- Email: [email protected]
A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments
BBRC Biochemical and Biophysical Research Communications 346 (2006) 479–483 www.elsevier.com/locate/ybbrc
A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments Robert B. White, Melanie R. Ziman
*
School of Exercise, Biomedical and Health Science, Edith Cowan University, Joondalup Drive, WA 6027, Australia Received 18 May 2006 Available online 2 June 2006
Abstract The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments. 2006 Elsevier Inc. All rights reserved. Keywords: Target gene identification; Pax7; T-PCR; ChIP
Chromatin immunoprecipitation (ChIP) has rapidly become a significant weapon in the arsenal of methodologies used for deducing transcription factor function. The majority of ChIP assays currently performed provide a rapid in vivo or cell culture-based verification of occupation of the promoters of known or predicted target genes by a given transcription factor. However, in more exploratory studies, ChIP assays are now being used for the identification of novel target genes of a transcription factor, in a genome-wide context. Identification of novel target genes using an unbiased genome-wide ChIP screen has taken two main forms to date—ChIP genomic fragments are cloned and sequenced [1–4] or used for microarray hybridisation [3,5,6]. An unbiased microarray (which must comprise the entire genome) has significantly enhanced our understanding of yeast
*
Corresponding author. Fax: +61 8 6304 5717. E-mail address: [email protected] (M.R. Ziman).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.145
genomic function [6,7], but has yet to be produced for mammalian genomes. Cloning of ChIP DNA fragments presents significant difficulties, particularly the technical challenge associated with shotgun cloning minute quantities of DNA obtained from ChIP experiments. While the presence of immunoprecipitated DNA can be checked by promoter-specific PCR for a bona fide target [4,8], this is not possible for transcription factors that have not previously been studied to that degree. Here we describe a protocol adapting a simple random PCR amplification of ChIP fragments which greatly enhances the efficiency of cloning of novel targets. ChIP fragments are subjected to tagged-random PCR (T-PCR), involving preamplification using primers designed with a random dodecameric sequence at the 3 0 end (allowing binding to an array of target sequences) and a constant 5 0 tail. After two rounds of extension, the unbound primers are removed, and PCR amplification is carried out with a primer specific to the 5 0 constant region of the initial primers, allowing exponential amplification of ChIP DNA,
480
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which is then cloned. We have adapted this method for use with mouse embryonic tissue and compare this method with a shotgun-cloning approach in terms of efficiency of cloning as well as efficiency of success in target gene identification (deduced from proximity of ChIP fragment to a known/predicted coding region). Materials and methods Immunoprecipitation—Western blot. Pre-cleared soluble lysates from whole embryos of C57BL/6J mice were immunoprecipitated with 1 lg anti-Pax7 monoclonal antibody (Developmental Studies Hybridoma Bank—DSHB, Iowa City, IA, USA) and protein G-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) using standard protocols (Santa Cruz). Equivalent volumes (10 lL) of immunoprecipitates, flowthroughs (lysates depleted by Pax7-MAb-proteinG-agarose IP), and embryo lysates were separated on 12% acrylamide/bis gels and transferred to a nitrocellulose membrane. Blocked membranes were probed with antiPax7 and detected with IgG-HRP using the ECL+ plus Western Blot Detection Kit (Amersham Biosciences UK Ltd., Buckinghamshire, England). Chromatin immunoprecipitation. This work was authorised by the Edith Cowan University Animal Ethics Committee and conducted in accordance with the guidelines set by the National Health and Medical Research Council of Australia. The ChIP procedure used was an optimised modification of several published protocols [9–11]. Whole embryonic day 16 (E16) embryos from C57BL/6J mice were minced to 1 mm-sized pieces, and immediately cross-linked in 1% paraformaldehyde in PBS for 15 min at room temperature then quenched by addition of 0.125 M glycine, washed once in PBS with 0.125 M glycine and twice with PBS. Minced, fixed tissue was Dounce homogenised (Bellco Glass, Inc., Vineland, NJ, USA) in cell lysis
