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Analysis of p53 binding to DNA by fluorescence imaging microscopy
Micron 43 (2012) 996–1000
Contents lists available at SciVerse ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
Analysis of p53 binding to DNA by fluorescence imaging microscopy Hwee Jing Ong a , Jia Wei Siau b , Jing Bo Zhang c , Minghui Hong c , Horst Flotow a , Farid Ghadessy b,∗ a
Experimental Therapeutics Centre, Agency for Science and Technology and Research, Singapore 138669, Singapore p53Lab, Agency for Science and Technology and Research, Singapore 138648, Singapore c Data Storage Institute, Agency for Science and Technology and Research, Singapore 117608, Singapore b
a r t i c l e
i n f o
Article history: Received 17 January 2012 Received in revised form 27 March 2012 Accepted 29 March 2012 Keywords: p53 Transcription factor DNA Fluorescence imaging Microscopy
a b s t r a c t Transcription factors play a central role in cell biology through binding to target DNA elements and regulating gene expression. In this study, we used the p53 tumour suppressor as a model transcription factor to develop an imaging based assay to measure DNA binding. The assay utilizes fluorescence imaging microscopy to detect labelled p53 bound to DNA coated on microbeads. We demonstrate the ability to multiplex the assay by interrogating simultaneous binding to variant DNA sequences present on tractable beads. Additionally, the assay measures activation of p53 for increased DNA binding by a known peptide in addition to reactivation of mutant p53 by a small molecule. It may therefore be adaptable to a highcontent imaging screen for compounds capable of restoring the function of mutant p53 associated with cancer. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction The p53 tumour suppressor plays a central role in governing cell fate (Vogelstein et al., 2000). Through primarily acting as a transcription factor, it binds to specific DNA response elements (REs) to regulate the expression of target genes (Yu et al., 1999). p53 REs typically comprise 2 decameric half-sites related to the consensus sequence RRRCA/T A/TGYYY (where R and Y represent purine and pyrimidine bases, respectively) (Funk et al., 1992). Mutation of p53 is seen in 50% of all cancers and most commonly results in abrogation of DNA binding (Hainaut et al., 1998). Commonly used methods to investigate p53–DNA binding include gel-shift, surface plasmon resonance, fluorescence anisotropy and chromatin immunoprecipitation. More recently an ELISA-based assay was described (Jagelska et al., 2002), and this has subsequently been adapted to bead-based assays utilizing either real-time PCR (Goh et al., 2010) or flow analysis (Noureddine et al., 2009) for signal read out. Whilst the latter methods represent improvements in terms of robustness and reduced technical burden, they are not particularly suited to high throughput screening applications. We therefore adapted the bead ELISA assay to enable fluorescence based imaging and quantification of p53 binding to DNA. The results demonstrate the
∗ Corresponding author at: p53Lab, 8A Biomedical Grove, #06-06, Immunos, Singapore 138648, Singapore. Tel.: +65 64070562; fax: +65 64642085. E-mail address: [email protected] (F. Ghadessy). 0968-4328/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2012.03.024
feasibility of this approach, which has potential to be used in high throughput screens for compounds able to modulate p53 activity.
2. Materials and methods 2.1. DNA constructs and protein expression Constructs for in vitro expression of wild type and mutant p53s have been described previously (Fen et al., 2007; Goh et al., 2010). The Y220C22 mutant template was made by Quickchange mutagenesis (Stratagene) using primers 5 CATAGTGTGGTGGTGCCCTGTGAGCCGCCTGAGGTTGGC-3 and 5 -GCCAACCTCAGGCGGCTCACAGGGCACCACCACACTATG-3 with parental WTp5322 template. Unless otherwise stated, fulllength p53 (and mutants thereof) were used in all assays. Protein synthesis was carried out using the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs) as per manufacturer’s protocol with the following modifications: in each 25 l of IVT reaction, 50 ng of appropriate in vitro expression construct and 0.5 M ZnCl2 were used, and reaction was carried out at 30 ◦ C for 90 min. Expression levels were evaluated by Western blot using DO-1 antibody. The 2CONA RE construct was made by inserting tandem copies of CONA (Noureddine et al., 2009) into the pet22 vector. PCR amplification using primers petF2 and biotin-petRC (Tay et al., 2010) was used to generate DNA for coating on streptavidin beads. Control DNA (−RE) was made by amplification of pet22 vector using same primers. Recombinant p53 was expressed in E. coli as a fusion protein with trigger factor (Takara Bio), purified using
H.J. Ong et al. / Micron 43 (2012) 996–1000
997
Fig. 1. (A) Schematic of bead based detection of p53–DNA binding. Microbeads are red and green circles. White bar represents DNA either with or without a p53 RE (positive and control beads respectively). Grey oval represents p53 bound to RE. Fluorescently labelled antibody depicted by blue starburst. (B) Fluorescence imaging of p53 bound to DNA on microbeads. Left panel shows binding to positive (+RE) beads. Right panel indicates significantly reduced binding to control (−RE) beads.
