Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo
- Ronaldo Schuch†1, 5Email author,
- Konstantin Agelopoulos†2,
- Anna Neumann1,
- Burkhard Brandt3,
- Horst Bürger4 and
- Eberhard Korsching1
© Schuch et al; licensee BioMed Central Ltd. 2012
Received: 14 November 2011
Accepted: 20 February 2012
Published: 20 February 2012
Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs.
The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue.
The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme digestion enables a reliable and individual detection option for densely arranged binding motifs in vivo not provided by e.g. EMSA or in vivo footprinting. Given the important function of transcription factors in neoplastic mechanism, our method enables a broad diversity of application options for clinical studies.
Transcription factors (TFs) are core elements of transcriptional regulation and play also an important role in the systems biology of cancer characterized by changes in the expression levels of certain genes . A complex of more than 20 TFs molecules is involved in RNA polymerase II initiation of transcription in the promoter region for the majority of the genes . The activity of the transcription machinery is based on the arrangement and the occupancy of transcription factor binding sites (TFBSs) along the 5'-region of the gene. Because of this dense arrangement and the necessity to analyze the individual occupancy of the TFBSs to establish regulation models, there is a strong demand for methods that enable this type of individual analysis.
So far, several methods have been developed for identification and analysis of TFBSs. A commonly used method to verify TFBSs is the electrophoretic mobility shift assay (EMSA) . Indeed, even though the gel mobility shift analysis provides a fast and easy identification of which nucleotides are required for TF binding, it does not work under in vivo conditions . On the other hand, the method of in vivo footprinting  enables the investigation of protein binding in living cells, but this technique is only capable of identifying DNA regions that are bound by protein, being not able to identify which protein is responsible for the observed footprint [4, 6].
Thus, the aim of this study was to develop and optimize a ChIP technique for the specific and individual analysis of neighboring TFBSs at once in both cell culture and tissue material.
In order to support the individual analysis of adjacent TFBSs, we have developed an improved ChIP assay by replacing the traditionally used random fragmentation step with a site-specific enzyme digestion. This site-specific ChIP allows the immunoprecipitation and enrichment of DNA fragments containing only one TFBS, thus permitting to distinguish between adjacent occupied and non- occupied binding sequences in vivo. The principle of the site-specific ChIP and its advantages over similar procedures like EMSA and in vivo footprinting is exemplified in Figure 1.
Results of Sp1-TFBSs verification with MatInspector
position [bp] ‡
core similarity §
matrix- similarity $
capitals: core sequence †
Primer used for analysis of site-specific ChIP products
Sp1 binding site a
Sp1 binding site b
Rev: 5'- AGGGCGGGAGGAGGAGGGAC-3
Sp1 binding site c
Sp1 binding site d
RsaI cutting site
BfaI cutting site
BfaI 2nd cutting site
BfaI 3rd cutting site
SacI cutting site
GAPDH unrelated region
Analysis of the allocation of Sp1 binding sites in EGFR expressing cells
Application of Sp1-directed site-specific ChIP on EGFR expressing tissue samples
Even in the complex sequence environment of the egfr, the site-specific ChIP proved to be an adequate and effective method for the individual analysis of the TFBSs in vivo. The site-specific ChIP provides an improvement towards the standard ChIP method and further techniques for TFBS analysis. It unites the benefits of both EMSA and in vivo DNase I footprinting--the specific detection and localization of neighboring occupied binding sites in vivo--in one assay. Hence, the usually performed verification of the obtained results by a second method is therefore unnecessary (Figure 1). In combination with the enzyme digestion control by PCR using primers targeting regions spanning each enzyme cutting site, the site-specific ChIP may be generally applied for the investigation of any transcription factor recognition site along the human genome.
Completeness in a molecular sense can only be achieved by analysis methods which in situ detect all possible realised combinations of truly existing binding sites. No modern technology (including next generation sequencing) can assure that up to now. So our approach reasonably approximates this completeness. Though, in combination with a preceded consensus analysis using an adequate algorithm (like MatInspector) our site-specific ChIP is an effective method for the selective identification of complex TFBSs structures.
For all restriction sites we used restriction enzymes (BfaI, RsaI and SacI) which exhibit exactly the same digestion and buffering conditions (incubation at 37°C and 100% activity in the following restriction buffer: 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM Magnesium Acetate, 1 mM Dithiothreitol pH 7.9 at 25°C). The concern that TFBSs might not be flanked by naturally occurring restriction enzyme sites and not separated by an appropriate distance from each other seems, in accordance to our experience, to be a rare event. At least in the case of the egfr, there were no complications.
The suitability of the used anti-Sp1-Antibody for an application in the ChIP-procedure has been checked and assured by the supplier (Merck Millipore Co., Billerica, MA). The utilized buffers are common to all antibody based ChIP assays.
By adjusting sample preparation and chromatin isolation procedures the technique is also applicable on tissue material, enabling a broad diversity of application options for clinical and molecular studies.
The site-specific ChIP is not dedicated to high throughput screening approaches but instead supports the functional analysis of a complex regulation scheme of a single gene in a systems biology view - i.g. the interaction pattern between occupied TFBSs. The focus of this work is develop a method for a) the detection of new binding sites in combination with a preceded consensus analysis and b) the individual examination of single TFBSs allocation in a complex system of neighboring binding motifs of the same type. Hence, the site-specific ChIP technique does not provide an assessment of whether the TF-binding is associated with gene transcription, what is a common limitation of all ChIP-variations. For answering the question of functional relevance further methods have to be employed. E.g. site directed mutagenesis or deletion analysis for TFBSs and promoter activity investigation in vivo. So, the main benefit of the site-specific ChIP lies in the investigation of specific regulatory regions in greater detail.