buffer (10 mM Tris, 10 mM NaCl, 0.2% NP-40, and 1 mM each of protease inhibitors PMSF, aprotinin, pepstatin A) and after centrifugation, the pellet was homogenised in nuclei lysis buffer (50 mM Tris–HCl, pH 8.1, 10 mM EDTA, 1% SDS, 10 mM sodium butyrate, and protease inhibitors). The remaining chromatin was sonicated to an empirically determined average length of 800 bp using a Sonifier 450 (power output 2, 10% duty cycle) (Branson, Danbury, CT, USA). Debris was removed by centrifugation and supernatant was diluted in IP dilution buffer (20 mM Tris–HCl, pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, 10 mM sodium butyrate, and proteinase inhibitors) and pre-cleared by addition of 200 lL protein G-agarose for 2 h at 4 C under rotation. Cleared chromatin was divided into 600 lL aliquots and incubated overnight at 4 C by rotation with 2 lg of anti-Pax7 monoclonal antibody, or no antibody for control treatment. Immunocomplexes were collected by rotation at 4 C for 3 h with 120 lL protein G-agarose. Beads were washed with low salt buffer (20 mM Tris–HCl, pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS), high salt buffer (10 mM Tris–HCl, pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, and 1% deoxycholic acid) and twice with TE buffer. Immunocomplexes were eluted with elution buffer (1% SDS, 100 mM NaHCO3), two identical samples were combined, and cross-links were reversed by incubation at 65 C for 8 h in the presence of 0.3 M NaCl, then 80 lg Proteinase K (Qiagen, Valencia, CA, USA) was added and samples were incubated at 45 C for 2 h. DNA was purified using QIAquick spin columns (Qiagen) and two identical samples were pooled. Shotgun ChIP cloning. ChIP fragments were cloned using the TOPO Shotgun Subcloning Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, a 45 lL ChIP solution was made bluntended (T4 and Klenow DNA polymerases) and dephosphorylated (calf intestinal phosphatase), phenol/chloroform/ethanol purified, cloned into pCR 4-Blunt-TOPO vector, and chemically transformed into OneShot TOP10 Escherichia coli cells. Tagged-random-PCR amplification cloning. Tagged-random PCR (T-PCR) was performed on ChIP-isolated DNA using tagged-random
Fig. 1. ChIP-cloning procedure. (A) Schematic diagram of ChIP procedure from embryos followed by cloning of immunoprecipitated DNA by either shotgun cloning or T-PCR cloning. (B) Western blot of Pax7 immunoprecipitates from whole embryos: lanes 1, IP-flow-through; 2, no antibody control IP; 3, cleared embryo lysate (no IP); 4, Pax7 IP. (C) T-PCR amplified ChIP-immunoprecipitated DNA: lanes 1, no first round PCR control; 2, no second round PCR control; 3, no DNA control; 4, sonicated input DNA; 5, T-PCR amplified ChIP products; M, 1 kb-plus DNA ladder (Invitrogen). (D) Screening of clones—agarose gels showing several representative clones obtained by either T-PCR or shotgun-cloning methods, and their relative size distributions; M, 1 kb-plus DNA ladder.
R.B. White, M.R. Ziman / Biochemical and Biophysical Research Communications 346 (2006) 479–483 primers to amplify DNA present in immunoprecipitates (to increase the efficiency of vector cloning) [6,12]. T-PCR consists of two rounds of amplification, the first of which uses specific tagged-random dodecamer primers to synthesise tagged copies of ChIP fragments, followed by second strand synthesis of these copies. The second round amplifies tagged products using primers complementary to the tags. The first round was performed in a 20 lL reaction containing 15.9 lL ChIP DNA, 200 lM dNTP, 3 lM primer A (GTTTCCCAGTCACGATCN12), and 1· Klenow polymerase reaction buffer (0.5 M Tris–HCl, pH 7.5, 0.1 M MgCl2, 10 mM DTT, and 0.5 mg/mL BSA). The reaction was heated to 94 C for 2 min, cooled to 4 C for 5 min, then 4 U Klenow polymerase (USB Biochemical, Cleveland, OH, USA) was added and the reaction was ramped at 0.1 C/min to 37 C and extended for 20 min. Second strand synthesis was then performed by heating the reaction to 94 C for 4 min, followed by cooling to 4 C for 5 min, addition of a further 4 U Klenow polymerase, ramping at 0.1 C/min to 37 C, and extension for 20 min. First round primers were removed using QIAquick spin columns, DNA was eluted with dH2O, and 1/10th of the elution volume (2 lL) was PCR amplified in a 20 lL PCR containing 0.5 U Taq polymerase (Qiagen), 1· Q-solution (Qiagen), 2 lM primer B (GTTTCCCAGTCACGATC), and 200 lM each dNTP for 30 cycles of 30 s at 94 C, 30 s at 40 C, 30 s at 50 C, and 2 min at 72 C, followed by a final extension of 72 C for 10 min. Second round T-PCR products were column purified, blunt ended by a 15 min room temperature incubation with 5 U Klenow polymerase in 1· Klenow reaction buffer with 0.5 mM each dNTP, column purified, spectrophotometrically quantified, and 100 ng was cloned into pCR 4-Blunt-TOPO vector (Invitrogen) and transformed into OneShot TOP10 E. coli cells (Invitrogen). All PCRs were performed in an MJ Research PTC-100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) with a heated lid. Clones were randomly selected and analysed by PCR with vector primers (T7 forward and reverse) and electrophoresed on 1.5% agarose gel. Clones carrying inserts >500 bp were sequenced from T7 forward priming sites using BigDye Terminator (Applied Biosystems, Foster City, CA, USA). Data analysis. Sequences had vector sequences removed using VecScreen and were located via megaBLAST of the mouse genome database (all assemblies, build 35.1) and nr databases, available at the National Centre for Biotechnology Information (NCBI) [13]. Chi-square tests were performed using Microsoft Excel 2003.