nickel beads (Invitrogen) and cleaved using PreScission protease (GE Healthcare). 2.2. Antibody labelling 1 g of DO-1 antibody was labelled using Zenon® Alexa Fluor® 488 Mouse IgG2a Labelling Kit (Invitrogen). The labelled antibody was then prepared in PBS with 100 mM KCl and 0.1% BSA, and aliquoted out accordingly for use in the binding assay. 2.3. DNA binding assay Streptavidin-coupled magnetic beads (Dynabeads® M-280 Streptavidin, Invitrogen) were washed as per manufacturer’s protocol before use. In each reaction, 1 g of RE (or control) construct was incubated with 10 l of beads (approximately 6–7 × 106 ) for 15 min, washed twice, blocked in a 3% BSA–PBS solution for 30 min, and washed once before use in the binding assay. All incubation was carried out at room temperature with gentle rotation. 5 l of 6× p53 binding buffer (PBS with 600 mM KCl and 30 mM DTT) was then added to 25 l in vitro expressed protein along with the DNAconjugated beads. The bead-reaction mixture was incubated at 4 ◦ C for 30 min with gentle rotation. The beads were then washed with 200 l of 0.1% BSA–PBST (PBS with 0.1% Tween 20) to remove nonspecifically bound proteins. 0.5 g of labelled DO-1 antibody was
added to the beads and incubated at 4 ◦ C for 20 min with gentle rotation, followed by four washes before resuspending the beads in 20 l of PBS with 100 mM KCl. For the reactivation assay, PK5176 was added at indicated concentrations to the bead-reaction mixture. Specific binding was determined by subtracting fluorescence from control DNA bead assay in presence of indicated amounts of PK5176. For the multiplex assay, 10 l of beads were labelled with 1 pmol fluorescent biotin (Atto-fluorescent biotin, Sigma–Aldrich) at room temperature for 15 min and washed twice before conjugating the appropriate DNA with the beads. Atto Rho101 and Atto 488 were used to label RE-containing and control beads, respectively. DO-1 antibody was labelled with Alexa Fluor® 350. The RE-containing and control beads were pooled together before adding the IVT reaction. The amount of IVT reaction and DO-1 antibody were increased by two fold, and the beads were resuspended in a final volume of 30 l. For the activation assay, recombinant p53 (∼20 ng) was incubated with peptide 46 (2 mM) at 4 ◦ C for 15 min before adding to the DNA-conjugated beads.
2.4. Fluorescence imaging microscopy Multiplex assay images were acquired on the IN Cell Analyzer 2000 (GE Healthcare) using a 10× objective to maximize bead count. The following filter combinations and exposure times were used: DAPI (350/50× and 455/50 m) with 3000 ms exposure for
998
H.J. Ong et al. / Micron 43 (2012) 996–1000
Fig. 2. (A) Multiplex assay showing differential binding of indicated p53s to co-incubated positive (Texas red channel) and control (FITC channel) 2.8 m beads. p53-binding detected in DAPI channel. Merged images (5th column) depict preferential binding by wild type and p53T123A to positive beads (magenta). Inset figures denote percentage of positive beads occupied ± SD. Bottom row shows binding of p53T123A to DNA coated on 1 m beads. (B) Western blot indicating expression levels of wild type and mutant p53 proteins.
