Overview of Epigenetics

Epigenetics is the study of changes in gene expression that modify DNA, RNA, and protein – but do not alter the primary sequence. The most common types of epigenetic modifications are methylation, acetylation, phosphorylation, and ubiquitination. These modifications lead to steric changes in chromatin structure that regulate various cellular processes such as transcription, replication, and DNA repair. In the following sections you will learn about histone modifications, writers, erasers, readers, polycomb repressive complexes, and transcription factors and why they are important to the study of epigenetics.

Explore Epigenetic Antibodies

• Histone modification
• Writers, erasers, and readers
• Polycomb repressive complexes
• Transcription factors

Figure 2. H3K9me2 antibody specifically recognizes di-methyl at Lys9. Cross-reactivity ELISA for demonstrating specificity towards di-methyl-histone H3 Lys9 (Histone H3K9me2) was performed using histone H3K9me2 recombinant rabbit monoclonal antibody (Cat. No. 701783). The antigens (H3K9me1, H3K9me2, H3K9me3, and H3K9) were coated at 0.004 µg/mL. Antibodies at 10, 2.5, 0.625, and 0.156 µg/mL were added and the signal was detected using goat anti–rabbit IgG (H+L) secondary antibody, HRP conjugate (Cat. No. G-21234, diluted 1:5,000). The plate was developed using Stabilized Chromogen, TMB (Cat. No. SB02) and detected at an absorbance of 488 nm.

Figure 3. Histone H3K4me1 detection with superior peptide specificity. Peptide arrays were performed using our H3K4me1 recombinant rabbit polyclonal antibody (Cat. No. 710795) and two other suppliers’ antibodies targeting H3K4me1. Arrays were incubated overnight with a 1:2,000 dilution of primary antibody. After washing, they were incubated with goat anti–rabbit IgG (H+L) superclonal secondary antibody (Cat. No. A27036) at a dilution of 1:5,000 for 1 hour. Arrays were incubated with SuperSignal West Pico PLUS substrate (Cat. No. 34578) and then visualized using the myECL Imager.

Figure 5. Epigenetic regulation includes writers, erasers, and readers. Writers, readers, and erasers dynamically associate with chromatin and regulate gene expression. Reprinted by permission of MacMillan Publishers Ltd: Nature Reviews Drug Discovery 13, 673–691 (2014). Visit http://www.nature.com/nrd/index.html.

Figure 7. Immunofluorescence analysis of PRMT5 performed on 70% confluent log-phase MCF-7 cells. Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with PRMT5 mouse monoclonal antibody (Cat. No. 730054) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and labeled with goat anti–mouse IgG (H+L) Superclonal secondary antibody, Alexa Fluor 488 conjugate (Cat. No. A28175) at a dilution of 1:2,000 for 45 minutes at room temperature (green, Panel A). Nuclei (blue, Panel B) were stained with SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938). F-actin (red, Panel C) was stained with Alexa Fluor 555 Rhodamine Phalloidin (Cat. No. R415, diluted 1:300). Panel D is a merged image showing cytoplasmic localization. Panel E is a control with no primary antibody. The images were captured at 60X magnification.

Figure 8. Enrichment of endogenous KDM4A (JMJD2A) protein at specific gene loci. Chromatin Immunoprecipitation (ChIP) was performed using Anti-KDM4A Recombinant Rabbit Monoclonal Antibody (Cat. No. 702449, 5 µg) on sheared chromatin from 2 million NTERA-2 cells using the MAGnify ChIP system kit (Cat. No. 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Cat. No. 4351106) with optimized PCR primer pairs for the promoters of the MYOG, MYOD, GAPDH region used as positive control target gene, and the region of the GAPDH intron, SAT2 satellite repeat used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

• Ring1a
• Ring1b
• CBX2
• CBX4
• CBX5
• RbAp46
• RbAp48

• CBX7
• PCGF2
• PCGF6
• HPH1
• HPH3
• EZH1
• EZH2

Figure 9. An example of how polycomb repressive complexes function. Illustration of polycomb repressive complexes (PRC1 and PRC2).

Figure 10.Enrichment of endogenous histone H2A-Ub protein using anti–histone H2A-Ub rabbit polyclonal antibody. Chromatin immunoprecipitation (ChIP) was performed using anti–histone H2A-Ub rabbit polyclonal antibody (Cat. No. 720148, 3 µg) on sheared chromatin from 2 x 10⁶ HeLa cells using the MAGnify Chromatin Immunoprecipitation System (Cat. No. 49-2024). Normal rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed on the Applied Biosystems 7500 Fast Real-Time PCR System (Cat. No. 4351106) with optimized PCR primer pairs for the region of the inactive SAT2 satellite repeat used as the positive-control target, and promoters of the active cFOS (FOS) beta-actin (ACTB) region used as the negative-control target. Results are presented as fold enrichment of the antibody signal compared to the negative control IgG, using the comparative Ct method.

Figure 11. Flow cytometry analysis of EZH2 on HCT 116 cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with EZH2 rabbit polyclonal antibody (Cat. No. 36-6300, red histogram) or with rabbit isotype control (pink histogram) at 3–5 µg per 106 cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with goat anti-rabbit secondary antibody, Alexa Fluor 488 conjugate (Cat. No. A-11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using the Applied Biosystems Attune Acoustic Focusing Cytometer. The purple histogram represents unstained control cells, and the green histogram represents a control with no primary antibody.

Figure 13. ChIP-qPCR analysis of NF-kappa-B (p65) with specific antibody. The experiment was performed with 3 µg/mL of NF-kappa-B p65 recombinant antibody (Cat. No. 710048) on sheared chromatin from 2 x 10⁶ HeLa cells treated with TNF-alpha (50 ng/mL for 1 hr) using the MAGnify Chromatin Immunoprecipitation System (Cat. No. 49-2024). Normal rabbit IgG (3 µg/mL) was used as a negative IP control. The purified DNA from each ChIP sample was analyzed using the Applied Biosystems StepOnePlus Real-Time PCR System (Cat. No. 4376600), with primers for the promoters of the IL-8 and IL-6 genes used as positive-control targets, and the GAPDH gene used as negative-control target. Results are presented as fold enrichment of the antibody signal compared to the negative control IgG, using the comparative Ctmethod.

Figure 14. Expression of SMAD2 detected after transfection of HeLa cells with siRNA. Cells were transfected with 50 nM Silencer SMAD2 siRNA (lane 3), transfected with 50 nM Silencer Negative Control siRNA (lane 2), or left untransfected (lane 1). Proteins from cells lysates were then separated by gel electrophoresis, transferred to membrane and detected by western blot using SMAD2 recombinant rabbit monoclonal antibody (Cat. No. 700048, 0.5 µg/mL) and goat anti–rabbit IgG (H+L) Superclonal secondary antibody, HRP conjugate (Cat. No. A27036, 0.4 µg/mL, 1:2,500 dilution). The blot was reprobed with actin antibody (Cat. No. PA5-16914) as a loading control. The relative densities of the bands normalized to actin confirm silencing of SMAD2 expression and the specificity of the recombinant rabbit monoclonal antibody.

Figure 15. Immunohistochemistry analysis of Phospho-STAT1 (pTyr701) showing staining in the nucleus and cytoplasm of paraffin-embedded human cervical carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 minutes. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 minutes at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-STAT1 pTyr701 monoclonal antibody (Cat. No. 33-3400) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.