Results and discussion Chromatin immunoprecipitation from mouse embryos As the monoclonal antibody utilised in this experiment had not previously been validated for use in immunoprecipitation, we first sought to determine whether Pax7 protein would be enriched in immunoprecipitates from mouse embryos. Western blots of mouse whole embryonic lysates immunoprecipitated with monoclonal Pax7 antibody show a substantial enrichment (Fig. 1B; lane 4) and the flow-through from these immunoprecipitates displays an almost complete depletion of Pax7 protein (Fig. 1B; lane 1), indicating high efficiency immunoprecipitation by this monoclonal antibody. T-PCR cloning The minute quantities of DNA obtained after ChIP make DNA quantification and thus optimisation of vector to insert DNA ratios difficult, necessitating substantial optimisation for each individual ChIP experiment. T-PCR
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amplification of ChIP-immunoprecipitated DNA prior to cloning alleviates this problem. Thirty cycles of T-PCR amplification produced sufficient quantities of DNA for accurate quantification by either densitometry or spectrophotometry (Fig. 1C, lane 5) allowing vector/input DNA ratios to be readily determined. The number of clones obtained using the T-PCR-cloning strategy greatly exceeded that of shotgun cloning. Moreover, a far greater proportion of T-PCR generated clones carried an insert (Fig. 1D); of the 150 colonies
Table 1 Pax7-ChIP-isolated genomic clone locations Entrez gene ID
Location of ChIP fragment
Fragment size (bp)
Number of hits
1 1 1 1 1 5 11
1000 800 600 650 600 1500 1000
1 1 1 3a 1 1 1
Proximal 16976 67487
368 bp upstream 7495 bp upstream
1200 1600
1 1
Distal 14302 621636
13001 bp downstream 98501 bp upstream
800 700
1 1
Shotgun cloned targets Intragenic 12804 Intron 1 231044 Intron 1 30794 Intron 1 271786 Intron 1 433507 Intron 1 328657 Intron 1 75033 Intron 2 16526 Intron 2 19414 Intron 3 67161 Intron 6 170711 Intron 7 238205 Exon 11 (non-coding) 240476 Intron 12 11303 3 0 UTR
1500 600 400 1000 400 650 700 900 800 700 600 400 400 700
1 1 1 1 1 1a 1 1 1 1 1 2 2 1
Proximal 113864 75064 18114 620927 15312 12739
1602 bp 1952 bp 2315 bp 2801 bp 2958 bp 6170 bp
800 400 500 1200 500 500
2 3 2 1 3 2
Distal 19359 14063 18933 67328 12040
11770 bp 20342 bp 41079 bp 52780 bp 64209 bp
850 500 600 400 800
1 1 1 1 1
T-PCR cloned targets Intragenic 14051 Intron 639025 Intron 68421 Intron 328657 Intron 434019 Intron 227541 Intron 20924 Intron
a
upstream downstream upstream upstream upstream upstream downstream upstream downstream upstream downstream
This region of gene 238657 was isolated by both techniques.
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randomly picked from each cloning reaction, 30.6% of shotgun cloned colonies carried inserts, whereas 68% of T-PCR colonies carried inserts. Thus inclusion of a T-PCR amplification step is certainly an effective strategy for increasing the efficiency of cloning of ChIP fragments. Comparison of T-PCR vs shotgun cloned target gene identification The ultimate aim of ChIP-cloning experiments is the identification of putative target genes. This is desirably achieved by the proximity of the isolated DNA fragments to a promoter unit, or within 10 kb of an identified coding region [14,15]. However, a considerable problem with the cloning of genome fragments obtained by ChIP is that often a high proportion of non-specific DNA is isolated [3–5]. We therefore sought to ascertain whether the T-PCR amplification step altered the proportion of ChIP fragments that are in close proximity to coding regions relative to those which are either unlocated within the genome or located distal to any particular coding region (thus considered non-specific). We sequenced all of the clones (N = 47) obtained by shotgun cloning of ChIP fragments and a randomly chosen representative sample (N = 30) of the T-PCR amplified ChIP fragment clones and used BLAST searches to locate the fragments within the mouse genome database (all assemblies) (Table 1). Criteria for annotating the locations of ChIP fragments were; intragenic—contained within coding region of a known or predicted transcription unit (gene), proximal—0 to 10 kb upstream or downstream of a gene, distal—10 to 100 kb upstream or downstream of a gene, or located in an intergenic desert—>100 kb from a gene (Fig. 2C) [15].