detection of Alexa Fluor® 350; FITC (490/20× and 525/36 m) with 80 ms exposure for detection of Atto 488; Texas Red (579/34× and 624/40 m) with 80 ms exposure for detection of Atto Rho101. 6–12 fields of view were acquired to ensure a sufficient bead count was achieved. Images were collected from 2 to 3 separate binding assays. Beads in single fluorescence assays were imaged with an inverted fluorescence microscope (Nikon Eclipse TE300, Nikon Corporation) equipped with a FITC filter cube (EX 465–495, DM 505, BA 515–555) and a 10× objective. 2.5. Image analysis Image analysis for the multiplex assay was performed using the IN Cell Developer Toolbox 1.8 (GE Healthcare). A user-defined
protocol was written to count the number of bead-specific signals. Images were segmented using the granular segmentation module with kernel size = 5 and sensitivity setting at 50 for DAPI and FITC channels and 60 for Texas Red channel. Images were post-processed using the sieve operation with filter size ≥ 3.65 pixels. The relational links between DO-1 antibody binding and bead target sets were established by specifying a linking criterion of 50% intersection. All image analysis parameters were determined empirically. The occupancy rate was defined as binding to a specific population of beads divided by the number of beads in the population. Beads in single fluorescence assays were analysed using ImageJ 1.45 (National Institutes of Health). The lower threshold of the original greyscale images was set at 300 and the integrated density of each bead was measured.
H.J. Ong et al. / Micron 43 (2012) 996–1000
1600 1400 1200
Fluorescence
The assay format is shown in Fig. 1A. Beads are coated with DNA comprising a p53 response element (RE). Recombinant or in vitro expressed p53 is incubated with the beads which are then incubated with a fluorescently labelled antibody against p53. The beads are next dispersed on a glass side and imaged. To gauge non-specific DNA binding, control beads are used in a separate reaction wherein the attached DNA does not contain the RE. Fig. 1B shows the results of a binding experiment using in vitro expressed p53 and two tandem copies of a high affinity RE (CONA) derived from the consensus p53 binding sequence. The presence of the response element shows a clear increase in p53 binding when compared to the control beads. We next multiplexed the assay by labelling RE-containing and control beads with Atto Rho101 and Atto 488 respectively. This enables the beads to be combined, and for both specific and non-specific p53 binding to be assayed simultaneously. As before, beads are dispersed on a glass side and imaged using the appropriate filters to measure antibody binding and bead type. The multiplex assay was carried out using wild type p53, an inactive mutant commonly seen in cancer (R273H) and a hyper-active mutant known to bind DNA more proficiently (T123A) (Fen et al., 2007; Hainaut et al., 1998). The results in Fig. 2A show negligible binding by p53R273H to positive beads (0.11 ± 0.1% occupancy rate) and no binding to control beads was detected (top row). In contrast, wild type p53 preferentially bound to positive beads (35.8 ± 8.9% occupancy rate) versus control beads (0.05 ± 0.005% occupancy rate, second row). The p53T123A super-binder displayed the expected phenotype, showing further increased binding to both positive (68.8 ± 3.9% occupancy rate) and control beads (0.13 ± 0.04% occupancy rate, third row). Quantification of the input protein levels by Western blot indicated similar expression levels of the wild type and p53T123A proteins and an elevated level of the inactive p53R273H (Fig. 2B). During development of the assay we used smaller beads (∼1 m diameter) for coating DNA. The results showed a striking aggregation phenomenon, wherein positive but not negative beads clumped together upon p53T123A binding (bottom row, Fig. 2A). Aggregation, presumably brought about by antibody crosslinking of p53 molecules bound to REs on separate beads, is clearly visible under the brightfield and DAPI (antibody-positive) channels. Aggregation does not occur when larger (2.8 m diameter) beads are used (row 3, last panel). Wild type p53 induced similar aggregation when the 1 m beads were used (data not shown). Therefore, further refinement using even smaller beads or higher affinity truncated wild type p53 (Hupp et al., 1992), may lead to sufficient