Vol.

174,

January

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 542-548

31, 1991

PURIFICATION NUCLEAR

OF A NOVEL DNA-BINDING

55 kDa HeLa

CELL

PROTEIN

Wei-Wei Zhang, JaumeFarms, andHarris Busch* Departmentof Pharmacology,Baylor College of Medicine, Houston, Texas 77030 Received

December

4,

1990

A novel 55 kDa DNA-binding protein (~55) waspurified from HeLa cell nuclearextracts to apparenthomogeneityby conventional chromatographycoupledwith DNA-affinity chromatography.The DNA-binding activity of ~55, followed by band mobility shift and Southwesternassays,wasenriched 800-fold. This relatively abundantprotein was shown to bind nonspecifically to DNA. When addedto nuclearextracts, ~55 enhanced2-fold the in vitro transcriptionof CAT reporter genedriven by the SV40 promoter. The sequenceof the N-terminal 20 amino acid residuesof purified ~55 wasdeterminedasAPSTPLLTV(P)G(S)EGLYMVNG. No homologiescould be found when comparedto protein sequences available in all databanks. 0 1991

Academic

Press.

Inc.

In the courseof studieson generegulationof the humanproliferation-associatedprotein ~120 (l-4), two upstreamregionsof the ~120 genewere demonstratedto be important for efficient transcription (5). To identify possibletrans-actingfactors, which might be responsiblefor the regulation of the ~120 genetranscription, HeLa cell nuclearextracts were fractionated and usedin bandmobility shift assays.Preliminary resultsshowedthat severalproteinswere able to bind to the region - 1430/-1327of the p 120gene(4). This report describesthe purification of a 55 kDa protein (~55) from HeLa cell nuclearextracts. Sequence-specificitystudiesdemonstratedthat ~55 bound to a DNA fragment within the region - 1430/-1327of the p 120 geneaswell asto non-related DNA probes.In vitro transcription assaysshowedthat ~55 enhancedtranscriptionof a construct containing the same~120 fragment insertedupstreamof a SV40 promoter-CAT reporter gene.Nterminal sequenceanalysisrevealedthat ~55 is a novel nuclearprotein from HeLa cells.

MATERIALS

AND

METHODS

The DNA probe for gel retardation assayswas a 125-bp fragment (Fl) including the 5’-flanking region (-1430/-1327) of the ~120 gene (4). The ~123 probe wasa 123-bpDNA fragment purified from the 123-bpDNA ladder (GIBCO BRL). Probeswere endlabeledusing the Klenow fragment of DNA polymeraseI and [a-szP] dATP. Plasmidsusedfor in virro transcription were pCAT-Basic, pCAT-Control, pCAT-Promoter from Promegaand pCATFl constructedby inserting the Fl fragment into the Xba I/Barn HI cloning sitesof the pCATPromoter vector.

DNA

probes

and plasmids.

*Corresponding author. Abbrevw CAT, chloramphenicolacetyl transferase;D’IT, dithiothreitol; sodiumdodecyl sulfate-polyacrylamidegel electrophoresis. 0006-291X/91 Copyright All righrs

$1.50

0 1991 by Academic Press. Inc. of reproduction it? any form reserved.

542

SDS-PAGE,

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Protein purification. HeLa cell nuclear extracts (Fraction I), prepared as previously described (6), were fractionated with 50% ammonium sulfate (pH 7.0). The pellet was resuspended in Buffer D [20 mM Hepes @H 7.9), 20% glycerol, 0.2 mM EDTA, 1 mM DIT, 1 mM phenylmethylsulfonyl fluoride, 100 mM KC11 and dialyzed extensively against the same buffer. The dialysate was centrifuged at 25,000 x g for 20 min. The supematant (Fraction II) was placed on a DEAE-Sepharose (Sigma) column, and the flow-through (Fraction III) was applied onto a Heparin-Sepharose (Pharmacia LKB) column, followed by elution with a KC1 gradient (100-600 mM). Fractions containing the DNA-binding activity were pooled, concentrated (Fraction IV), and placed on a Sephacryl S-300 (Pharmacia LKB) column, which was equilibrated with 400 mM KC1 in Buffer D. The pooled active fractions were dialyzed against Buffer D (Fraction V) and loaded onto a DNA-affinity column containing the concatenated oligonucleotide (- 1385/- 1344 of the p 120 gene), according to the method of Kadonaga and Tjian (7). The bound proteins were eluted with a KC1 gradient (O.l- 1 M). The active fractions were pooled, dialyzed and rechromatographed on the same affinity column, resulting in Fraction VI. Band mobility shift assays. Band mobility shift assays (8) were performed using 20 pl reactions containing nuclear extract or chromatographic fractions with 0.1 ug poly dI.dC (Pharmacia LKB) and 1 ng 32P-labeled DNA probe. SDS-PAGE and Southwestern assay. Protein concentrations were determined by Coomassie Protein Assay (Pierce). SDS-PAGE was carried out according to Laemmli (9). Southwestern blotting was performed as described by Mangalam et al. (10). In vitro transcription assay. Transcription assays were performed according to Jones et al. (11). The primer used to detect RNA products was a 22-mer oligonucleotide that hybridizes to sequences - 18 /+4 of CAT gene, which generates 95 nt long primer-extension products. Protein microsequencing. N-terminal sequencing of ~55 was carried out as described by Matsudaira (12). Ten pmol of p55 were fractionated by SDS-PAGE and transferred to Immobilon (Millipore).The protein band corresponding to ~55 was cut out and sequenced on an Applied Biosystems model 477A protein sequencer with a 120A phenylthiohydantoin analyzer. Computer databank searches. Sequence comparisons were carried out using the FASTA program (13). The National Biomedical Research Foundation Protein Database (PIR) was searched using the EuGene 3.2 interface in a SUN workstation, at the Molecular Biology Information Resource, Baylor College of Medicine. Swiss-Prot (release 14.0) and GenPept (release 63.0) databanks were accessed through the GenBank on-line service.

