Vol. 180, No. 2, 1991

October 31, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 805-8]2

PURIFICATION OF A GROUP OF HeLa NUCLEAR PROTEINS THAT BIND TO A REGULATORY ELEMENT (-1430/-1327) OF THE HUMAN

PROLIFERATING CELL NUCLEOLAR PROTEIN PI20 GENE

Amitava Chatterjee, Rose K. Busch, David Jung, Wei-Wei Zhang and Harris Busch

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

Received September 13, 1991

SUMMARY: A group of proteins was purified from HeLa nuclear extract by DNA affinity chromatography which bind to an important regulatory element (-1430/-1327) of the PI20 gene. The DNA binding activity was enriched 1075 fold. By silver staining three major polypeptides (50, 40, 37 kDa) were detected in the purified fraction. The band shift assay and the southwestern assay showed that the 50 kDa protein (P50) binds to the F1 (-1430/-1327) DNA fragment. The binding specificity of the group of proteins with F1 DNA in the presence of non-specific competitor DNA is much higher than that of P50 alone. On the basis of molecular weight and specific antibody binding, the 37 kDa protein appears to be the B23 nucleolar protein. ©1991AcademicPress, Inc.

PI20 is a nucleolar protein expressed early in G I phase of the

cell cycle and associated with the proliferating state of the cell

(i). Previous studies (2-5) defined two important regulatory

elements in the upstream region (-1426/-1223 and -537/-280) of the

PI20 gene. Preliminary results showed that several proteins bind

to the -1430/-1327 region of the PI20 gene (4). Recently a 55 kDa

nuclear protein was reported to bind non-specifically to

-1430/-1327 region of the PI20 gene (6). Our present study

describes the purification of a group of proteins from HeLa nuclear

extracts. The DNA binding activity of the group of proteins with

the F1 fragment in the presence of non specific "competitor" DNA

is greater than the binding of P50 alone to the F1 probe in the

presence of same amount of competitor DNA.

*To whom correspondence should be addressed.

Abbreviation s- SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; DTT, dithiothreitol.

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Vol. 180, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS

DNA probes. For band shift assay a 125-bp fragment (FI) from the 5' flanking region (-1430/-1327) of the PI20 gene was used as the DNA probe. A similar size DNA fragment (I00 bp) from the ECoRI digest of lambda DNA was used as competitor DNA. Protein purification. HeLa cell nuclear extracts were prepared following the method of Dignam et al (7). The nuclear extracts were first made to 40% saturated with (NH4)2S04. The precipitated proteins were removed by centrifugation at 25,000xg for 20 mins at 40 . The clear supernatant was then made to 80% saturation with (NH4)2S04. The precipitated proteins were then collected by centrifugation at 25,000xg for 30 mins at 40 . The pellet was suspended in buffer D (20 mM Hepes pH 7.9, 50 mM KCI, 1 mM PMSF, 1 mM DTT, 0.2 mM EDTA, 20% glycerol) and dialysed overnight against buffer D. The dialysate was centrifuged at 25,000xg for 20 mins. The proteins precipitated from HeLa nuclear extract at 40-80% (NH4)2S04 was then loaded onto a DNA affinity column containing the concatenated oligonucleotide (-1415/-1375) of the PI20 gene according to the method of Kadonaga and Tjian (8). The proteins were allowed to bind to the DNA for 2-3 hrs at 4~ The column was then washed with 50 volumes of buffer D and the proteins bound specifically to the column were eluted with a KCI gradient (0.i M- 2.0 M). The active fractions were pooled, dialyzed and rechromatographed on the same DNA affinity column. The protein which bind to DNA was finally purified by DEAE 52 ion exchange chromatography with a KCl gradient of 0.1-0.3M. SDS-PAGE, Southwestern assay and Immunoblotting. SDS-PAGE was carried out following the Laemmlis method (9). Southwestern assay was performed according to Mangalam et al (i0). Western transfer and immunoblotting was performed according to Towbin et al (11). Band mobility shift assay. Band mobility shift assays (12) were performed in 30 ~i of reaction mixture containing different protein fractions with 1 ng of 32p labeled DNA probe (F1) and 1 ~g poly dI.dC (Pharmacia LKB). Development of antibody to PS0. Polyclonal antibodies to P50 were raised in rabbits using gel purified P50. Determination of amino acid composition of PS0. The amino acid composition of P50 was determined by Waters Pico Tag amino acid analysis system (13),

RESULTS

Proteins both in HeLa cytosol and nuclear extract bind to the

F1 fragment as evidenced by band shift assays (Fig. I.IB, C).