increases in sensitivity to use aggregation as a reliable proxy for DNA binding. Several compounds and peptides have been described that are capable of potentiating the specific binding of p53 to REs (reviewed in Brown et al., 2011). We therefore tested the previously described peptide 46 (Hupp et al., 1995) for its ability to modulate DNA binding to the high affinity CONA sequence and two endogenous REs regulating the PUMA and p21 genes (Fig. 3A). Peptide 46 is derived from the p53 C-terminus and is responsible for allosteric control of DNA binding capacity. In the absence of peptide 46, binding of recombinant p53 was only observed for the CONA RE. The addition of peptide 46 increased binding to all REs, with this being most pronounced for the PUMA-2 RE (∼2.5 fold increase in binding). An increase in binding to control DNA was also observed, although this was considerably less (∼1.3 fold), indicating preferential activation of p53 for RE binding as previously described (Goh et al., 2010; Weinberg et al., 2005). We additionally investigated reactivation of the cancer-associated Y220C p53 mutant by the compound PK5176 (Wilcken et al., 2012) (Fig. 3B). C-terminally truncated Y220C (Y220C22) was used to mimic cellular activation by
A
1000 800 600 400 200 0 Control
P21-2
PUMA-2
2CONA
35
B
30
Specific binding (x103)
3. Results and discussion
999
25 20 15 10 5 0
Y220C 22 PK5176( M) :
10
50
WT
100
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Fig. 3. (A) Activation of p53 for increased DNA binding by peptide 46. Grey and black bars respectively indicate binding in absence or presence of peptide 46 to control DNA and denoted REs. The dotted line shows level of binding to control (−RE) in absence of peptide 46. (B) Reactivation of mutant p53 (Y220C22) by PK5176. Graph shows specific binding of mutant p53 to DNA in the presence of increasing amounts (10–100 M) of PK5176. Specific binding of WT p53 is included as positive control.
post-translational modification (Hupp et al., 1992). The data show the ability of PK5176 to increase specific binding of the Y220C22 mutant to the CONA RE in a dose dependent manner. The observations reported above show the feasibility of an imaging-based assay to measure p53–DNA binding. The multiplex assay can potentially be used to gauge relative binding of either wild type or mutant p53 to different endogenous REs by further differential bead labelling. The measurement of p53 activation by a known agent indicates potential use of the assay to screen for compounds capable of reactivating cancer-associated mutant p53s for DNA binding activity. This screen could be made high throughput by using microfabricated chip devices capable of segregating microbeads into discrete addressable reaction chambers (Kan et al., 2011). Acknowledgements Robert Graves (GE Life Sciences) for help with developing the image analysis algorithms in Developer. Alan Fersht for kindly providing PK5176.
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References Brown, C.J., Cheok, C.F., Verma, C.S., Lane, D.P., 2011. Reactivation of p53: from peptides to small molecules. Trends in Pharmacological Sciences 32, 53–62. Fen, C.X., Coomber, D.W., Lane, D.P., Ghadessy, F.J., 2007. Directed evolution of p53 variants with altered DNA-binding specificities by in vitro compartmentalization. Journal of Molecular Biology 371, 1238–1248. Funk, W.D., Pak, D.T., Karas, R.H., Wright, W.E., Shay, J.W., 1992. A transcriptionally active DNA-binding site for human p53 protein complexes. Molecular and Cellular Biology 12, 2866–2871. Goh, W., Lane, D., Ghadessy, F., 2010. Development of a novel multiplex in vitro binding assay to profile p53–DNA interactions. Cell Cycle 9, 3030–3038. Hainaut, P., Hernandez, T., Robinson, A., Rodriguez-Tome, P., Flores, T., Hollstein, M., Harris, C.C., Montesano, R., 1998. IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools. Nucleic Acids Research 26, 205–213. Hupp, T.R., Meek, D.W., Midgley, C.A., Lane, D.P., 1992. Regulation of the specific DNA binding function of p53. Cell 71, 875–886. Hupp, T.R., Sparks, A., Lane, D.P., 1995. Small peptides activate the latent sequencespecific DNA binding function of p53. Cell 83, 237–245. Jagelska, E., Brazda, V., Pospisilova, S., Vojtesek, B., Palecek, E., 2002. New ELISA technique for analysis of p53 protein/DNA binding properties. Journal of Immunological Methods 267, 227–235.