BLAST searches of the sequences from these clones identified a pronounced correspondence in the distribution of location of ChIP fragments (relative to genes) between cloning strategies (Fig. 2). Of the fragments that could be singularly located within the mouse genome (build 35.1) by BLAST, the proportion of ChIP fragments located within recognised transcription units (genes) or proximal to them (the best criteria for target gene identification [14]) was 66.7% using shotgun cloning, and 68.8% for clones derived from T-PCR cloning. The composition of locations of the other clones also showed no significant distortion between techniques, with shotgun cloning resulting in 11.9% of clones located distally to genes and 21.4% located in gene deserts (Fig. 2A), and T-PCR cloning resulting in 12.5% of clones located distally, and 18.8% located in gene deserts (Fig. 2B). Chi-square testing confirmed that the distributions of these fragments did not significantly differ between cloning strategies (v2 ¼ 0:90; NS). The only clear distinction between these data was the substantially greater proportion of fragments derived from T-PCR cloning that could not be singularly located within build 35.1 of the mouse genome by BLAST. Only 10.6% of the ChIP fragments obtained by shotgun cloning were not singularly located by BLAST, whereas 46.7% of the T-PCR cloned ChIP fragments tested were not able to be singularly located. To ascertain the identity of the unlocated fragments, fragments were BLAST searched from the non-organism-specific NCBI nr database (representing all GenBank, EMBL, DBJ, and PDB sequences) and all sequences were found to represent multiple hits within the mouse genome, indicating that they were of mouse origin and not the result of contamination. Discontiguous megaBLAST searches were performed on all sequences to check for possible recombination during PCR, and vector
Fig. 2. Comparison of genomic distributions of ChIP clones obtained by T-PCR or shotgun cloning. (A) Positional distribution of T-PCR cloned ChIP fragments and (B) positional distribution of shotgun cloned ChIP fragments. (C) Schematic illustration of positional annotation of ChIP fragments relative to a transcription unit.
R.B. White, M.R. Ziman / Biochemical and Biophysical Research Communications 346 (2006) 479–483
contamination was screened for using VecScreen [13] but did not successfully identify any single location for ChIP fragments. Moreover, to determine whether the primers utilised for T-PCR were selectively amplifying complementary sequences within the mouse genome, the primer sequences were searched for within the mouse genome using short, nearly exact matches BLAST limited to the mouse genome. The best matches obtained by this were three locations matching 15 of the 17 nucleotides of the primer; none of these were in locations identified for ChIP fragments. Thus the majority of ChIP fragments not located within the mouse genome constituted repetitive DNA regions resulting in myriad hits when BLAST searched. It is possible that future further refinements of genome databases may eliminate or mitigate this problem. In summary, T-PCR cloning may be of considerable use in the cloning of ChIP fragments, as it rapidly overcomes the significant methodological challenges presented by the minute quantities of DNA obtained via ChIP. We advise use of this technique to researchers experiencing difficulty with shotgun cloning of ChIP fragments, with the caveat that the sequences obtained may require greater scrutiny. This is, of course, tempered by the fact that T-PCR makes available a vastly increased number of clones from which to choose. Acknowledgments This work was supported by an Edith Cowan University Postgraduate Research Scholarship. The monoclonal antibody developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. References [1] D.E. Phelps, G.R. Dressler, Identification of novel Pax-2 binding sites by chromatin precipitation, J. Biol. Chem. 271 (1996) 7978–7985. [2] A.S. Weinmann, S.M. Bartley, T. Zhang, M.Q. Zhang, P.J. Farnham, Use of chromatin immunoprecipitation to clone novel E2F target promoters, Mol. Cell. Biol. 21 (2001) 6820–6832.