Formation of the DNA protein complexes was inhibited by unlabeled

F1 probe used as competitor DNA (Fig. I.ID). When nuclear proteins

fractionated at different (NH4)zS04 saturation were tested by band

shift assays more than 95% of the DNA binding activity was detected

in the 40-80% (NH4)2S04 precipitated proteins (Fig. 1.2A, B).

The DNA binding activity was purified from 40-80% (NH4)2S04

fractionated proteins through a sequence specific affinity column.

The proteins specifically bound to the column were eluted at 0.5

M and 1.0 M KCI concentration (Fig. 2D, E). The purified fraction

contained three major polypeptides (50, 40, 37 kDa) in a silver

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V o l . 1 8 0 , N o . 2, 1991 B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

i 2

Q A B C D A B ~ A B G D E F

Fig. i. Band mobility shift assay. Radiolabeled FI fragment was incubated, panel i, (A) without any protein, (B) with cytosol extract, (C) with nuclear extract, (D) with nuclear extract in the presence of 20ng of unlabeled F1 fragment. Panel 2, (A) with 40% (NH4)2S04 fraction, (B) with 40-80% (NH4)2S04 fraction. Approximately 1 ng of labeled DNA probe in the presence of 1 #g of poly dI.dC was used per assay.

Fig. 2. Band mobility shift assay of different fractions of DNA affinity chromatography. Approximately 1 ng of labeled F1 fragment was incubated with 20 ~i each of flow through, 0.1M, 0.25M, 0.5M, 1.0M, and 2.0M KCI fractions (lanes A, B, C, D, E, and F respectively) in the presence of 1 ~g of poly dI.dC.

stained SDS-PAGE (Fig. 3A). In southwestern assays of the purified

fraction only a 50 kDa protein was detected by the labeled F1 DNA

(Fig. 3B). The 50kDa F1 DNA binding protein was purified from the

200

97

6 8

43"

30"

A B G

Fig. 3. SDS-PAGE and DNA binding activity of the protein complex purified through DNA affinity chromatography. (A) Silver stained protein profile of the purified fraction, (B) southwestern assay of the purified fraction and (C) silver stained pure P50.

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A B C D E F S

Fig. 4. Band mobility shift assay. Labeled F1 fragment was incubated (A) without any protein, (B,E) with PS0 and purified group of proteins, with P50 and purified group of proteins preincubated with i000 ng (C,F) and 2000 ng (D,G) of i00 bp DNA fragment as competitor DNA. Approximately, 1 ng of labeled F1 DNA with 1 ~g of poly dI.dC was used per assay.

group of protein (DNA affinity purified fraction) through DE-52 ion

exchange chromatography at 0.1M-0.3M KCI concentration (Fig. 3C).

In the presence of unrelated competitor DNA, the group of

proteins had greater F1 DNA binding activity than P50 alone (Fig.

4A-F) as shown by band mobility shift assay. The DNA binding

activity was increased 1075 fold in the purified fraction over the

Table i. Recovery of P50 Activity at Different Stages of Purification

Total Total Specific Yield Purification Fractions Protein Activity Activity % (Fold)

(mg) (Unit)~ (Unit/mg)

Nuclear extract 115 10695 93 i00 1.0 40-80% (NH4)2SO 4 41 7953 194 74 2.1 DNA-Affinity 1 1.2 4800 4000 45 43.0 DNA-Affinity 2 0.ii 3100 28182 29 303.0 DE-52 purified P50 0.028 2800 100,000 26 1075.3

a One Unit of activity is the amount of protein needed to shift 50% of 32P Labeled F1 DNA (i ng) in the presence of 1 ~g poly dI-dC in band mobility shift assays.