Kan, C.W., Rivnak, A.J., Campbell, T.G., Piech, T., Rissin, D.M., Mosl, M., Peterca, A., Niederberger, H.P., Minnehan, K.A., Patel, P.P., et al., 2011. Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies. Lab on a Chip. Noureddine, M.A., Menendez, D., Campbell, M.R., Bandele, O.J., Horvath, M.M., Wang, X., Pittman, G.S., Chorley, B.N., Resnick, M.A., Bell, D.A., 2009. Probing the functional impact of sequence variation on p53–DNA interactions using a novel microsphere assay for protein–DNA binding with human cell extracts. PLoS Genetics 5, e1000462. Wilcken, R., Liu, X., Zimmermann, M.O., Rutherford, T.J., Fersht, A.R., Joerger, A.C., Boeckler, F.M., 2012. Halogen-enriched fragment libraries as leads for drug rescue of mutant p53. Journal of the American Chemical Society, http://dx.doi.org/10.1021/ja301056a, in press. Tay, Y., Ho, C., Droge, P., Ghadessy, F.J., 2010. Selection of bacteriophage lambda integrases with altered recombination specificity by in vitro compartmentalization. Nucleic Acids Research 38, e25. Vogelstein, B., Lane, D., Levine, A.J., 2000. Surfing the p53 network. Nature 408, 307–310. Weinberg, R.L., Veprintsev, D.B., Bycroft, M., Fersht, A.R., 2005. Comparative binding of p53 to its promoter and DNA recognition elements. Journal of Molecular Biology 348, 589–596. Yu, J., Zhang, L., Hwang, P.M., Rago, C., Kinzler, K.W., Vogelstein, B., 1999. Identification and classification of p53-regulated genes. Proceedings of the National Academy of Sciences of the United States of America 96, 14517–14522.
Contents lists available at SciVerse ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
Analysis of p53 binding to DNA by fluorescence imaging microscopy Hwee Jing Ong a , Jia Wei Siau b , Jing Bo Zhang c , Minghui Hong c , Horst Flotow a , Farid Ghadessy b,∗ a
Experimental Therapeutics Centre, Agency for Science and Technology and Research, Singapore 138669, Singapore p53Lab, Agency for Science and Technology and Research, Singapore 138648, Singapore c Data Storage Institute, Agency for Science and Technology and Research, Singapore 117608, Singapore b
a r t i c l e
i n f o
Article history: Received 17 January 2012 Received in revised form 27 March 2012 Accepted 29 March 2012 Keywords: p53 Transcription factor DNA Fluorescence imaging Microscopy
a b s t r a c t Transcription factors play a central role in cell biology through binding to target DNA elements and regulating gene expression. In this study, we used the p53 tumour suppressor as a model transcription factor to develop an imaging based assay to measure DNA binding. The assay utilizes fluorescence imaging microscopy to detect labelled p53 bound to DNA coated on microbeads. We demonstrate the ability to multiplex the assay by interrogating simultaneous binding to variant DNA sequences present on tractable beads. Additionally, the assay measures activation of p53 for increased DNA binding by a known peptide in addition to reactivation of mutant p53 by a small molecule. It may therefore be adaptable to a highcontent imaging screen for compounds capable of restoring the function of mutant p53 associated with cancer. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction The p53 tumour suppressor plays a central role in governing cell fate (Vogelstein et al., 2000). Through primarily acting as a transcription factor, it binds to specific DNA response elements (REs) to regulate the expression of target genes (Yu et al., 1999). p53 REs typically comprise 2 decameric half-sites related to the consensus sequence RRRCA/T A/TGYYY (where R and Y represent purine and pyrimidine bases, respectively) (Funk et al., 1992). Mutation of p53 is seen in 50% of all cancers and most commonly results in abrogation of DNA binding (Hainaut et al., 1998). Commonly used methods to investigate p53–DNA binding include gel-shift, surface plasmon resonance, fluorescence anisotropy and chromatin immunoprecipitation. More recently an ELISA-based assay was described (Jagelska et al., 2002), and this has subsequently been adapted to bead-based assays utilizing either real-time PCR (Goh et al., 2010) or flow analysis (Noureddine et al., 2009) for signal read out. Whilst the latter methods represent improvements in terms of robustness and reduced technical burden, they are not particularly suited to high throughput screening applications. We therefore adapted the bead ELISA assay to enable fluorescence based imaging and quantification of p53 binding to DNA. The results demonstrate the
∗ Corresponding author at: p53Lab, 8A Biomedical Grove, #06-06, Immunos, Singapore 138648, Singapore. Tel.: +65 64070562; fax: +65 64642085. E-mail address: [email protected] (F. Ghadessy). 0968-4328/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2012.03.024
feasibility of this approach, which has potential to be used in high throughput screens for compounds able to modulate p53 activity.