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[3] A.S. Weinmann, P.J. Farnham, Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation, Methods 26 (2002) 37–47. [4] H. Wang, W. Tang, C. Zhu, S.E. Perry, A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos, Plant J. 32 (2002) 831–843. [5] A.S. Weinmann, P.S. Yan, M.J. Oberley, T.H. Huang, P.J. Farnham, Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis, Genes Dev. 16 (2002) 235–244. [6] J.D. Lieb, Genome-wide mapping of protein–DNA interactions by chromatin immunoprecipitation and DNA microarray hybridization, Methods Mol. Biol. 224 (2003) 99–109. [7] S. Mukherjee, M.F. Berger, G. Jona, X.S. Wang, D. Muzzey, M. Snyder, R.A. Young, M.L. Bulyk, Rapid analysis of the DNAbinding specificities of transcription factors with DNA microarrays, Nat. Genet. 36 (2004) 1331–1339. [8] K.E. Boyd, P.J. Farnham, Coexamination of site-specific transcription factor binding and promoter activity in living cells, Mol. Cell. Biol. 19 (1999) 8393–8399. [9] K.D. Johnson, E.H. Bresnick, Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation, Methods 26 (2002) 27–36. [10] N.M. Tsankova, A. Kumar, E.J. Nestler, Histone modifications at gene promoter regions in rat hippocampus after acute and chronic electroconvulsive seizures, J. Neurosci. 24 (2004) 5603–5610. [11] J. Wells, P.S. Yan, M. Cechvala, T. Huang, P.J. Farnham, Identification of novel pRb binding sites using CpG microarrays suggests that E2F recruits pRb to specific genomic sites during S phase, Oncogene 22 (2003) 1445–1460. [12] D. Grothues, C.R. Cantor, C.L. Smith, PCR amplification of megabase DNA with tagged random primers (T-PCR), Nucleic Acids Res. 21 (1993) 1321–1322. [13] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [14] L.A. Boyer, T.I. Lee, M.F. Cole, S.E. Johnstone, S.S. Levine, J.P. Zucker, M.G. Guenther, R.M. Kumar, H.L. Murray, R.G. Jenner, D.K. Gifford, D.A. Melton, R. Jaenisch, R.A. Young, Core transcriptional regulatory circuitry in human embryonic stem cells, Cell 122 (2005) 947–956. [15] Y.H. Loh, Q. Wu, J.L. Chew, V.B. Vega, W. Zhang, X. Chen, G. Bourque, J. George, B. Leong, J. Liu, K.Y. Wong, K.W. Sung, C.W. Lee, X.D. Zhao, K.P. Chiu, L. Lipovich, V.A. Kuznetsov, P. Robson, L.W. Stanton, C.L. Wei, Y. Ruan, B. Lim, H.H. Ng, The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells, Nat. Genet. 38 (2006) 431–440.
A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments Robert B. White, Melanie R. Ziman
*
School of Exercise, Biomedical and Health Science, Edith Cowan University, Joondalup Drive, WA 6027, Australia Received 18 May 2006 Available online 2 June 2006
Abstract The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments. 2006 Elsevier Inc. All rights reserved. Keywords: Target gene identification; Pax7; T-PCR; ChIP
Chromatin immunoprecipitation (ChIP) has rapidly become a significant weapon in the arsenal of methodologies used for deducing transcription factor function. The majority of ChIP assays currently performed provide a rapid in vivo or cell culture-based verification of occupation of the promoters of known or predicted target genes by a given transcription factor. However, in more exploratory studies, ChIP assays are now being used for the identification of novel target genes of a transcription factor, in a genome-wide context. Identification of novel target genes using an unbiased genome-wide ChIP screen has taken two main forms to date—ChIP genomic fragments are cloned and sequenced [1–4] or used for microarray hybridisation [3,5,6]. An unbiased microarray (which must comprise the entire genome) has significantly enhanced our understanding of yeast
*
Corresponding author. Fax: +61 8 6304 5717. E-mail address: [email protected] (M.R. Ziman).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.145
genomic function [6,7], but has yet to be produced for mammalian genomes. Cloning of ChIP DNA fragments presents significant difficulties, particularly the technical challenge associated with shotgun cloning minute quantities of DNA obtained from ChIP experiments. While the presence of immunoprecipitated DNA can be checked by promoter-specific PCR for a bona fide target [4,8], this is not possible for transcription factors that have not previously been studied to that degree. Here we describe a protocol adapting a simple random PCR amplification of ChIP fragments which greatly enhances the efficiency of cloning of novel targets. ChIP fragments are subjected to tagged-random PCR (T-PCR), involving preamplification using primers designed with a random dodecameric sequence at the 3 0 end (allowing binding to an array of target sequences) and a constant 5 0 tail. After two rounds of extension, the unbound primers are removed, and PCR amplification is carried out with a primer specific to the 5 0 constant region of the initial primers, allowing exponential amplification of ChIP DNA,