RESULTS Purification

of the ~55 protein. Throughout the purification

using SDS-PAGE,

process, ~55 was followed

band mobility shift and Southwestern assays. In the Sephacryl S-300

chromatography, the ~55 activity was eluted with an apparent molecular weight of about 140 kDa (Fig. l), suggesting that during this purification step either ~55 may be associated with other proteins or it may be a multimer. A multimeric structure is often observed in proteins that bind to DNA (14). Figure 2 shows the activity and protein profiles of ~55 in fractions eluted from the first DNA-affinity column. The overall protein profile of ~55 purification is shown in Figure 3. The enrichment of ~55 activity was estimated about 800-fold (Table 1). N-terminal

sequencing

of the ~55 protein.

Protein microsequencing

was performed

on

purified ~55. The N-terminal amino acid sequence is as follows: Ala-Pro-Ser-Thr-Pro-Leu-LeuThr-Val-(Pro)-Gly-(Ser)-Glu-Gly-Leu-Tyr-Met-Val-Asn-Gly, with uncertain residues indicated in parentheses. Accordingly, the N-terminus of ~55 is relatively hydrophobic. Sequence searches of the data bases did not reveal any significant homologies. No sequence identities were found when ~55 was compared to other known DNA-binding proteins of similar molecular weight. 543

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

F&J, DNA-binding activity and apparent molecular weight of ~55 in fractions from Sephacryl S300 column. (A) Band mobility shift assay. Elution fractions used in different lanes are indicated. Each lane contained 1 ml of 5-fold diluted fractions that had been incubated with 1 ng of 32P-labeled Fl probe and 100 ng of poly dI.dC. (B) Protein molecular weight and DNA-binding activity plotted against fraction number. Molecular weight standards used were Blue dextran, 2000 kDa; Thyroglobulin, 670 kDa; Gamma globulin, 158 kDa; Ovalbumin, 44 kDa; Myoglobin, 17 kDa; and Vitamin B12, 1.35 kDa.

DNA-binding activity of the ~55 protein. Multiple

bandsof various intensities were seen

in bandmobility shift assayswhen the Fl probe wasbound to partially purified or purified protein, suggestingthat the protein forms different molecularweight complexeswith DNA (Fig. 2A). Fig. 2B showsthat the main polypeptide in fractions 24-34 had an approximatemolecularweight of 55 kDa. Only the 55 kDa bandwas detectedby the Fl probe in Southwesternassays(Fig. 2C). An unrelatedDNA fragment (~123) alsocaused band-shiftswhen assayedwith purified ~55 (Fig. 4), showingthat the DNA-binding activity of ~55 lacks specificity. Transcription-enhancing

activity

of the ~55 protein. As shown in Fig. 5, the Fl

fragment enhancedCAT transcription under the control of the SV40 promoter (lanes2 and4), althoughthe enhancingactivity was lessthan that of the SV40 enhancer(lane 3). This is consistent with the results obtained

previously

in our laboratory

using the thymidine

kinase promoter

in CAT

assays(5). When 0.5 or 2 pg of ~55 protein wasaddedto the systemcontaining pCAT-Fl (lane 4), transcription wasenhancedabout 2 fold (lanes6 and 9). This effect was competedby adding 1 pg of Fl fragment (lane 7), but not by the sameamount of poly dI.dC (lane 8). The flow-through of the DNA-affinity column, which did not contain ~55, did not have an enhancingeffect on pCAT544

Vol.

