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. 2011 Nov 18:12:49.
doi: 10.1186/1471-2199-12-49.

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

Jingjing Yu  1 , Qinghua Feng, Yusong Ruan, Radko Komers, Nancy Kiviat, Karol Bomsztyk
Affiliations

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

Jingjing Yu et al. BMC Mol Biol. .

Abstract

Background: The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription.

Results: To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease.

Conclusion: The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease.

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Figures

Figure 1
Figure 1
Diagram of the microplate-based Matrix ChIP-MeDIP method. Batches of polypropylene plates are coated overnight with protein A (room temperature) and are then stored sealed up to 2 weeks (4°C) and used when needed. Starting with protein A-coated plates the ChIP-MeDIP assay takes 4-5 hours to generate PCR-ready DNA. Detailed protocol is described in "Method" section. The red and yellow lines exemplify rows of wells with ChIP and MeDIP antibodies, respectively.
Figure 2
Figure 2
Optimization and testing the microplate-based MeDIP. HeLa cells were treated with 200 nM of DAC for 3 days and 300 nM of TSA for the last 24 hours. Media containing fresh drugs was replaced daily. Control cells (vehicle) treated identically with drug solvents (PBS and 100% ethanol). Cells were trypsinized and washed with PBS. For MeDIP input DNA was deproteinized using proteinase K, sheared by ultrasound (Bioruptor), RNase treated and denatured. Assays were done using in-lab made protein A-coated polypropylene 96-well plates. A, MeDIP was tested either when anti-5mC antibody (Diagenode) was first attached to wells and then DNA was added (No) or the DNA was preincubated (Yes) with the antibody first and then the mixture was added to protein A-coated 96-well plates and binding was done in ultrasonic bath. After the binding step, wells were washed, DNA was eluted and used in real-time PCR using primers to ALU and LINE elements as wells as the SFRP1 gene. B, MeDIP was done with the preincubation step using monoclonal anti-5mC antibodies from either Diagenode (5mC-D) or Aviva (5mC-A) companies. Monoclonal anti-Flag tag antibody was used as control. Results are expressed as % Input, mean ± SEM of three independent biological replicates.
Figure 3
Figure 3
Validation of microplate-based MeDIP assay. A, Denatured HeLa cell DNA was preincubated with either monoclonal anti-5mC or Flag (mock control) monoclonal antibody (0.5 μg each) in binding buffer (60 μl, 60 minutes at 4°C) in ultrasonic bath. After antibody preincubation, the mixture was divided into two 30 μl aliquots, one was used in microplate MeDIP (Microplate MeDIP) and the other in protein A-agarose beads MeDIP (Beads MeDIP). For each step the same buffer volumes were used in microplate- and beads-based assays. The X-axis shows total DNA input used per IP (nanograms DNA/30 μl IP reaction). After binding, wells and beads were washed with IP buffer and TE buffer. DNA was purified from well walls using 60 μl elution buffer/proteinase K and from beads using 60 μl 10% Chelex/proteinase K [46]. Eluted DNA was used in real-time PCR using primers to ALU and LINE elements. Results are expressed as % Input, mean ± SEM of three independent biological replicates. B, As in (A) microplate and beads immunoprecipitated DNA was used in real-time PCR using primers to either the methylated H19 imprinted control region (H19 ICR) or the unmethylated promoter region of the house keeping gene UBE2B [18]. Data are expressed as ratio of the 5mC signal to the Flag mock (5mC/Flag). The red horizontal line represents background binding, 5mC/Flag ratio = 1.
Figure 4
Figure 4
Comparison of MethyLight and Matrix ChIP-MeDIP assay of normal cervix tissue and cervical cancer. MethyLight, DNA purified from clinical cancer and normal tissues was bisulfite-converted and amplified using SPARC gene primer in Taqman PCR [43]. Methylight results are expressed as PMR values for each individual sample. ChIP-MeDIP, frozen (-80°C) archived cervical tissue samples histologically classified as normal and cancer were thawed, minced and fixed with formaldehyde. After glycine treatment and washes samples were treated with high energy ultrasound to shear the chromatin. The lysates were cleared by centrifugation, aliquoted and stored (-80°C). For MeDIP the input was deproteinized, RNase-treated and denatured DNA. MeDIP was done using anti-5mC antibody (Aviva) and ChIP assays with anti-Pol II CTD, H3K9/14Ac, H2A.Z and H3K27m3 antibodies. MeDIP and ChIP DNA were analyzed at the indicated site of the SPARC gene in real-time PCR. Data represent values for each individual sample (five normal and five cancer), expressed as % input.
Figure 5
Figure 5
Combined ChIP-MeDIP analysis at a single locus is sufficient to demarcate normal cervical tissues from cervical cancer. 5mC data for five normal and five cancer samples is compared (Figure 4) to either 5mC/Pol II or 5mC/H3K27m3 ratio. The red horizontal line demarcates the highest value for the normal tissues.
Figure 6
Figure 6
Combined ChIP-MeDIP analysis reveals changes in methylation and Pol II levels at a pro-inflammatory gene locus in diabetic mouse kidney. Sheared cross-linked kidney chromatin from two-month-old diabetic OVE26 (on FVB background) (A), and two-month-old leptin resistant insulin resistant obese C57BL/6J ob/ob mice (B) and their respective normal age-matched controls were assayed using anti-5mC, Pol II CTD and H3K9,14Ac antibodies. The same cross-linked chromatin samples were used for both MeDIP and ChIP assays. MeDIP and ChIP DNA were analyzed at the indicated sites of the MCP-1 and TGF-β1 genes in real-time PCR. Data represent mean ± SEM (6 animals from each strain), expressed either as % Input (panels 1-3) or 5mC/Pol II ratio (panel 4).
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