480
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which is then cloned. We have adapted this method for use with mouse embryonic tissue and compare this method with a shotgun-cloning approach in terms of efficiency of cloning as well as efficiency of success in target gene identification (deduced from proximity of ChIP fragment to a known/predicted coding region). Materials and methods Immunoprecipitation—Western blot. Pre-cleared soluble lysates from whole embryos of C57BL/6J mice were immunoprecipitated with 1 lg anti-Pax7 monoclonal antibody (Developmental Studies Hybridoma Bank—DSHB, Iowa City, IA, USA) and protein G-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) using standard protocols (Santa Cruz). Equivalent volumes (10 lL) of immunoprecipitates, flowthroughs (lysates depleted by Pax7-MAb-proteinG-agarose IP), and embryo lysates were separated on 12% acrylamide/bis gels and transferred to a nitrocellulose membrane. Blocked membranes were probed with antiPax7 and detected with IgG-HRP using the ECL+ plus Western Blot Detection Kit (Amersham Biosciences UK Ltd., Buckinghamshire, England). Chromatin immunoprecipitation. This work was authorised by the Edith Cowan University Animal Ethics Committee and conducted in accordance with the guidelines set by the National Health and Medical Research Council of Australia. The ChIP procedure used was an optimised modification of several published protocols [9–11]. Whole embryonic day 16 (E16) embryos from C57BL/6J mice were minced to 1 mm-sized pieces, and immediately cross-linked in 1% paraformaldehyde in PBS for 15 min at room temperature then quenched by addition of 0.125 M glycine, washed once in PBS with 0.125 M glycine and twice with PBS. Minced, fixed tissue was Dounce homogenised (Bellco Glass, Inc., Vineland, NJ, USA) in cell lysis
buffer (10 mM Tris, 10 mM NaCl, 0.2% NP-40, and 1 mM each of protease inhibitors PMSF, aprotinin, pepstatin A) and after centrifugation, the pellet was homogenised in nuclei lysis buffer (50 mM Tris–HCl, pH 8.1, 10 mM EDTA, 1% SDS, 10 mM sodium butyrate, and protease inhibitors). The remaining chromatin was sonicated to an empirically determined average length of 800 bp using a Sonifier 450 (power output 2, 10% duty cycle) (Branson, Danbury, CT, USA). Debris was removed by centrifugation and supernatant was diluted in IP dilution buffer (20 mM Tris–HCl, pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, 10 mM sodium butyrate, and proteinase inhibitors) and pre-cleared by addition of 200 lL protein G-agarose for 2 h at 4 C under rotation. Cleared chromatin was divided into 600 lL aliquots and incubated overnight at 4 C by rotation with 2 lg of anti-Pax7 monoclonal antibody, or no antibody for control treatment. Immunocomplexes were collected by rotation at 4 C for 3 h with 120 lL protein G-agarose. Beads were washed with low salt buffer (20 mM Tris–HCl, pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS), high salt buffer (10 mM Tris–HCl, pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, and 1% deoxycholic acid) and twice with TE buffer. Immunocomplexes were eluted with elution buffer (1% SDS, 100 mM NaHCO3), two identical samples were combined, and cross-links were reversed by incubation at 65 C for 8 h in the presence of 0.3 M NaCl, then 80 lg Proteinase K (Qiagen, Valencia, CA, USA) was added and samples were incubated at 45 C for 2 h. DNA was purified using QIAquick spin columns (Qiagen) and two identical samples were pooled. Shotgun ChIP cloning. ChIP fragments were cloned using the TOPO Shotgun Subcloning Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, a 45 lL ChIP solution was made bluntended (T4 and Klenow DNA polymerases) and dephosphorylated (calf intestinal phosphatase), phenol/chloroform/ethanol purified, cloned into pCR 4-Blunt-TOPO vector, and chemically transformed into OneShot TOP10 Escherichia coli cells. Tagged-random-PCR amplification cloning. Tagged-random PCR (T-PCR) was performed on ChIP-isolated DNA using tagged-random
Fig. 1. ChIP-cloning procedure. (A) Schematic diagram of ChIP procedure from embryos followed by cloning of immunoprecipitated DNA by either shotgun cloning or T-PCR cloning. (B) Western blot of Pax7 immunoprecipitates from whole embryos: lanes 1, IP-flow-through; 2, no antibody control IP; 3, cleared embryo lysate (no IP); 4, Pax7 IP. (C) T-PCR amplified ChIP-immunoprecipitated DNA: lanes 1, no first round PCR control; 2, no second round PCR control; 3, no DNA control; 4, sonicated input DNA; 5, T-PCR amplified ChIP products; M, 1 kb-plus DNA ladder (Invitrogen). (D) Screening of clones—agarose gels showing several representative clones obtained by either T-PCR or shotgun-cloning methods, and their relative size distributions; M, 1 kb-plus DNA ladder.
