Chromatrap® Technology for Studying Complex Epigenetic Mechanisms

Introduction

Chromatin immunoprecipitation (ChIP) is a widely used technique for mapping the interactions between intracellular proteins and their DNA targets in gene regulation. The epigenetic mechanisms that affect chromatin formation and control the availability of the DNA backbone to transcription factor regulators is an important area of research. ChIP is a technology that allows researchers a glimpse into the regulatory mechanisms that gene expression.

Although a number of platforms are available to perform ChIP, Porvair's new Chromatrap® platform is the first solid phase chromatin immunoprecipitation assay. The system enables transcription factors to be captured with high sensitivity and selectivity, providing an exciting new pathway in the analysis of global gene expression.

The capture of these protein– DNA complexes is accelerated with the use of the solid phase Chromatrap® system when compared with techniques that use magnetic or agarose beads. The system also offers the advantage of low background signal, therefore allowing low abundance targets to be detected.

This article demonstrates the selectivity, sensitivity, and reproducibility of solid phase Chromatrap®, as an assay for ChIP from low cell numbers.

Epigenetic Landscape

The epigenetic landscape is a complex balance of closed and open chromatin configurations that determine active and inactive gene expression patterns. Understanding this balance could unlock the regulatory mechanisms by which normal versus abhorrent cell function develops. Detection of these can prove challenging, especially with low abundance transcription factors in low volume samples. The Chromatrap® system overcomes this hurdle, providing the capture of both high and low abundance transcription factors with an exciting change in assay technology.

To demonstrate the capability of the Chromatrap® platform, a low volume and high sensitivity ChIP assay was conducted using two in vitro cell line models, K562 and HepG2. The binding of RNA pol II onto the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene locus was used for positive expression.

The balance of histone H3 tri-methylation at lysine 27 and 4 in the tails of H3 was employed here to indicate the presence of closed (low H3K4 and high H3K27) and/or open (high H3k4 and low H3k27) chromatin conformation, respectively. β-globin was utilized as a negative gene for RNA pol II and the presence of H3K4me3. The negative target  gene β-globin is only active in erythroid cells and is inactivated in non-erythroid tissues. For low abundance transcription factor targets, the Enhancer of zeste homologue 2 (EZH2) and histone de-acetylase 1 (HDAC1) were selected.

Methodology

Chromatin Preparation

For this study, a hepatocellular carcinoma cell line, Hep G2 and K562 - obtained from chronic myeloid leukaemia  were selected. Cells were grown and prepared for IP as per the standard Chromatrap® protocol. Cells were fixed in 1% formaldehyde, lysed and the chromatin isolated by centrifugation. The chromatin was sheared to produce fragments of 100 to 500 bp in length, as shown in Figure 1. A NanoDrop spectrophotometer was used to quantify the concentration of the chromatin, and chromatin aliquots were stored at -80°C.

Figure 1. Chromatin was sheared using a Bioruptor for 30-second bursts with 30-second intervals on ice at a power setting of 3 for 15min so that desired fragment lengths between 1 and 500bp were obtained.

Figure 1. Chromatin was sheared using a Bioruptor for 30-second bursts with 30-second intervals on ice at a power setting of 3 for 15min so that desired fragment lengths between 1 and 500bp were obtained.

Immunoprecipitation and identification

500ng of chromatin was precipitated with 1ug of antibody for each slurry preparation.. Table 1 shows the antibodies used in each reaction. Chromatin aliquots were set aside as the input for qPCR analysis. After each IP slurry was incubated on the columns at 4°C for 1 hour, the columns were washed with buffers and DNA and protein complexes were eluted. Both the eluted samples and input samples were subjected to proteinase K digestion and reverse cross linking before direct  qPCR analysis.

Table 1. Antibody targets for ChIP and the gene loci at which targets are positive or negative.

