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.
Methylation, acetylation, phosphorylation, and ubitquitination modifications on histones are key players in gene expression. These modifications serve as signals for opening as well as compacting of the chromatin, and also as recruiting factors that promote and antagonize transcription. See figure 1 for important sites of histone post-translational modifications (PTMs) that effect epigenetics.
While doing epigenetics research it is essential to use an antibody that is specific to an individual histone modification because each one represents a unique signal for gene expression. For example, Lys9 on H3 can be acetylated or methylated. Acetylation is an activating modification and methylation has different signals depending upon the number of methyl groups. H3K9me1 is found enriched at transcription start sites, whereas H3K9me2 and H3K9me3 are associated with gene repression. Further, H3K9me2 is specifically associated with chromosome X inactivation. Thus, each modification on H3K9 has a distinct effect on the cell. Knowing the identity of the modification is essential for accurately characterizing expression. In figure 2, a cross reactivity ELISA was run to demonstrate the specificity of an antibody for a di-methyl-histone – histone H3K9me2.
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.
Using H3K4me1 as another example, the H3K4me1 antibody must only recognize a single methyl group on Lys4 of H3 and not a di- or trimethylation. Invitrogen ChIP-validated H3K4me1 antibody has greater specificity for that single modification and yields overall higher signal compared to other antibodies tested in a peptide array (figure 3). Specificity of this antibody has also been tested in a biological setting. SETD7 mono-methylates H3 at Lys4. PFI-2 is a potent inhibitor of SETD7. Using this antibody we were able to detect a decrease in H3K4me1 in nuclear lysates of cells treated with PFI-2 (figure 4).
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 4. Identification of a decrease in H3K4me1 in PFI-2 treated cells using H3K4me1 recombinant antibody, Cat. No. 710795. HeLa cells were treated with varying concentrations of SETD7 inhibitors as indicated for 2 hours. Cells were collected, and chromatin-bound nuclear extracts were prepared. Western blot analysis of H3K4me1 (upper panel) was performed by loading 10 µg of acid chromatin-bound nuclear extract in reducing sample buffer (Cat. No. 39000) and protein ladder (Cat. No. 26619) onto a 4–20% Tris-glycine polyacrylamide gel. Proteins were transferred to nitrocellulose membrane (Cat. No. 88018) with Pierce 1-Step Transfer Buffer (Cat. No. 84731) using a semi-dry blotter. Membrane was blocked in StartingBlock T20 (Cat. No. 37543) for 30 minutes at room temperature. H3K4me1 was detected at approximately 17 kDa using a histone H3K4me1 recombinant rabbit antibody (Cat. No. 710795) at a dilution of 1:2,000 in StartingBlock T20 overnight at 4°C on a rocking platform, followed by a goat anti–rabbit Superclonal IgG-HRP secondary antibody (Cat. No. A27036) at a dilution of 1:5,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico PLUS substrate (Cat. No. 34578) and the myECL Imager. A loading control (lower panel) was performed by stripping the blot with Restore PLUS Western Blot Stripping Buffer (Cat. No. 46430) and re-probing with histone H3 recombinant rabbit antibody (Cat. No. 711055) at a dilution of 1:2,000 in StartingBlock T20 overnight at 4°C on a rocking platform, followed by goat anti–rabbit Superclonal IgG-HRP secondary antibody (Cat. No. A27036) at a dilution of 1:5,000.
Writers, erasers, and readers
Epigenetic regulation is dynamic and includes writers, erasers, and readers. Writers place a mark on a specific amino acid on histones or other proteins. These include histone acetyltransferases (HATs), histone methyltransferases (HMTs), protein arginine methyltransferases (PRMTs), and kinases. Erasers remove such marks and include histone deacetylases (HDACs), lysine demethylases (KDMs), and phosphatases. Readers bind to the epigenetic marks and include proteins with bromodomains, chromodomains, and Tudor domains (see figure 5 for a visual representation).
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.
The writing, reading, and erasing of these posttranslational marks leads to changes in chromatin structure that can promote or antagonize gene expression. This highly dynamic process regulates transcription, DNA replication, and DNA repair. Mutations in many of these proteins are associated with disease. Figure 6 shows immunohistochemistry analysis of HP1 alpha, a reader that binds to H3K9. HP1 alpha is commonly found in multiple types of cancer. Figure 7 shows an example of PRMT5, an irreversible post translational modification writer, detection in MCF-7 cells. Figure 8 shows enrichment of KDM4A, an eraser, at a specific gene loci.
Figure 6. Immunohistochemistry was performed on human cerebellum and human liver. Tissue was de-paraffinized with xylene, followed by rehydration in sequential washes of 100% ethanol, 95% ethanol, 80% ethanol, and water. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and heated for 20 minutes in Lab Vision PT Module (Cat. No. A80400012). Following antigen retrieval, tissues were blocked in a 10% goat serum (Cat. No. 50-062Z) in wash buffer solution for 30 minutes at room temperature and endogenous peroxidase activity quenched with Peroxidase Suppressor (Cat. No. 35000). Tissue was then probed with a HP1 alpha mouse monoclonal antibody (Cat. No. MA1-218) at a dilution of 1:100 in 10% goat serum in wash buffer for 1 hour at room temperature in a humidified chamber. Tissues were washed extensively with PBST, and detection was performed using a SuperBoost goat anti-mouse Poly HRP secondary antibody reagent (Cat. No. B40961) followed by colorimetric detection using DAB Quanto (Cat. No.TA-125-QHDX). Tissues were then counterstained with hematoxylin (Cat. No. TA-125-MH), mounted and imaged on an EVOS FL Auto Imaging Station.
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.