174,

No.

2, 1991

BIOCHEMICAL

A

B

UL

hi BL Fl- W I2

C

ULFi-W

FT

W

12

AND

16

20

24

BIOPHYSICAL

26

32

36

RESEARCH

40

44

14 16 I8 20 22 24 26 26 30 32 34 36 36 40 42

12

1416

48

COMMUNICATIONS

52

44 46 48 50 52 M

18202224262830323436384042444648

>

w Analysis of ~55 in the fractions from the first DNA-affinity column. (A) Band mobility shift assay, (B) 7.5% SDS-PAGE, and (C) Southwestern assay. Lanes: BL, before loading. Ff, flow-through. W, washing. M, prestained molecular weight markers (27-180 kDa range, Sigma). Other lanes contain different elution fractions from the cohnnn. Arrows indicate the position of the ~55 protein band. For the band mobility shift assay, 0.5 ~1 each of the indicated fractions were used. Starting material (BL) was diluted 10 fold. For SDS-PAGE and Southwestern assav. each ’ iane was loadid with 20.~1 of sample. Starting material (BL) was diluted 5 fold.

Fl transcription

(datanot shown). Since ~55 hadlittle additionaleffect on pCAT-Control

transcription (lane 5), ~55 activity may be dependenton a specific sequencewithin the Fl fragment.

DISCUSSION We have purified a novel DNA-binding protein (~55) from HeLa cell nuclearextracts and determinedits N-terminal sequence.The purified protein appearsto be nonspecificin DNAbinding asassessed by bandmobility shift assaysusing competitor poly dI-dC andnonrelated DNA probes.When addedto nuclearextracts, ~55 stimulatedtranscriptionof a templatecontaining 545

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

- 180

u

- 116 - 84 ,58 - p55 - 48.5

*,

- 36.5 - 26.6

F&& Protein profile of p55 purification. Fractions I-VI (nuclear extract, ammonium sulfate precipitation, DEAE-Sepharose, Heparin-Sepharose, Sephacryl S-300, and second DNA-affinity chromatography) were analyzed on a 7.5% SDS-polyacrylamide gel electmphoresis followed by Coomassie-Blue staining. M, prestained molecular weight markers (Sigma).

the enhancer

element Fl. Although ~55 binds nonspecifically to the Fl fragment, it may assist

other sequence-specificfactors to perform their functions, perhapsthrough protein-protein interactions. In this respect,the synergisticeffects betweenc-fos and c-jun proteinsin their DNAbinding and transcription activities is one suchexample(15). There are alsoother examplesof abundantand nonspecificDNA-binding proteinsthat may be relevant to the transcriptionprocess. Poly (ADP-ribose) polymerase,a multifunctional enzyme that bindsto nicked DNA, was identified asTFIIC, a transcription factor that preventsrandom transcriptionby RNA polymerasesI and II (16, 17). Recently, it was suggestedthat the Ku autoantigen(18, 19), which had been shownto be associatedwith active chromatin andto bind nonspecifically to free DNA termini, may be a sequence-specifictranscription factor (20). Also, a specific binding sitehasbeenidentified for cmyc protein, which was previously found to bind randomly to DNA (21). Future studieswill involve further characterizationof ~55 andpossibleinteractionsof ~55 with sequence-specific trans-actingfactors.

Table

1. Recovery of p55 Activity

Fractions Nuclear Extract 50% (NH4)2SO4 DEAESepharose Heparin-Sepharose Sephacryl s-300 DNA-Afllntty

Volume [ml) 111 52 60 30 30 0.5

Protein (mg/ml) 9.1 4.8 2.5 1.1 0.2 0.8

at Different

z:Elln crng) 1000 550 216 41 6.6 0.4

Stages of Purification

z$$y 12000 10850 10440 9600 6720 3840

Yield 12 20 48 234 1020 9600

a One unit is the amount of protein needed to shift 50% of 32P-labeled in the presence of 100 ng poly dl-dC in band mobility shift assays.