The site-specific ChIP, which uses an endonuclease-based TFBSs specific DNA fragmentation followed by a PCR-based enzyme digestion control, opens new possibilities for the functional investigation of complex neighboring TFBSs systems of genes, even within GC-rich regions. In combination with methods for TFBSs prediction [16–18], chip-techniques and/or sequencing, it is a specific and sensitive tool for the detailed characterization of the activity of neighboring binding motifs at once. The site-specific ChIP enables the individual and reliable analysis of known and predicted binding motifs in vivo, on both cultured cells and mammalian tissue material. Hence, our method enables the detection of differences in the interaction of transcription factors and promoter regions of genes in normal and neoplastic tissues, thereby opening new possibilities for the investigation of the transcriptional regulation of genes involved in cancer biology.
Site-specific Chromatin immunoprecipitation
For the cell line experiments we used 107 osteosarcoma cells which were fixated by a 1% formaldehyde solution. The Nuclei were isolated using cell lysis buffer, separated by centrifugation and resuspended in adequate standard restriction buffer (New England Biolabs Inc., Ipswich, MA). Chromatin was fragmented by subjecting the nuclei to restriction enzyme digestion according to Kang et al. , including the following modifications. A simultaneous application of three restriction endonucleases - BfaI, RsaI and SacI (New England Biolabs Inc., Ipswich, MA) - was performed to cleave the DNA at positions flanking each Sp1 binding site within egfr intron 1. A complete DNA digestion was achieved by chromatin treatment with 200 U of each enzyme for 4 h at 37°C and further 100 U of each enzyme for additional 16 h at 37°C. The nuclei were then incubated with and 200 U aliquot of each enzyme and 200 U of RNase for 2 h at 37°C. Completion of restriction enzyme fragmentation was verified by electrophoresis separation on a 1.5% agarose gel. The optimization of the specific enzyme digestion based DNA fragmentation is shown on Additional file 1: Figure S1. Chromatin was isolated using nuclei lysis buffer. The lysate was diluted 10-fold in ChIP dilution buffer and equal aliquots of cleaved chromatin (equivalent to 2 million cells) from a single cell lysate were used for immunoprecipitation with antibodies against Sp1 and IgG (mock IP) as well as a control for the amount of input DNA used in precipitations (input control). 3 μg of the antibodies against Sp1 (Merck Millipore Co., Billerica, MA) and IgG (Sigma-Aldrich Co., St. Louis, MO) were used. The antibody was captured with protein A/G agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After washing the beads-antibody-chromatin complexes under stringent conditions, reverse crosslinking and purification of ChIP DNA was performed using Chelex-100 according to Nelson et al. . DNA from the non-antibody whole-cell control supernatant was isolated as described in literature  and processed in the same way as the IP samples. All steps not mentioned here were performed according to established standards .
The tissues samples were processed as follows. 0.03 g frozen kidney carcinoma tissue was chopped into small pieces and thawed in freshly prepared PBS containing 1% formaldehyde for crosslinking. The tissue was homogenized to avoid cell clumps and the ChIP was performed as described above for the cell culture experiments.
PCR based detection of immunoprecipitated TFBSs
The detection of the immunoprecipitated Sp1 binding sites was done by PCR. For each sample (DNA extracted from either the input chromatin, the normal rabbit IgG or the anti-Sp1-immunoprecipitated chromatin): 2.5 μl ChIP-DNA template, 5 pmol of Sp1 binding site specific primer, 1 U of Taq DNA Polymerase, 1X PCR reaction buffer II, 400 μM of each dNTP (Applied Biosystems) and 1.3 mM MgCl2 were mixed for PCR amplification in a 25 μl reaction volume. A defined amount of 4 ng lymphocyte DNA was used for the internal control of each PCR reaction approach. The PCR was started with an initial denaturation of 95°C for 9 min, followed by a secondary denaturation at 98°C for 1 min. Next 40 cycles of denaturation at 98°C for 10 sec, annealing at 65°C for 2 min, and a single product extension step at 72°C for 7 min followed. The PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining.
PCR based controls of site-specific ChIP
The control of completeness of the enzyme digestion control was also performed by PCR as described and carried out on DNA templates prepared from the Sp1 immunoprecipitated chromatin sample and the whole-cell sample (input control), as well as uncleaved HOS cells DNA from the same lysate (positive control).
For the negative control, primers spanning an unrelated genomic region within the 3'-UTR of the GAPDH gene were used yielding a 97-bp product.
Availability and requirements
Project name: Site-specific chromatin immunoprecipitation: A selective method to individually analyze neighboring transcription factor binding sites in vivo
Project home page: none
Operating systems: none
Programming language: none
Other requirements: MatInspector V7.4.8 (http://www.genomatix.de)
Any restriction to use by non-academics: none
Data were analyzed anonymously. Nonetheless we have performed the study according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of Muenster. We have acquired tissue samples only with the informed consent of the patients or patients' next of kin with the understanding by all parties that it may well be used for research. All patients provided written informed consent for the collection of samples and subsequent analysis.
- Egfr :
Epidermal Growth Factor Receptor gene
transcription factor binding sites.
Special thanks go to Ricardo Schuch, for critical review of the manuscript. This study was funded in part by the Federal German Ministry of Research (Grant No. 01GM0869 TranSaRNet) and the European Union-European Network of Excellence for studying the pathology and genetics of bone tumors (Grant No. LSHCCT-2006-018 814 EuroBoNeT).
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