Histone modifications and their associated writers, erasers, and readers
There is a vast array of options when it comes to writers, erasers, and readers. Find the options for histones, their amino acid sites, the type of modification, and the writers/erasers/readers of interest below.
|H2A||K5||ac||Tip60, p300, CBP, KAT1, KAT5|
|H2A||T120||ph||Bub1, VprBP, NHK-1|
|H2A.X||S139||ph||ATM, ATR, DNA-PK||PP4||MDC1, MDC1, NBS1, 53BP1, TDRr, BRCA1|
|H2B||K12||ac||p300, CBP, ATF2|
|H2B||K15||ac||p300, CBP, ATF2|
|H2B||K120||ub||UBE2E1, RNF20, RNF40, UBE2A, UBE2B|
|H3||K4||ac||SIRT1, SIRT2, SIRT3, HDAC1, HDAC2, HDAC3|
|H3||K4||me||MLL1, MLL2, MLL3, MLL4, MLL5, SETD1A, SETD1B, ASH1, SETD7, NSD3||LSD1, LSD2, KDM2B, JARID1A, JARID1B, JARID1C, JARID1D, PHF8, NO66||CHD1, MRG15, PHF20L1, TAF3, ING1, ING2, ING3, ING4, ING5, BPTF, RAG2, ATRX|
|H3||K9||ac||GCN5, PCAF, ELP3||SIRT6, SIRT1||BRD4, BAZ1B|
|H3||K9||me||Suv39H1, SUV39H2, G9a, SETDB1, Ash1, KMT1D, CLL8, RIZ1||LSD1, KMD3A, KMD3B, KMD4A, KMD4B, KMD4C, KMD4D, TRIP8, PHF8||L3MBTL1, Tip60, SFMBT, HP1, CDY1, PC1, MPP8, CBX1, CBX2, CBX3, CBX4, CBX5, CBX6, CBX7, CBX8, Np95, JARID1C, ATRX|
|H3||S10||ph||Aurora-B, MSK1, IKK-alpha, Snf1, MSK2, Pim1||PPF||14-3-3|
|H3||K14||ac||GCN5, PCAF, CBP, p300, Tip60, SRC-1, Elp3, KAT12, TAF1, MOZ, MORF||BRD4, BAZ1B, BRG1|
|H3||K18||ac||GCN5, p300, CBP, PCAF, KAT12|
|H3||K23||ac||GCN5, Sas3, p300, CBP, KAT3A, KAT3B|
|H3||K27||ac||GCN5, p300, CBP|
|H3||K27||me||EZH2, G9a, EZH1, NSD3||UTX, JMJD3, PHF8||PC1, CBX2, CBX4, CBX6, CBX7, CBX8, EED|
|H3||S28||ph||Aurora-B, MSK1, MSK2||14-3-3|
|H3||K36||me||SETD2, NSD1, SMYD2, NSD2, ASH1, SETMAR||KDM2A, KDM2B, KDM4A, KDM4B, KDM4C, NO66||MSL3, MRG15, BRPF1, PHF19, PHF1|
|H3||K56||ac||GCN5, CBP, p300||HDAC1, HDAC2, SIRT2, SIRT6|
|H4||R3||me||PRMT 1, PRMT5, PRMT6||JMJD6||TDRD3|
|H4||K5||ac||Hat1, Tip60, ATF2, p300, CBP, HBO1||BRD4|
|H4||K8||ac||GCN5, Tip60, ATF2, Elp3, p300, CBP, HBO1||BRD2, BRD4|
|H4||K12||ac||Hat1, Tip60, p300, CBP||BRD2, BRD4|
|H4||K16||ac||Gcn15, MOF, Tip60, ATF2||SIRT2, SIRT1|
|H4||K20||me||PR-SET7, SUV4-20H1, SUV20-H2, ASH1, NSD1, SETD8||PHF20L1, L3MBTL1, SFMBT, MBTD1, 53BP1|
Polycomb repressive complexes
Polycomb group (PcG) proteins function in polycomb repressive complexes (PRC1 and PRC2). PRC complexes modify histones, as well as other proteins, and are generally associated with the silencing of gene expression. Figure 9 shows an example of how a polycomb repressive complex functions. These complexes play key roles, not only in epigenetic regulation of transcription, but also in stem cell identity, differentiation, and disease.
Here is a list of PcG proteins:
Figure 9. An example of how polycomb repressive complexes function. Illustration of polycomb repressive complexes (PRC1 and PRC2).
Figure 10 shows ChIP analysis H2A-Ub. PCR1 ubiquitinates H2A contributing to gene silencing. Figure 11 shows flow cytometry analysis of EZH2, a PcG protein, in HCT 116 cells. EZH2 is abundant in embryonic stem cells and plays a major role in forming H3K27me3 which is required for embryonic stem cells identity and proper differentiation.
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.
Transcription factors recognize and bind to specific sequences on DNA. They activate or repress gene expression by altering RNA polymerase recruitment. Transcription factors can act alone or bind together to form a complex. Some transcription factors bind unmodified proteins, while others require modification such as phosphorylation to bind. Some transcription factors are basal, while others respond to environmental or intracellular signals. Other transcription factors are essential during development or differentiation. Transcription factors can also be involved in pathogenesis- these types of transcription factors include oncogenes and tumor suppressors.
Figures 12, 13, and 14 show examples of transcription factors p65, SMAD2, and STAT1. p65 (NF-kappa-beta) is activated via phosphorylation and upregulates expression. It is involved in developmental processes, cellular growth, apoptosis, and immune/inflammatory responses. SMAD2 interacts with TGF-beta receptors and is phosphorylated. It regulates multiple processes such as cell proliferation, apoptosis, and differentiation. STAT1 mediates cellular response to IFNs, cytokines, and growth factors.
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.
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