2. Materials and methods 2.1. DNA constructs and protein expression Constructs for in vitro expression of wild type and mutant p53s have been described previously (Fen et al., 2007; Goh et al., 2010). The Y220C22 mutant template was made by Quickchange mutagenesis (Stratagene) using primers 5 CATAGTGTGGTGGTGCCCTGTGAGCCGCCTGAGGTTGGC-3 and 5 -GCCAACCTCAGGCGGCTCACAGGGCACCACCACACTATG-3 with parental WTp5322 template. Unless otherwise stated, fulllength p53 (and mutants thereof) were used in all assays. Protein synthesis was carried out using the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs) as per manufacturer’s protocol with the following modifications: in each 25 l of IVT reaction, 50 ng of appropriate in vitro expression construct and 0.5 M ZnCl2 were used, and reaction was carried out at 30 ◦ C for 90 min. Expression levels were evaluated by Western blot using DO-1 antibody. The 2CONA RE construct was made by inserting tandem copies of CONA (Noureddine et al., 2009) into the pet22 vector. PCR amplification using primers petF2 and biotin-petRC (Tay et al., 2010) was used to generate DNA for coating on streptavidin beads. Control DNA (−RE) was made by amplification of pet22 vector using same primers. Recombinant p53 was expressed in E. coli as a fusion protein with trigger factor (Takara Bio), purified using
H.J. Ong et al. / Micron 43 (2012) 996–1000
997
Fig. 1. (A) Schematic of bead based detection of p53–DNA binding. Microbeads are red and green circles. White bar represents DNA either with or without a p53 RE (positive and control beads respectively). Grey oval represents p53 bound to RE. Fluorescently labelled antibody depicted by blue starburst. (B) Fluorescence imaging of p53 bound to DNA on microbeads. Left panel shows binding to positive (+RE) beads. Right panel indicates significantly reduced binding to control (−RE) beads.
nickel beads (Invitrogen) and cleaved using PreScission protease (GE Healthcare). 2.2. Antibody labelling 1 g of DO-1 antibody was labelled using Zenon® Alexa Fluor® 488 Mouse IgG2a Labelling Kit (Invitrogen). The labelled antibody was then prepared in PBS with 100 mM KCl and 0.1% BSA, and aliquoted out accordingly for use in the binding assay. 2.3. DNA binding assay Streptavidin-coupled magnetic beads (Dynabeads® M-280 Streptavidin, Invitrogen) were washed as per manufacturer’s protocol before use. In each reaction, 1 g of RE (or control) construct was incubated with 10 l of beads (approximately 6–7 × 106 ) for 15 min, washed twice, blocked in a 3% BSA–PBS solution for 30 min, and washed once before use in the binding assay. All incubation was carried out at room temperature with gentle rotation. 5 l of 6× p53 binding buffer (PBS with 600 mM KCl and 30 mM DTT) was then added to 25 l in vitro expressed protein along with the DNAconjugated beads. The bead-reaction mixture was incubated at 4 ◦ C for 30 min with gentle rotation. The beads were then washed with 200 l of 0.1% BSA–PBST (PBS with 0.1% Tween 20) to remove nonspecifically bound proteins. 0.5 g of labelled DO-1 antibody was
added to the beads and incubated at 4 ◦ C for 20 min with gentle rotation, followed by four washes before resuspending the beads in 20 l of PBS with 100 mM KCl. For the reactivation assay, PK5176 was added at indicated concentrations to the bead-reaction mixture. Specific binding was determined by subtracting fluorescence from control DNA bead assay in presence of indicated amounts of PK5176. For the multiplex assay, 10 l of beads were labelled with 1 pmol fluorescent biotin (Atto-fluorescent biotin, Sigma–Aldrich) at room temperature for 15 min and washed twice before conjugating the appropriate DNA with the beads. Atto Rho101 and Atto 488 were used to label RE-containing and control beads, respectively. DO-1 antibody was labelled with Alexa Fluor® 350. The RE-containing and control beads were pooled together before adding the IVT reaction. The amount of IVT reaction and DO-1 antibody were increased by two fold, and the beads were resuspended in a final volume of 30 l. For the activation assay, recombinant p53 (∼20 ng) was incubated with peptide 46 (2 mM) at 4 ◦ C for 15 min before adding to the DNA-conjugated beads.
