Solid-state ChIP Platform for High Throughput Analysis of Gene Regulation

Introduction

In recent years, epigenetics has become an increasingly important focus in the worlds of academia and industry as researchers try to better understand the fundamental s of transcription regulation. Developments in high throughput ChIP together with the use of automated liquid handing and next generation sequencing is laying the foundations for analysis of global gene expression and understanding the epigenetic landscape in a huge number of cell types across a variety of diseases and treatments.

To this end, chromatin immunoprecipitation (ChIP) has emerged as an important assay in understanding how protein regulators interact with their DNA targets. This article shows the remarkable potential of Chromatrap® spin column technology in the field of epigenetics. The technology uses a novel solid phase technique to provide a microplate platform with 96 wells for large scale analysis using high throughput chromatin immunoprecipitation (ChIP).

ChIP Platform vs. Solid Phase ChIP Platform

Most current ChIP platforms use magnetic or agarose beads, which presents difficulties such as washing techniques that cause sample loss, low specificity in antibody binding, and high background.  Solid phase immunoprecipitation of the chromatin using the Chromatrap® spin system offers highly sensitive and rapid analysis at low sample volume to allow targeting of epigenetic regulatory mechanisms in the presence of low abundance transcription factors.

In addition to the technical advantages over bead-based assays, Chromatrap 96 HT enables 96 samples to be processed in a single day and can be combined with automatic liquid handling to reduce processing time. Moreover, the 96-well platform allows parallel testing of multiple cell types together with treatments of interest. Various antibody targets can also be enriched on the same plate, providing a large spectrum of data and allowing simultaneous observation of effects within one single experiment.

The Chromatrap 96 HT system can also be used with ChIPseq techniques, which has the particular advantage in pharmaceutical and industrial settings of producing large amounts of reliable data in a timely and cost effective way.

In order to demonstrate the sensitivity and efficiency of Chromatrap 96 HT, an experiment was designed to perform the simultaneous immunoprecipitation of various cell lines alongside low abundance transcription factors and associated methylation marks.

Experimental Framework

Chromatin was prepared from five different cancer cell lines (Table 1) according to the Chromatrap® protocol and using the kit reagents. Yield and shearing efficiency were tested using aliquots form each isolated chromatin, enabling the chromatin input to be standardized and aiding comparison of cross- sample signals.

The enriched DNA was then analysed using qPCR analysis and data generated for both negative and positive gene targets. The results in figure 3 demonstrate reliability and reproducibility of the ChIP assay.

Table 1: Cell lines included in the study

Cell Line Cell Type ReferenceCell Line

Cell Type

HepG2

 Human liver hepatocellular carcinoma cell

K562

 Human chronic myelogenous leukemia cell line

HeLa

 Human cervical adenocarcinoma cell line

Ishikawa

 Human endometrial adenocarcinoma cell line

MCF7

Human breast adenocarcinoma cell line

Using the Chromatrap 96 HT system, the chromatin from the five cell lines was immunoprecipitated against epigenetic marks H3K4me3 and H3K27me3 alongside the low abundance transcription factor EZH2. Each combination of antibody and chromatin was performed three times to show the reproducibility of the enrichment.

Table 2 shows the details of the target antibodies and the related gene loci. H3K27me3 is associated with repressing gene transcription while H3K4me3 is associated with enhancing transcription.

Table 2: Antibody targets and gene loci included in this study

Antibody

Positive Gene Targets

Negative Gene Targets

RNA Pol II

GAPDH

ß-globin

H3K4me3

 GAPDH

 ß-globin

H3K27me3

 ß-globin, MYT1

 GAPDH, ZNF333

EZH2

 MYT1

 ZNF333

Negative control gene targets as well as non-specific IgG from the same source as the antibodies tested were included in the study to demonstrate the low non-specific background achieved with the Chromatrap® system.

A major advantage of the Chromatrap® 96 HT solid phase system over traditional kits is the ability to proceed straightaway with downstream processing without the need for DNA cleanup.

Results and Discussion

To show the use of the Chromatrap® 96 HT solid phase system in producing reliable, and reproducible ChIP results quickly, common transcription factors and epigenetic marks from five cancer cell lines underwent enrichment using the platform. The data shown here provide only a glimpse of the data that can be achieved using this system.

Figure 3 shows the value of parallel immunoprecipitation of chromatin from different cell lines or treatments utilizing high throughput ChIP

Figure 3. Positive and negative GAPDH promoter enrichment of chromatin obtained from five different human cancer cell lines using antibodies directed against H3K4me3 and H3K27me3.

Figure 3. Positive and negative GAPDH promoter enrichment of chromatin obtained from five different human cancer cell lines using antibodies directed against H3K4me3 and H3K27me3.

Figure 4. Histone methylation signal precipitated at the B-globin promoter region of five different human cancer cell lines using antibodies directed against H3K4me3 and H3K27me3.

Figure 4. Histone methylation signal precipitated at the B-globin promoter region of five different human cancer cell lines using antibodies directed against H3K4me3 and H3K27me3.

The excellent enrichment of MYT1 that was achieved after immunoprecipitation with EZH2 and H3K27me3 , with very little change between repeated cycles, and no enrichment of the negative gene target for EZH2, H3K4me3, and H3K27me3 shows the sensitivity, selectivity, and reproducibility of the Chromatrap 96® HT platform.

Conclusion

Chromatrap® 96 HT offers an excellent tool for the large-scale, highly selective and high throughput analysis of gene regulation which depends on transcription factor interaction and vast epigenetic modifications. The system provides vast amounts of robust and highly sensitive, comparable data in just one single experiment.

ChIP using Chromatrap® 96 HT paves the way for reliable and accurate analysis of global gene expression and examination of various regulatory pathways after different treatments and/or in the parallel examination of several different cell lines.

Acknowledgment

Produced from articles provided by Chromatrap®.

About 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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Last updated: Jun 26, 2014 at 11:30 PM

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