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crude nuclear extract and the specific activity was i00,000

units/mg of proteins (Table i).

Polyclonal antibodies to PS0 were raised in rabbits using

gel purified P50. The specificity of the antibody to P50 was

tested by Western blot (Fig. 5) and gel retardation assay. When

P50 polyclonal antibody was reacted to the purified fraction and

used for gel retardation assay the DNA protein complexes migrated

more slowly than that of the control (Fig. 6A-C). When the

proteins in the purified fraction were immunoreacted with B23

monoclonal antibody, a strong immunopositive band was found that

co-migrated with purified B23 (Fig. 7A, B). When the purified

fraction was allowed to react with B23 monoclonal antibody and used

for band shift assay the DNA protein complexes migrated more slowly

than that of the control (Fig. 8B,C). When B23 monoclonal antibody

was immunoreacted with purified B23 protein and then added to the

purified fraction for band shift assays, the antibody was found

20C

97

68

43 %

3 E

30

® A B Q A 8 C

Fiq. 5. Immunoblot analysis of PS0 antibody (A) molecular weight markers, (B) purified fraction. Proteins were run in a 10% SDS-PAGE, transferred to nitrocellulose membrane and incubated withl:100 diluted PS0 antibody. The strip was then treated with alkaline phosphatase labeled anti-rabbit second antibody. The band was detected using NBT/BCIP.

Fig 6. Band mobility shift assay. Labeled F1 fragment was incubated (A) without any protein, with purified fraction reacted (B) with C 23 monoclonal antibody, (C) with P50 antibody for 1 hr. at 40 . Approximately 1 ng of F1 DNA with i ~g of poly dl.dC was used per assay.

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2O0

97-

68-

43

30-

( ~ A B ( ~ A B C D E

Fig. 7. Western blot assay of (A) the purified fraction and (B) pure B 23 with B 23 monoclonal antibody. Proteins were dissolved in Laemmli buffer, ran in a 7.5% gel, transferred to nitrocellulose membrane and reacted with B 23 monoclonal antibody as discribed in materials and methods.

Fig. 8. Band mobility shift assay. Labeled F1 fragment was incubated (a) without any protein, (B) with~urified fraction reacted with C 23 monoclonal antibody for 1 hr. at 4 , (C) with purified fraction reacted with B 23 monoclonal antibody for 1 hr. at 40 , (D) with purified fraction reacted with B 23 adsorbed anti B 23 monoclonal antibody, (E) with pure B 23. i ng of labeled probe was used with 1 ~g poly dI.dC per assay.

to be ineffective (Fig. 8D). Band shift assays with purified

protein B23 showed that it did not bind to F1 fragment (Fig. BE).

These results demonstrate that B23 or one of its forms is in the

purified group of proteins.

DISCUSSION

A group of proteins that bind to the -1430/-1327 fragment of

the PI20 gene was purified from HeLa nuclear extracts. The

activity was in both cytosol and nuclear extracts. In silver

stained SDS gel the purified fraction contained three major

polypeptides (50, 40 and 37 kDa). In Southwestern and band shift

assays only the 50 kDa protein bound to the F1 DNA fragment. In

the presence of unrelated competitor DNA the group of proteins had

greater binding activity to F1 DNA fragment than the P50 alone as

shown by band mobility shift assays. These results indicate that

P50 binds to the F1 fragment and other two proteins (P40 and P37)

associated with P50 may produce increased binding of P50 to F1 DNA

fragment. The 37 kDa protein of the purified fraction appears to

be protein B23, a well characterized nucleolar protein. Protein

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Table 2

Amino acid mole % # residues

ASX 9.5 52.7 GLX 12.8 71.0 SER 8.2 45.6 GLY 12.0 64.8 HIS 1.6 8.9 ARG 3.16 17.5 THR 3.00 16.6 ALA 6.15 34.1 PRO 4.10 22.7 TYR 1.77 9.7 VAL 5.10 28.3 MET 2.60 14.4 CYS 0.35 2.0 ILE 4.05 22.0 LEU 9.10 50.4 PHE 3.17 17.6 LYS 6.35 35.2