Antibody targets

 Positive gene targets

Negative gene targets

H3K27me3

 MYT1 ß-globin

 ZNF333 GAPDH

H3K4me3

 GAPDH

 ß-globin

EZH2

 MYT1

 ZNF333

RNA polymerase II

 GAPDH

 ß-globin

HDAC1

 Cyclin 2A p21

 GAPDH

qPCR Analysis

To provide a negative control, each IP sample was compared to a corresponding loading of nonspecific IgG antibodyfrom the same species. Positive and negative IP were precipitated and assessed simultaneously and then compared against the input sample to give the percentage of real signal relative to input. The standard error was calculated from a minimum of three replicate procedures to demonstrate the reproducibility of results.

Table 2. Specific and non-specific antibodies used in ChIP.

Antibody target

Type

H3K27me3

pAb / rabbit

H3K4me3

 pAb / rabbit

EZH2

 mAB/mouse

RNA polymerase II

 pAb / rabbit

HDAC1

 mAb/rabbit

Unspecific IgG, 1

 rabbit serum

Unspecific IgG, 2

 mouse serum

Results and Discussion

Using 500ng of input chromatin, chromatin from human hepatocellular carcinoma cell line HepG2, and K562 cells derived from chronic myeloid leukaemia were immunoprecipitated. The presence of RNA pol II at the GAPDH gene was identified along the tri-methylation of lysine 4 in the H3 tail, indicating open and actively transcribed chromatin (Figure 2).

Figure 2. ChIP results in Hep G2 cell line demonstrating the relationship of methylation marks on potentiallytranscribed and silenced gene targets.

Figure 2. ChIP results in Hep G2 cell line demonstrating the relationship of methylation marks on potentiallytranscribed and silenced gene targets.

The positive gene control that was used, MYT1, demonstrated a high percentage real signal for the presence of low abundance transcription factor EZH2, as shown in Figure 3.

Figure 3. EZH2 induced presence of H3K27me3 is evident on MYT1 promoter. The % real signal (input) values are very low on the negative gene ZNF333 as anticipated, highlighting the sensitivity of the Chromatrap® assay.

Figure 3. EZH2 induced presence of H3K27me3 is evident on MYT1 promoter. The % real signal (input) values are very low on the negative gene ZNF333 as anticipated, highlighting the sensitivity of the Chromatrap® assay.

Similar to the Hep G2 cells, there was positive signal amplification relative to background, demonstrating that RNA pol II was recruited to the GAPDH promoter (Figure 4). A positive amplification signal of 1% for EZH2 recruitment to the MYT1 gene loci was identified using Chromatrap®, which was twelve times higher than the 0.08% detected for the negative gene loci.

This data indicates that EZH2 is a negative regulator of MYT1 gene expression through its recruitment of histone methyl-transferase activity. As with the Hep G2 cells, the H3K4me3 and RNA pol II signals were extremely low at the ZNF333 and MYT1 gene loci (Figure 5).

Figure 4. Chromatin Immunoprecipitation of methylation marks and transcription factors at multiple gene loci in K562 cell line.

Figure 4. Chromatin Immunoprecipitation of methylation marks and transcription factors at multiple gene loci in K562 cell line.

Figure 5. Detected precipitation of low abundance EZH2 at the MYT1 locus; the % real signal (input) values are extremely low on the negative gene ZNF333 as expected.

Figure 5. Detected precipitation of low abundance EZH2 at the MYT1 locus; the % real signal (input) values are extremely low on the negative gene ZNF333 as expected.

Conclusion

Porvair’s Chromatrap® is a solid phase chromatin immunoprecipitation assay, which is based on true filtration of antibody-targeted chromatin fragments from interactions ofunspecific antibodies and chromatin. Other assays kits often feature high concentrations of starting material or use extended incubation times to increase sensitivity. Chromatrap®, on the other hand, overcomes these problems without compromising on the level of sensitivity achieved, thus providing a valuable tool for enhancing the understanding of epigenetic gene regulation.

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 11:29 PM

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