2.4. Fluorescence imaging microscopy Multiplex assay images were acquired on the IN Cell Analyzer 2000 (GE Healthcare) using a 10× objective to maximize bead count. The following filter combinations and exposure times were used: DAPI (350/50× and 455/50 m) with 3000 ms exposure for
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H.J. Ong et al. / Micron 43 (2012) 996–1000
Fig. 2. (A) Multiplex assay showing differential binding of indicated p53s to co-incubated positive (Texas red channel) and control (FITC channel) 2.8 m beads. p53-binding detected in DAPI channel. Merged images (5th column) depict preferential binding by wild type and p53T123A to positive beads (magenta). Inset figures denote percentage of positive beads occupied ± SD. Bottom row shows binding of p53T123A to DNA coated on 1 m beads. (B) Western blot indicating expression levels of wild type and mutant p53 proteins.
detection of Alexa Fluor® 350; FITC (490/20× and 525/36 m) with 80 ms exposure for detection of Atto 488; Texas Red (579/34× and 624/40 m) with 80 ms exposure for detection of Atto Rho101. 6–12 fields of view were acquired to ensure a sufficient bead count was achieved. Images were collected from 2 to 3 separate binding assays. Beads in single fluorescence assays were imaged with an inverted fluorescence microscope (Nikon Eclipse TE300, Nikon Corporation) equipped with a FITC filter cube (EX 465–495, DM 505, BA 515–555) and a 10× objective. 2.5. Image analysis Image analysis for the multiplex assay was performed using the IN Cell Developer Toolbox 1.8 (GE Healthcare). A user-defined
protocol was written to count the number of bead-specific signals. Images were segmented using the granular segmentation module with kernel size = 5 and sensitivity setting at 50 for DAPI and FITC channels and 60 for Texas Red channel. Images were post-processed using the sieve operation with filter size ≥ 3.65 pixels. The relational links between DO-1 antibody binding and bead target sets were established by specifying a linking criterion of 50% intersection. All image analysis parameters were determined empirically. The occupancy rate was defined as binding to a specific population of beads divided by the number of beads in the population. Beads in single fluorescence assays were analysed using ImageJ 1.45 (National Institutes of Health). The lower threshold of the original greyscale images was set at 300 and the integrated density of each bead was measured.