R.B. White, M.R. Ziman / Biochemical and Biophysical Research Communications 346 (2006) 479–483 primers to amplify DNA present in immunoprecipitates (to increase the efficiency of vector cloning) [6,12]. T-PCR consists of two rounds of amplification, the first of which uses specific tagged-random dodecamer primers to synthesise tagged copies of ChIP fragments, followed by second strand synthesis of these copies. The second round amplifies tagged products using primers complementary to the tags. The first round was performed in a 20 lL reaction containing 15.9 lL ChIP DNA, 200 lM dNTP, 3 lM primer A (GTTTCCCAGTCACGATCN12), and 1· Klenow polymerase reaction buffer (0.5 M Tris–HCl, pH 7.5, 0.1 M MgCl2, 10 mM DTT, and 0.5 mg/mL BSA). The reaction was heated to 94 C for 2 min, cooled to 4 C for 5 min, then 4 U Klenow polymerase (USB Biochemical, Cleveland, OH, USA) was added and the reaction was ramped at 0.1 C/min to 37 C and extended for 20 min. Second strand synthesis was then performed by heating the reaction to 94 C for 4 min, followed by cooling to 4 C for 5 min, addition of a further 4 U Klenow polymerase, ramping at 0.1 C/min to 37 C, and extension for 20 min. First round primers were removed using QIAquick spin columns, DNA was eluted with dH2O, and 1/10th of the elution volume (2 lL) was PCR amplified in a 20 lL PCR containing 0.5 U Taq polymerase (Qiagen), 1· Q-solution (Qiagen), 2 lM primer B (GTTTCCCAGTCACGATC), and 200 lM each dNTP for 30 cycles of 30 s at 94 C, 30 s at 40 C, 30 s at 50 C, and 2 min at 72 C, followed by a final extension of 72 C for 10 min. Second round T-PCR products were column purified, blunt ended by a 15 min room temperature incubation with 5 U Klenow polymerase in 1· Klenow reaction buffer with 0.5 mM each dNTP, column purified, spectrophotometrically quantified, and 100 ng was cloned into pCR 4-Blunt-TOPO vector (Invitrogen) and transformed into OneShot TOP10 E. coli cells (Invitrogen). All PCRs were performed in an MJ Research PTC-100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) with a heated lid. Clones were randomly selected and analysed by PCR with vector primers (T7 forward and reverse) and electrophoresed on 1.5% agarose gel. Clones carrying inserts >500 bp were sequenced from T7 forward priming sites using BigDye Terminator (Applied Biosystems, Foster City, CA, USA). Data analysis. Sequences had vector sequences removed using VecScreen and were located via megaBLAST of the mouse genome database (all assemblies, build 35.1) and nr databases, available at the National Centre for Biotechnology Information (NCBI) [13]. Chi-square tests were performed using Microsoft Excel 2003.
Results and discussion Chromatin immunoprecipitation from mouse embryos As the monoclonal antibody utilised in this experiment had not previously been validated for use in immunoprecipitation, we first sought to determine whether Pax7 protein would be enriched in immunoprecipitates from mouse embryos. Western blots of mouse whole embryonic lysates immunoprecipitated with monoclonal Pax7 antibody show a substantial enrichment (Fig. 1B; lane 4) and the flow-through from these immunoprecipitates displays an almost complete depletion of Pax7 protein (Fig. 1B; lane 1), indicating high efficiency immunoprecipitation by this monoclonal antibody. T-PCR cloning The minute quantities of DNA obtained after ChIP make DNA quantification and thus optimisation of vector to insert DNA ratios difficult, necessitating substantial optimisation for each individual ChIP experiment. T-PCR
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amplification of ChIP-immunoprecipitated DNA prior to cloning alleviates this problem. Thirty cycles of T-PCR amplification produced sufficient quantities of DNA for accurate quantification by either densitometry or spectrophotometry (Fig. 1C, lane 5) allowing vector/input DNA ratios to be readily determined. The number of clones obtained using the T-PCR-cloning strategy greatly exceeded that of shotgun cloning. Moreover, a far greater proportion of T-PCR generated clones carried an insert (Fig. 1D); of the 150 colonies
Table 1 Pax7-ChIP-isolated genomic clone locations Entrez gene ID
Location of ChIP fragment
Fragment size (bp)
Number of hits
1 1 1 1 1 5 11
1000 800 600 650 600 1500 1000
1 1 1 3a 1 1 1
Proximal 16976 67487
368 bp upstream 7495 bp upstream
1200 1600
1 1
Distal 14302 621636
13001 bp downstream 98501 bp upstream
800 700
1 1
Shotgun cloned targets Intragenic 12804 Intron 1 231044 Intron 1 30794 Intron 1 271786 Intron 1 433507 Intron 1 328657 Intron 1 75033 Intron 2 16526 Intron 2 19414 Intron 3 67161 Intron 6 170711 Intron 7 238205 Exon 11 (non-coding) 240476 Intron 12 11303 3 0 UTR
1500 600 400 1000 400 650 700 900 800 700 600 400 400 700
1 1 1 1 1 1a 1 1 1 1 1 2 2 1
Proximal 113864 75064 18114 620927 15312 12739
1602 bp 1952 bp 2315 bp 2801 bp 2958 bp 6170 bp
800 400 500 1200 500 500
2 3 2 1 3 2
Distal 19359 14063 18933 67328 12040
11770 bp 20342 bp 41079 bp 52780 bp 64209 bp
850 500 600 400 800
1 1 1 1 1
T-PCR cloned targets Intragenic 14051 Intron 639025 Intron 68421 Intron 328657 Intron 434019 Intron 227541 Intron 20924 Intron
a
upstream downstream upstream upstream upstream upstream downstream upstream downstream upstream downstream
This region of gene 238657 was isolated by both techniques.
