CTCF is a highly conserved eleven zinc finger transcription factor, which specially binds to various divergent DNA sequences. This multifunctional and ubiquitously expressed transcription factor is a main factor in regulating cell growth, as it controls major regulators of cell proliferation, for instance, activates p14ARF/p19ARF and represses c-myc genes. The CTCF gene itself is re-arranged in several clinical tumour samples and cancer cell lines. Therefore, indirect evidence implies that CTCF could be a new tumour suppressor gene.
In order to address the role of CTCF in tumorigenesis, it is vital to explore the binding of CTCF to its DNA targets in clinical tissue samples. For this purpose, chromatin immunoprecipitation (ChIP) assays can be utilized. This article examines the use of the Chromatrap® Pro-A 1ml columns based on a novel porous polyethylene solid matrix, BioVyon™ Protein A, for ChIP assays from complex breast tissues.
Method and Materials
ChIP assays were performed as per the Chromatrap® Pro-A 1ml column protocol using gravity feed. In short, fresh paired breast tissues were crossed-linked with 1% of formaldehyde, quenched and chromatin was trimmed until fragments of roughly 500 to 750bp were achieved. The chromatin was sonicated and its diluted lysates were incubated overnight with me3H3K27, me3H3K4, CTCF, Pol 2, CTCF, c-MYC, PARP1, SP1, WT1 and Egr1 antibodies.
As a negative control, an immunoprecipitation reaction without antibodies was run separately under the same experimental conditions. Following the incubation, Chromatrap® Pro-A 1ml columns were used to capture the complexes, reverse cross-linked, and DNA was isolated with chloroform and phenol.
Real-time PCR was performed using 300nM primers diluted to 25µl in SensiMix Plus SYBR (Quantace) and 2µl of the immunoprecipitated DNA sample or input DNA. Percentage of precipitated DNA was quantified as follows:
input= AEA(Ctinput - CtChIP) x Fd x 100% (AE is amplification efficiency; Ctinput and CtChIP are threshold values acquired from exponential phase of qPCR; Fd is a dilution factor utilized for normalisation)
Enrichment folds were determined as the percentage of input chromatin precipitated at the area of interest and presented as fold change corresponding to the control ChIP experiment without antibody (designated as 1.0). Real-time PCR experiments were carried out three times and the mean value was demonstrated. Error bars signify standard deviations.
Results and Discussion
Figure 1. CT5 in p/4AR7 is characterized by enrichment in CTCF, PARP1 and Pol2 binding and higher levels of me3H3K4 in normal tissue (1492N, Normal) compared to tumour tissue (1492 T, Tumour).
Figure 2. (a) Real Time -PCR analyses using primers to amplify CTS-1; (b) Real Time -PCR analyses using primers to amplify CTS-2.
Chromatrap® Pro-A 1ml columns were used to perform the ChIP experiments to evaluate the occupancy of RNA Polymerase II (Pol 2), CTCF, ERG1, me3H3K4, me3H3K27, PARP1, SP1, c-MYC and WT1 antibodies at the CTCF target sites (CTSs) in the p14ARF (one CTS) and Bax (two CTSs, CTS-1 and CTS-2) promoter regions. The samples of ChlP and DNA collected from tumour and normal tissues were compared for the enrichment of the three CTSs utilizing Real-time PCR with results shown in Figures 1 and 2.
In Figures 2a and 2b, the CTS-1 and CTS-2 in Bax are characterized by enrichment in c-MYC, EGR1 and SP1 antibodies in normal tissue (N1094, Normal), while WT1 and CTCF are enriched in tumour tissues (T1094, Tumour).
The ChIP experiments detailed in this article show that Chromatrap® Pro-A 1ml columns can be easily utilized and offer superior enrichment with complex breast tissue samples. Due to the inert solid BioVyon™ Protein A matrix, background binding is reduced. This porous polyethylene solid matrix ensures excellent sample mixing and washing during the assay. The Chromatrap® Pro-A 1ml columns can be effectively used with gravity flow or vacuum for faster throughput.
Produced from articles provided by Chromatrap®.
Chromatrap® is a product of Porvair Sciences, a wholly owned subsidiary of Porvair plc. We are one of the largest manufacturers of Ultra-Clean microplates, 96 well well filtration plates and Microplate handling equipment for life science and synthetic chemistry. With offices and Class VIII clean room manufacturing located in the UK, combined with a world-wide network of distributors and dedicated distribution hub in the USA, we pride ourselves on our continuous innovation, research and flexibility to meet customer demands. We offer OEM production and contract manufacturing through our North Wales facility.
Our porous polymeric material, BioVyon™, whose chemical functionalisation can endow it with internal surface properties individually configured to capture and separate target species out of difficult mixtures, has opened up many possibilities in the field of BioSciences where molecules of interest such as DNA, RNA, proteins etc can be selectively pulled out of complex mixtures of biological origin. The materials have proven to be a remarkably good substrate for accepting novel chemistries such as the organically bound Protein A and Protein G in Chromatrap®.
Using our 25 years experience of microplate manufacturing, Porvair Sciences has now developed a high-throughput bead-free ChIP assay based on our filtration plates containing our Chromatrap chemistry. Chromatrap-96 enables large scale epigenetic screening to become a reality in many laboratories and eliminates many of the long and laborious steps previously undertaken in such work.
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