H.J. Ong et al. / Micron 43 (2012) 996–1000
1600 1400 1200
Fluorescence
The assay format is shown in Fig. 1A. Beads are coated with DNA comprising a p53 response element (RE). Recombinant or in vitro expressed p53 is incubated with the beads which are then incubated with a fluorescently labelled antibody against p53. The beads are next dispersed on a glass side and imaged. To gauge non-specific DNA binding, control beads are used in a separate reaction wherein the attached DNA does not contain the RE. Fig. 1B shows the results of a binding experiment using in vitro expressed p53 and two tandem copies of a high affinity RE (CONA) derived from the consensus p53 binding sequence. The presence of the response element shows a clear increase in p53 binding when compared to the control beads. We next multiplexed the assay by labelling RE-containing and control beads with Atto Rho101 and Atto 488 respectively. This enables the beads to be combined, and for both specific and non-specific p53 binding to be assayed simultaneously. As before, beads are dispersed on a glass side and imaged using the appropriate filters to measure antibody binding and bead type. The multiplex assay was carried out using wild type p53, an inactive mutant commonly seen in cancer (R273H) and a hyper-active mutant known to bind DNA more proficiently (T123A) (Fen et al., 2007; Hainaut et al., 1998). The results in Fig. 2A show negligible binding by p53R273H to positive beads (0.11 ± 0.1% occupancy rate) and no binding to control beads was detected (top row). In contrast, wild type p53 preferentially bound to positive beads (35.8 ± 8.9% occupancy rate) versus control beads (0.05 ± 0.005% occupancy rate, second row). The p53T123A super-binder displayed the expected phenotype, showing further increased binding to both positive (68.8 ± 3.9% occupancy rate) and control beads (0.13 ± 0.04% occupancy rate, third row). Quantification of the input protein levels by Western blot indicated similar expression levels of the wild type and p53T123A proteins and an elevated level of the inactive p53R273H (Fig. 2B). During development of the assay we used smaller beads (∼1 m diameter) for coating DNA. The results showed a striking aggregation phenomenon, wherein positive but not negative beads clumped together upon p53T123A binding (bottom row, Fig. 2A). Aggregation, presumably brought about by antibody crosslinking of p53 molecules bound to REs on separate beads, is clearly visible under the brightfield and DAPI (antibody-positive) channels. Aggregation does not occur when larger (2.8 m diameter) beads are used (row 3, last panel). Wild type p53 induced similar aggregation when the 1 m beads were used (data not shown). Therefore, further refinement using even smaller beads or higher affinity truncated wild type p53 (Hupp et al., 1992), may lead to sufficient increases in sensitivity to use aggregation as a reliable proxy for DNA binding. Several compounds and peptides have been described that are capable of potentiating the specific binding of p53 to REs (reviewed in Brown et al., 2011). We therefore tested the previously described peptide 46 (Hupp et al., 1995) for its ability to modulate DNA binding to the high affinity CONA sequence and two endogenous REs regulating the PUMA and p21 genes (Fig. 3A). Peptide 46 is derived from the p53 C-terminus and is responsible for allosteric control of DNA binding capacity. In the absence of peptide 46, binding of recombinant p53 was only observed for the CONA RE. The addition of peptide 46 increased binding to all REs, with this being most pronounced for the PUMA-2 RE (∼2.5 fold increase in binding). An increase in binding to control DNA was also observed, although this was considerably less (∼1.3 fold), indicating preferential activation of p53 for RE binding as previously described (Goh et al., 2010; Weinberg et al., 2005). We additionally investigated reactivation of the cancer-associated Y220C p53 mutant by the compound PK5176 (Wilcken et al., 2012) (Fig. 3B). C-terminally truncated Y220C (Y220C22) was used to mimic cellular activation by
A
1000 800 600 400 200 0 Control
P21-2
PUMA-2
2CONA
35
B
30
Specific binding (x103)
3. Results and discussion
999
25 20 15 10 5 0
Y220C 22 PK5176( M) :
10
50
WT
100
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Fig. 3. (A) Activation of p53 for increased DNA binding by peptide 46. Grey and black bars respectively indicate binding in absence or presence of peptide 46 to control DNA and denoted REs. The dotted line shows level of binding to control (−RE) in absence of peptide 46. (B) Reactivation of mutant p53 (Y220C22) by PK5176. Graph shows specific binding of mutant p53 to DNA in the presence of increasing amounts (10–100 M) of PK5176. Specific binding of WT p53 is included as positive control.
post-translational modification (Hupp et al., 1992). The data show the ability of PK5176 to increase specific binding of the Y220C22 mutant to the CONA RE in a dose dependent manner. The observations reported above show the feasibility of an imaging-based assay to measure p53–DNA binding. The multiplex assay can potentially be used to gauge relative binding of either wild type or mutant p53 to different endogenous REs by further differential bead labelling. The measurement of p53 activation by a known agent indicates potential use of the assay to screen for compounds capable of reactivating cancer-associated mutant p53s for DNA binding activity. This screen could be made high throughput by using microfabricated chip devices capable of segregating microbeads into discrete addressable reaction chambers (Kan et al., 2011). Acknowledgements Robert Graves (GE Life Sciences) for help with developing the image analysis algorithms in Developer. Alan Fersht for kindly providing PK5176.
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