Asp + GLu = 22.3 mole% ARG + LYS = 9.5 mole%

B23 itself did not bind to the F1 fragment but may enhance binding

specificity of P50 to F1 DNA. Protein B23 is involved in the

transport or assembly of preribosomal particles (14). Protein B23

or one of its forms may also affect P120 gene expression working

as a co-factor for P50 in an analogous fashion of cooperation of

c-fos with c-jun protein (15). Previously, Zhang et al (6) has

reported a 55 kDa nuclear protein which binds nonspecifically to

the F1 fragment. When PS0 was compared to P55 in SDS gels they

migrated at different positions (data not shown). Microsequencing

of P50 was attempted but it has a blocked N-terminal. Amino acid

composition analysis (Table 2) shows that P50 is an acidic protein.

Future work will involve further characterization and functional

analysis of P50 and the associated P40 and P37 to determine their

specific roles in P120 gene regulation.

ACKNOWLEDGMENTS

This investigation was supported by the Cancer Research Center Grant CA-I0893, awarded by the National Cancer Institute, Department of Health and Human Services, USPHS: The DeBakey Medical Foundation; H. Leland Kaplan Cancer Research Endowment Fund and the William S. Farish Fund. The authors wish to thank Dr. P.K. Chan for the generous gift of pure B23 and B23 monoclonal antibody, and Ms. M. Finley for preparation of HeLa cells.

i.

REFERENCES

Freeman, J.W., Busch, R.K., Gyorkey, F., Gyorkey, P., Ross, B.E., and Busch, H. (1988) Cancer Res. 48, 1244-1251.

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2. Fonagy, A., Henning, D., Jhiang, S.M., Haidar, M.A., Busch, R.K., Larson, R.G., Valdez, B.C., and Busch, H. (1989) Cancer Commun. i, 243-251.

3. Hazlewood, J., Fonagy, A., Henning, D., Freeman, J.W., Busch, R.K., and Busch, H. (1989) Cancer Commun. i, 29-34.

4. Larson, R.G., Henning, D., Haider, M.A., Jhiang, S.M., Lin, W.L., Zhang, W.W., and Busch, H. (1990) Cancer Commun. 2, 63- 71.

5. Haider, M.A., Henning, D., and Busch, H. (1990) Mol. Cell. Biol. i0, 3253-3255.

6. Zhang, W.W., Farres, J., and Busch, H. (1991) B.B.R.C. 174, 542-548.

7. Dignam, J.D., Lebowitz, R.M., and Roeder, R.G. (1983) Nucleic Acid Res. ii, 1475-1489.

8. Kadonaga, J.T. and Tjian, R. (1986) Proc. Natl. Acad. Sci. USA 83, 5889-5893.

9. Laemmli, U.K. (1970) Nature (London) 227, 680-685. i0. Mangalam, H.J., Albert, V.R., Ingraham, H.A., Kapiloff, M.,

Wilson, L., Nelson, C., Elsholtz, H., and Rosenfield, M.G. (1989) Genes Develop. 3, 946-958.

ii. Towbin, H., Stachelin, T., and Gordan, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354.

12. Prywes, R. and Roeder, R.G. (1987) Mol. Cell. Biol. 7, 3482- 3489.

13. Bidlingmeyer, B.A., Cohen, S.A. and Travin, T.L. (1984) J. chromatogra. 336, 93-104.

14. Chan, P.K., Aldrich, M., Cook, R.G., and Busch, H. (1986) J. Biol. Chem. 261, 1868-1872.

15. Allegretto, E.A., Smeal, T., Angel, P., Spiegelman, B.M., and Karin, M. (1990) Jour. Cell Biochem. 42, 193-206.

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