Chromatrap Immunoprecipitation Assay Enriches Epigenetic Histone Methylation Marks from Primary Biopsy Samples

Introduction

The uterus is a pear-shaped organ that consists of a lower barrel shaped cervix and a large upper body with the 'fundus' over the uterine tubes. It has a thick muscular wall, which houses the human fetus from conception to birth.

The endometrium lining of the uterine cavity includes a layer of epithelial cells overlying a stroma, which comprises immune cells, blood vessels, and endometrial stromal cells.

During the menstrual cycle, the endometrium upon initial exposure to oestrogen becomes a glandular tissue layer that is rich in thick blood vessels. Precisely controlled differentiation, proliferation, and degeneration stages characterised by distinct functional and morphological changes follow this process during a normal menstrual cycle.

The cycle ends in the window of receptivity, wherein decidualization of the stromal cell layer is crucial for effective implantation and is important for establishing and supporting pregnancy.

Only limited data is available on the epigenetic regulation of the endometrium. Clinical interpretation of endometrial epigenetic research depends on ex vivo analysis of patient biopsy samples acquired from female reproductive organs.

Chromatin Immunoprecipitation (ChIP) is a commonly employed method to study the interactions between epigenetic marks and regulatory proteins, with a specific target gene or genomic DNA region.

The universal occurrence of epigenetic changes in cellular differentiation make ChIP assays useful for examining the molecular mechanisms involved in endometrial development and shedding. This article describes the isolation of endometrial stromal cells from pipelle biopsy samples obtained from patients attending clinics for infertile pathologies.

Methodology

Endometrial Biopsy

First, endometrial biopsies were acquired and processed for isolation of stromal cells as per the standard protocols. Then, fertile control biopsy samples were briefly taken from women with established fertility and regular menstrual cycles.

Biopsy samples were then incised to 1mm pieces and placed in Dulbecco's modified eagle media (DMEM) containing antibiotic-antimycotic solution. The tissue was enzymatically digested by adding deoxyribonuclease type I and collagenase at 37°C for 1h. Samples were rotated at 500 rpm for 5min.

Following this, the supernatant was removed and the pellet was re-suspended in complete culture medium DMEM supplemented with antibiotic-antimycotic solution, fetal bovine serum, sodium pyruvate, and sodium bicarbonate. Cells were later transferred to a falcon flask and cultured overnight with 5% CO2 at 37°C.  

Chromatin Collection

Cells were grown to confluency and fixed, chromatin was obtained using the Chromatrap® V5 spin column protocol as standard. Isolated chromatin was sonicated to create chromatin fragments in the region of 100-600 base pairs (Figure 1). Aliquots of the chromatin stock were obtained, reverse cross linked, and protease digested before the DNA concentration was measured using a NanoDrop spectrophotometer.

Figure 1. Qualitative analysis of Chromatin from endometrial stromal cells.

Immunoprecipitation

As per the Chromatrap® V5 spin column protocol, immunoprecipitation was carried out as standard. The antibodies investigated in this study comprise the epigenetic histone marks H3 and H4 alonside the modified histones H3K4me3 and H3K27me3. A non-specific rabbit IgG antibody was utilized as a negative antibody control.

Slurries containing the antibodies of interest were incubated on the column at 4OC for 1h. All inputs and samples were reverse cross linked and proteinase K digested before downstream processing. ChIP was carried out in triplicate, with three ChIPs per antibody.

To determine the precipitation efficiency of each antibody real time PCR (qPCR) was utilized.. Table 1 illustrates the antibody and gene targets employed in this study; both negative and positive gene targets were examined.

Table 1. Antibody and positive and negative gene targets used in this study.

Results and Discussion

In order to show the efficiency, utility, and reproducibility of the Chromatrap® Immunoprecipitation assay on primary tissue, an experiment was performed to enrich positive and  negative gene loci using antibodies directed against common epigenetic marks.

The level of amplification and the balance of epigenetic marks at these gene loci distinctly show that the assay is selective and sensitive. Demonstrated here is efficient enrichment of both low abundant and high abundant genes.from endometrial pipelle biopsy samples.

Increased real signal relative to the input chromatin was identified for H3K27 and H3K4 tri methylation at each of the gene loci examined in this study, while the assay’s efficiency was monitored through highly abundant H3 screening as a positive control (Figure 2).

Figure 2. Highly abundant epigenetic marks on positive and negative gene targets in primary stromal cells, illustrating % real signal relative to input.

Immunoprecipitation of stromal cell chromatin with the Histone H3 antibody displays enriched signal for the PABPC1, B-globin, and GAPDH loci, while Immunoprecipitation with the non-specific rabbit IgG resulted in extremely low background enrichment at each gene locus.

When compared to the negative IgG control (β-globin 9x, PABPC1 10x and GAPDH 11x enrichment), a minimum of 9fold increased enrichment was observed with the positive H3 antibody. Therefore, the Chromatrap® protocol was suitable for isolating superior quality chromatin and for sensitive amplification of positive target signal for histones on the DNA backbone. The speed and ease of handling of the IP process was an additional benefit against competitor kits available.

Conclusion

The above data clearly shows the usefulness of the Chromatrap® assay in the IP processing of epigenetic histone methylation marks from primary biopsy material.

Chromatrap® allows rapid analysis and elucidation of DNA/protein complexes in clinically relevant material. The assay’s reproducibility allows the targeting of both low abundant and highly abundant marks at many target gene loci.

The assay has also been employed successfully at 100ng, equivalent to a low number of cells, of input chromatin , emphasizing the opportunity for ChIP enabled insight into the endometrial epigenetic landscape and its contribution toward the control of complex cellular differentiation systems seen in endometrial cancer and infertile pathology.

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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