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randomly picked from each cloning reaction, 30.6% of shotgun cloned colonies carried inserts, whereas 68% of T-PCR colonies carried inserts. Thus inclusion of a T-PCR amplification step is certainly an effective strategy for increasing the efficiency of cloning of ChIP fragments. Comparison of T-PCR vs shotgun cloned target gene identification The ultimate aim of ChIP-cloning experiments is the identification of putative target genes. This is desirably achieved by the proximity of the isolated DNA fragments to a promoter unit, or within 10 kb of an identified coding region [14,15]. However, a considerable problem with the cloning of genome fragments obtained by ChIP is that often a high proportion of non-specific DNA is isolated [3–5]. We therefore sought to ascertain whether the T-PCR amplification step altered the proportion of ChIP fragments that are in close proximity to coding regions relative to those which are either unlocated within the genome or located distal to any particular coding region (thus considered non-specific). We sequenced all of the clones (N = 47) obtained by shotgun cloning of ChIP fragments and a randomly chosen representative sample (N = 30) of the T-PCR amplified ChIP fragment clones and used BLAST searches to locate the fragments within the mouse genome database (all assemblies) (Table 1). Criteria for annotating the locations of ChIP fragments were; intragenic—contained within coding region of a known or predicted transcription unit (gene), proximal—0 to 10 kb upstream or downstream of a gene, distal—10 to 100 kb upstream or downstream of a gene, or located in an intergenic desert—>100 kb from a gene (Fig. 2C) [15].
BLAST searches of the sequences from these clones identified a pronounced correspondence in the distribution of location of ChIP fragments (relative to genes) between cloning strategies (Fig. 2). Of the fragments that could be singularly located within the mouse genome (build 35.1) by BLAST, the proportion of ChIP fragments located within recognised transcription units (genes) or proximal to them (the best criteria for target gene identification [14]) was 66.7% using shotgun cloning, and 68.8% for clones derived from T-PCR cloning. The composition of locations of the other clones also showed no significant distortion between techniques, with shotgun cloning resulting in 11.9% of clones located distally to genes and 21.4% located in gene deserts (Fig. 2A), and T-PCR cloning resulting in 12.5% of clones located distally, and 18.8% located in gene deserts (Fig. 2B). Chi-square testing confirmed that the distributions of these fragments did not significantly differ between cloning strategies (v2 ¼ 0:90; NS). The only clear distinction between these data was the substantially greater proportion of fragments derived from T-PCR cloning that could not be singularly located within build 35.1 of the mouse genome by BLAST. Only 10.6% of the ChIP fragments obtained by shotgun cloning were not singularly located by BLAST, whereas 46.7% of the T-PCR cloned ChIP fragments tested were not able to be singularly located. To ascertain the identity of the unlocated fragments, fragments were BLAST searched from the non-organism-specific NCBI nr database (representing all GenBank, EMBL, DBJ, and PDB sequences) and all sequences were found to represent multiple hits within the mouse genome, indicating that they were of mouse origin and not the result of contamination. Discontiguous megaBLAST searches were performed on all sequences to check for possible recombination during PCR, and vector
Fig. 2. Comparison of genomic distributions of ChIP clones obtained by T-PCR or shotgun cloning. (A) Positional distribution of T-PCR cloned ChIP fragments and (B) positional distribution of shotgun cloned ChIP fragments. (C) Schematic illustration of positional annotation of ChIP fragments relative to a transcription unit.
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contamination was screened for using VecScreen [13] but did not successfully identify any single location for ChIP fragments. Moreover, to determine whether the primers utilised for T-PCR were selectively amplifying complementary sequences within the mouse genome, the primer sequences were searched for within the mouse genome using short, nearly exact matches BLAST limited to the mouse genome. The best matches obtained by this were three locations matching 15 of the 17 nucleotides of the primer; none of these were in locations identified for ChIP fragments. Thus the majority of ChIP fragments not located within the mouse genome constituted repetitive DNA regions resulting in myriad hits when BLAST searched. It is possible that future further refinements of genome databases may eliminate or mitigate this problem. In summary, T-PCR cloning may be of considerable use in the cloning of ChIP fragments, as it rapidly overcomes the significant methodological challenges presented by the minute quantities of DNA obtained via ChIP. We advise use of this technique to researchers experiencing difficulty with shotgun cloning of ChIP fragments, with the caveat that the sequences obtained may require greater scrutiny. This is, of course, tempered by the fact that T-PCR makes available a vastly increased number of clones from which to choose. Acknowledgments This work was supported by an Edith Cowan University Postgraduate Research Scholarship. The monoclonal antibody developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. References [1] D.E. Phelps, G.R. Dressler, Identification of novel Pax-2 binding sites by chromatin precipitation, J. Biol. Chem. 271 (1996) 7978–7985. [2] A.S. Weinmann, S.M. Bartley, T. Zhang, M.Q. Zhang, P.J. Farnham, Use of chromatin immunoprecipitation to clone novel E2F target promoters, Mol. Cell. Biol. 21 (2001) 6820–6832.
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