.::j 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 21 5855 -5856
In vitro transcription complementation assay with miniextracts of transiently transfected GaS-1 cells Hans-Peter Gerber, Oleg Georgiev, Keith Harshman and Walter Schaffner Institut fur Molekularbiologie 11, Universitat Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Received September 7, 1992; Accepted October 7, 1992 We previously described the quick preparation of small amounts of nuclear extracts ('miniextracts') from transfected COS cells for binding studies of cloned transcription factors (1). The procedure has been modified for functional studies of transcription factors by complementation of an in vitro transcription system. Treatment with nonionic detergent is replaced by mechanical lysis with a syringe (2). An ammonium sulfate precipitation step is also included. Cytoplasmic extracts can be obtained in parallel to nuclear extracts. Briefly, 100 mm culture dishes with COS-1 cells grown at 37°C are transfected (calcium phosphate precipitation) with 7 yg of expression vector encoding, for example, a GALA fusion protein (for detailed description of constructs see (3)). After 48 hours, cells are harvested by addition of 1.5 ml TBS, 5 mM EDTA, washed twice in TBS and swelled for 15 min in 700 1l buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM PMSF and 5 ytg/ml leupeptin). All steps are performed at 4°C. Cells are lysed in a 50 ml Falcon tube using a precooled 1 ml syringe and a 25 gauge needle. Cells are drawn into the syringe and ejected with rapid strokes 8 to 12 times. Samples are stained with Hoechst dye (H33258) and the proportion of free nuclei is monitored under a fluorescence microscope. The crude extract is transferred into a 1.5 ml tube and nuclei are separated by centrifugation in a microfuge at 14000 rpm for 30 sec. The pellet containing nuclei is resuspended in 600 yl buffer A and centrifugation is repeated. For cytoplasmic extract, 0.05 volume 3 M KCl is added to the pooled supernatants in a 2.5 ml Eppendorf tube, which is centrifuged 45 min in a microfuge. The supernatant is treated with ammonium sulfate and further processed as described below for nuclear extract, yielding about 250 ,ul extract with a protein concentration of 10-15 mg/ml. For nuclear extracts, nuclei are resuspended with a syringe (5 strokes) in 600 ,d buffer C (20 mM Hepes pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF and 5 itg/ml leupeptin), and transferred to a 5 ml Falcon tube. Nuclear proteins are eluted for 30 min by gently stirring the extracts with a magnetic stirrer (3 x 5 mm) on an ice water bath. The stirrer is removed and and nuclear debris are removed by centrifugation of the 5 ml Falcon tube for 10 minutes in a Sorvall SS 34 rotor at 8000 rpm. The supernatant is transferred to a 5 ml Falcon tube and 1.5 volumes of 4 M ammonium sulfate pH 7.6 are added in three steps over 60 minutes, with gentle stirring. The precipitate is centrifuged in an SS 34 rotor for 10 min at 8000 rpm. The supematant fraction is discarded and the pellet is resuspended in 100 yd buffer D (20 mM Hepes, 20% glycerol, 60 mM KCI, 2 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT). The extract is dialyzed for 10-16 h against buffer D in a microdialyzer (BRL)
COS nuclear extract C,
0
PM
oo6&
0
0
0
0
ccf,4 --- read
.-76_76 _f67
_W
-+*-- 55GAL/ ,B-globin
(pol II)
V-\A,(pol III)
**4w
1 2 3
through
4 5 6
7 8 9
Figure 1. Activation by transcription factors prepared from COS nuclear
miniextracts. The glutamine-rich domain of Oct-2A (GAL930ct2Q, lanes 3 and 6), stimulates transcription 9 fold compared to GAL93 (lanes 2 and 8). Neither
GAL93 nor overexpressed wild type Oct-2A (lanes 1 and 7), which cannot bind to the GAL4 site, enhanced transcription from this promoter. Addition of a-amanitin (1 jtg/ml, lanes 4-6) shows that transcription is dependent on RNA polymerase II. The addition of GAL93 or GAL930ct2Q did not increase incorrectly initiated transcripts ('mad through') as shown in lanes 1-3 and 7-8. Lanes 1-6, 34 iLg HeLa nuclear extract prepared as in (4) complemented with - 15 ytg of COS nuclear miniextracts. Lanes 7-9, 7.4 ytg HeLa nuclear extract prepared as described in (5) complemented with - 15 sg COS nuclear miniextracts. 140 ng 5 GAL/(-globin (5 GAL4 binding sites at position -50 of ,3-globin TATAbox (3)) and 10 ng pVAI, encoding the Adenovirus VA RNA, gene transcribed by polymerase Im (6) were added to a reaction volume of 25 ul. In vitro transcription, RNA preparation and Si mapping are performed as described in (7). Lane M, size marker, pBR322 digested with HpaIl. Lane P, full length SI probes.