546

Purification (Fold)

100 90 87 80 56 32

1.0 1.6 4.0 19.5 85.0 800.0

Fl (1 ngl

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A %YpCAT-Basic

I

pCAT-homoter

I

pCAT-Control

--mTv

1

CE-p

I+ SV40

enhancer

P

pCAT-Fl

1

2

3

4

95nt-b

Lanes

p55

(@

polydI.dC

04

LI-

Fl

D123

05

(pg,

12 -

-

3 -

4 -

-

-

-

-

Fl(yg)

----

FXR

0

(%)

5 0.5

6 0.5

7 0.5

8 0.5

9 2.0

-

-

-

l.0

-

26

80

100

--l.o-22

78

48

85

95

FBand mobility shift assay to determine binding of ~55 to Fl (125 bp) and ~123 (123 bp). Approximately 1 ng of each radioactive-labeled DNA probe was incubated with 10 ng of ~55 in the presence of 100 ng poly dI.dC. Lanes 1 and 3 are probes alone, lanes 2 and 4 are probes with p55. FM In vitro transcription assay. (A) Diagram representing pCAT templates and the position of the oligonucleotide used for primer extension. The SV40 promoter (pSV40) is indicated by a rectangular hatched box, the SV40 enhancer by a squared light-hatched box, and the Fl fragment by a squared dark-hatched box. The CAT coding region is depicted by an open box. The transcription start site is represented by an angled arrow. Covalently closed plasmids were used as templates. (B) Autoradiogram showing the 95nt primer-extension products. Templates used are indicated at the top of each lane. pCAT-B stands for pCAT-Basic, pCAT-C for pCAT-Control, and pCAT-P for pCAT-Promoter. Different amounts of protein and competitor DNA added to transcription reactions are indicated under the corresponding lanes. RTR (relative transcription rate) was quantitated by densitometer scanning from each lane in the autoradiogram.

ACKNOWLEDGMENTS This work was supportedby Public Health Service grant CA-10893 from the National CancerInstitute CancerResearchCenter,The DeBakey Medical Foundation, H. Leland Kaplan CancerResearchEndowment, Linda & Ronny Finger CancerResearchEndowment Fund, and The William S. Farish Fund.The authorswish to thank Dr. R.G. Larson for oligonucleotide synthesis, Ms. M. Finley for preparationof HeLa cells, and Mr. S. W. Yang for his assistance with Sephacryl S-300 chromatography.

REFERENCES 1. 2.

J. W., Busch, R. K., Gyorkey, F., Gyorkey, P., Ross, B. E., and Busch, H. (1988) Cancer Res.48, 1244-1251. 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. 1, 243-251. Freeman,

541

Vol.

3. 4. 5. 6. 7. 8. 9. 10. ::: ::: 15. 16. 17. 18. 19. 20. 21.

174,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Hazlewood, J., Fonagy, A., Henning, D., Freeman, J. W., Busch, R. K., and Busch, H. (1989) Cancer Commun. 1, 29-34. Larson, R. G., Henning, D., Haidar, M. A., Jhiang, S. M., Lin, W. L., Zhang, W.-W., and Busch, H. (1990) Cancer Commun. 2,63-7 1. Haidar, M. A., Henning, D., and Busch, H. (1990) Mol. Cell. Biol. 10, 3253-3255. Dignam, J. D., Lebowitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 14751489. Kadonaga, J. T., and Tjian, R. (1986) Proc. Natl. Acad. Sci. USA 83, 5889-5893. Prywes, R., and Roeder, R. G. (1987) Mol. Cell. Biol. 7, 3482-3489. Laemmli, U. K. (1970) Nature (London) 227,680-685. Mangalam, H. J., Albert, V. R., Ingraham, H. A., Kapiloff, M., Wilson, L., Nelson, C., Elsholtz, H., and Rosenfeld, M. G. (1989) Genes Develop. 3, 946-958. Jones, K. A., Yamamoto, K. R., and Tjian, R. (1985) Cell 42, 559-572. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038. Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448. Hai, T., Liu, F., Coukos, W. J. and Green, M. R. (1989) Genes Develop. 3, 3083-3090. Abate, C., Luk, D., Gagne, E., Roeder, R. G. and Curran, T. (1990) Mol. Cell. Biol. 10, 5532-5535. Slattery, E., Dignam, J. D., Matsui, T., and Roeder, R. G. (1983) J. Biol. Chem. 258, 5955-5959. Kurl, R. N. and Jacob, S. T. (1985) Nucleic Acids Res. 13, 89-101. Yaneva, M., Ochs, R., McRorie, D. K., Zweig, S., and Busch, H. (1985) B&him. Biophys. Acta 841, 22-29. Yaneva, M. and Busch, H. (1986) Biochemistry 25,5057-5063. Knuth, M. W., Gunderson, S. I., Thompson, N.E., Strasheim, L. A. and Burgess, R. (1990) J. Biol. Chem. 265, 17911-17920. Blackwell, T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N. and Weintraub, H. (1990) Science 250, 1149-1151.

548

Purification of a novel 55 kDa HeLa cell nuclear DNA-binding protein.

A novel 55 kDa DNA-binding protein (p55) was purified from HeLa cell nuclear extracts to apparent homogeneity by conventional chromatography coupled w...
1MB Sizes 0 Downloads 0 Views