- binding protein.

NF-a was found not only in inflamed rat liver but also in normal rat liver and kidney (data not shown), and its biological

role remains to be elucidated. Recently, Treisman has identified a DNA-binding protein which binds specifically to the St-activating element of c-fos gene (15). The protein binding site contains the nucleotide sequence GTCCATATTAGGACA, which shares 80% homology with topo II site (14). Interestingly, it was shown that topoisomerase II actually cuts the same site in - vitro (16). These results suggest the existence of nuclear proteins which interact with the same sites as topoisomerase II. NF-a may be a member of such a protein family.

ACKNOWLEDGEMENTS

We would like to thank Drs. Y. Tsuchiya and M. Hattori for providing the nucleotide sequence data, and Misses H. Hamada and Y. Hayashi for assistance in preparation of the manuscript. This work was supported in part by the Grant-in-Aid for Cancer Research from the Ministry of Education, Culture and Sciences (Japan).

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