using Spectrapor No. 1 tubing. During dialysis (monitored by conductivity measurement), the sample volume increases. Extracts are centrifuged for 5 min in a microfuge, the protein concentration of the remaining supernatant is determined by Bradford assay and samples are frozen in liquid nitrogen. Preparation of 10 culture dishes yields approximately 140 1l nuclear extract with a protein concentration of 3-5 mg/ml, sufficient for 28 in vitro transcription assays. The relative amount of fusion protein in each extract is determined by electrophoretic band shift assay (data not shown) and normalized to equal bandshift units by dilution with mock extract. For our in vitro transcription analyses we mostly used the GAL4 DNA binding domain, amino acids 1-93 ('GAL93'), fused to the activation domain of various transcription factors
5856 Nucleic Acids Research, Vol. 20, No. 21 (see Figure). We believe that this complementation assay should be useful for many experiments involving the characterization of transcription factors and other regulatory proteins.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Schreiber,E. et al. (1989) Nucleic Acids Res. 17, 6419. Lee,K. et al. (1988) Gene Anal. Techn. 5, 22-31. Seipel,K., Georgiev,O. and Schaffner,W. (1992) submitted. Muller,H.P., Sogo,J.M. and Schaffner,W. (1989) Cell 58, 767-777. Abmayr,S.M. et al. (1988) Genes Develop. 2, 542-553. Svensson,C. and Akusjarvi,G. (1984) Mol. Cell Biol. 4, 736-742. Westin,G. et al. (1987) Nucleic Acids Res. 15, 6787 -6798.
Nucleic Acids Research, Vol. 20, No. 21 5855 -5856
In vitro transcription complementation assay with miniextracts of transiently transfected GaS-1 cells Hans-Peter Gerber, Oleg Georgiev, Keith Harshman and Walter Schaffner Institut fur Molekularbiologie 11, Universitat Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Received September 7, 1992; Accepted October 7, 1992 We previously described the quick preparation of small amounts of nuclear extracts ('miniextracts') from transfected COS cells for binding studies of cloned transcription factors (1). The procedure has been modified for functional studies of transcription factors by complementation of an in vitro transcription system. Treatment with nonionic detergent is replaced by mechanical lysis with a syringe (2). An ammonium sulfate precipitation step is also included. Cytoplasmic extracts can be obtained in parallel to nuclear extracts. Briefly, 100 mm culture dishes with COS-1 cells grown at 37°C are transfected (calcium phosphate precipitation) with 7 yg of expression vector encoding, for example, a GALA fusion protein (for detailed description of constructs see (3)). After 48 hours, cells are harvested by addition of 1.5 ml TBS, 5 mM EDTA, washed twice in TBS and swelled for 15 min in 700 1l buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM PMSF and 5 ytg/ml leupeptin). All steps are performed at 4°C. Cells are lysed in a 50 ml Falcon tube using a precooled 1 ml syringe and a 25 gauge needle. Cells are drawn into the syringe and ejected with rapid strokes 8 to 12 times. Samples are stained with Hoechst dye (H33258) and the proportion of free nuclei is monitored under a fluorescence microscope. The crude extract is transferred into a 1.5 ml tube and nuclei are separated by centrifugation in a microfuge at 14000 rpm for 30 sec. The pellet containing nuclei is resuspended in 600 yl buffer A and centrifugation is repeated. For cytoplasmic extract, 0.05 volume 3 M KCl is added to the pooled supernatants in a 2.5 ml Eppendorf tube, which is centrifuged 45 min in a microfuge. The supernatant is treated with ammonium sulfate and further processed as described below for nuclear extract, yielding about 250 ,ul extract with a protein concentration of 10-15 mg/ml. For nuclear extracts, nuclei are resuspended with a syringe (5 strokes) in 600 ,d buffer C (20 mM Hepes pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF and 5 itg/ml leupeptin), and transferred to a 5 ml Falcon tube. Nuclear proteins are eluted for 30 min by gently stirring the extracts with a magnetic stirrer (3 x 5 mm) on an ice water bath. The stirrer is removed and and nuclear debris are removed by centrifugation of the 5 ml Falcon tube for 10 minutes in a Sorvall SS 34 rotor at 8000 rpm. The supernatant is transferred to a 5 ml Falcon tube and 1.5 volumes of 4 M ammonium sulfate pH 7.6 are added in three steps over 60 minutes, with gentle stirring. The precipitate is centrifuged in an SS 34 rotor for 10 min at 8000 rpm. The supematant fraction is discarded and the pellet is resuspended in 100 yd buffer D (20 mM Hepes, 20% glycerol, 60 mM KCI, 2 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT). The extract is dialyzed for 10-16 h against buffer D in a microdialyzer (BRL)
COS nuclear extract C,
0
PM
oo6&
0
0
0
0
ccf,4 --- read
.-76_76 _f67
_W
-+*-- 55GAL/ ,B-globin
(pol II)
V-\A,(pol III)
**4w
1 2 3
through
4 5 6
7 8 9
Figure 1. Activation by transcription factors prepared from COS nuclear
miniextracts. The glutamine-rich domain of Oct-2A (GAL930ct2Q, lanes 3 and 6), stimulates transcription 9 fold compared to GAL93 (lanes 2 and 8). Neither
GAL93 nor overexpressed wild type Oct-2A (lanes 1 and 7), which cannot bind to the GAL4 site, enhanced transcription from this promoter. Addition of a-amanitin (1 jtg/ml, lanes 4-6) shows that transcription is dependent on RNA polymerase II. The addition of GAL93 or GAL930ct2Q did not increase incorrectly initiated transcripts ('mad through') as shown in lanes 1-3 and 7-8. Lanes 1-6, 34 iLg HeLa nuclear extract prepared as in (4) complemented with - 15 ytg of COS nuclear miniextracts. Lanes 7-9, 7.4 ytg HeLa nuclear extract prepared as described in (5) complemented with - 15 sg COS nuclear miniextracts. 140 ng 5 GAL/(-globin (5 GAL4 binding sites at position -50 of ,3-globin TATAbox (3)) and 10 ng pVAI, encoding the Adenovirus VA RNA, gene transcribed by polymerase Im (6) were added to a reaction volume of 25 ul. In vitro transcription, RNA preparation and Si mapping are performed as described in (7). Lane M, size marker, pBR322 digested with HpaIl. Lane P, full length SI probes.
using Spectrapor No. 1 tubing. During dialysis (monitored by conductivity measurement), the sample volume increases. Extracts are centrifuged for 5 min in a microfuge, the protein concentration of the remaining supernatant is determined by Bradford assay and samples are frozen in liquid nitrogen. Preparation of 10 culture dishes yields approximately 140 1l nuclear extract with a protein concentration of 3-5 mg/ml, sufficient for 28 in vitro transcription assays. The relative amount of fusion protein in each extract is determined by electrophoretic band shift assay (data not shown) and normalized to equal bandshift units by dilution with mock extract. For our in vitro transcription analyses we mostly used the GAL4 DNA binding domain, amino acids 1-93 ('GAL93'), fused to the activation domain of various transcription factors
5856 Nucleic Acids Research, Vol. 20, No. 21 (see Figure). We believe that this complementation assay should be useful for many experiments involving the characterization of transcription factors and other regulatory proteins.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Schreiber,E. et al. (1989) Nucleic Acids Res. 17, 6419. Lee,K. et al. (1988) Gene Anal. Techn. 5, 22-31. Seipel,K., Georgiev,O. and Schaffner,W. (1992) submitted. Muller,H.P., Sogo,J.M. and Schaffner,W. (1989) Cell 58, 767-777. Abmayr,S.M. et al. (1988) Genes Develop. 2, 542-553. Svensson,C. and Akusjarvi,G. (1984) Mol. Cell Biol. 4, 736-742. Westin,G. et al. (1987) Nucleic Acids Res. 15, 6787 -6798.
