Looking into chromatin can be a complicated story. Transcriptional control is dependent on a huge network of epigenetic modifications.

For the last 9 years, it’s been Diagenode and academic partners full time job to establish standards in this field (and still is).

So, if you’re looking to start your epigenetic research and generatea number of hitting data, you'll want to consider the most validatedreagents, antibodies, DNA or Chromatin shearing devices or automated workstations for performing consistent ChIP assays.

ChIP/ChIP-seq grade antibodies, the critical key to Chromatin Immoprecipitationsuccess

When asked about how they choose their ChIP antibody of interest, researchers often point to their prior experiences with the antibody, comparative results, and well-cited products. However, researchers may not necessarily be satisfied with antibody performance. Oftentimes, they must optimize each and every ChIP assay with a suboptimal antibody simply to maintain "consistency" in future experiments even if better antibodies become available.

Diagenode has manufactured, quality-verified, and validated hundreds of polyclonal or monoclonal antisera and selected a fraction of them as actual high quality standards. Over time Diagenode became an expert aid for the ‘production and validation’ in many active epigenetic consortiums such as the recent EU project “Blueprint”. Production of ChIP-Seq grade reagents and methods is one of our main challenges today.

The most “popular couple” of Histone modifications: H3K27me3 and H3K4me3

These histone modifications have been largely reported to be closely associated with gene transcription activity and are therefore among the most frequently interrogated histone modifications.
H3K4me3 is associated with highly expressed and/or housekeeping genes while H3K27me3 is associated with under-expressed and/or repressed tissue-specific genes. Both modifications can be recognized by different chromatin factors through specific protein domains (iex  Plant Homeodomain (PHD) of ING2 (Inhibitor of Growth 2) can bind to H3K4me3 and the chromodomain of Polycomb proteins in animal cells and Like Heterochromatin Protein1 (LHP1) in Arabidopsis can bind to H3K27me3).Deregulation of H3K4me3 and H3K27me3 is associated with cancer development.

H3K27me3is silencing expression very efficiently. Trimethylation of lysine 27 on H3 has been stronglyrelated to transcriptional repression in numerousimportant processes in animals and plants such as during development and disease.This modification is maintained by Polycomb Repressive Complex 2 (catalyzed by EZH2 methyltransferase) and can be taken out by lysine demethylases like JMJD3.

H3K4me3, as presented in the introduction, is viewed as the opposite mark to H3K27me3.This modification is activating the transcription of a number of genes and marking meiotic and V(D)J recombination sites.The COMPASS complex in yeast, aka MLL/hSet1A and B complexes in mammals can mono-, di-, or trimethylate histone H3 at lysine 4. In plants, H3K4me3 can capture the homeodomain finger of TAF3 to bind TFIID in plants and get the transcription machinery started.

Although association with silenced or activated genes is well documented, there is still a lot to discover regarding how H3K27 or H3K4methylation interacts with the other players in gene regulation. As an example, there is a relationship between H3K27me3 and DNA methylation where they were found mutually exclusive and antagonistic.
A study on T Cells shown that the relative abundance of H3K4me3 and H3K27me3 generates 4 different interactions with gene expression: repressed, active, poised, and bivalent. For example, high H3K4me3 versus less H3K27me3 corresponds to genes that are “poised” for quick activation in resting cells. High level of both modifications corresponds to “Bivalent” genes. A 2011 ChIP-Seq experiment also shows 3 distinct enrichments patterns. One in the body of genes with expected transcription inhibition. A second, around TSS transcription start site (TSS) classically associated with ‘bivalent’ genes, where H3K4me3 also marks the TSS. A third enrichment peak in the promoter of genes that is associated with active transcription.

Definitely, antibodies for thesetwo marks should be in every fridge. Check out Diagenode’s

H3K27me3 , validated for ChIP/ChIP-seq ; ELISA, Dot blotting, Western blotting

H3K4me3 , validated for ChIP/ChIP-seq ; ELISA, Dot blotting, Western blotting

See also:

1. Bioruptor®, the most cited tool for Chromatin Shearing
2. Manual Low Cell ChIPand automatedChIP-Seq kit, the best kit for Low Cell ChIP/ChIP seq assays
3. IP-Star Compact®The only available automated workstation for consistent ChIP/ChIP-seq assay
4. ChIP/ChIP-Seq Grade antibodies, all categories:

• Chromatin modifying and/or associated proteins
• Chromatin modifying proteins: Histone Deacetylase
• Chromatin modifying proteins: Histone Demethylase
• Chromatin modifying proteins: Histone Methyl Transferase
• DNA Methylation
• Histone H3 and modified H3
• Histone H4 and modified H4
• m-RNA degradation
• Nuclear receptor
• Protein-tag
• Transcription / Transcription factor
• Exclusive customized epigenetic antibodies
• Peptides
• Others antibodies

The widespread adoption of emerging epigenetics technologies has provided the scientific community with new tools for understanding genetic inheritance phenomena. These advanced tools include kits and equipment for DNA or chromatin shearing, chromatin immunoprecipitation (ChIP, ChIP-Seq), methylated DNA immunoprecipitation (MeDIP), methyl binding domain (MethylCap, MBD) studies, bisulfite conversion, automation (IP-Star, SX8G Compact), and Next Generation sample preparation.

In recent years, interest in DNA methylation modifications has increased, as abnormal DNA methylation status is often correlated with disease. One particular DNA modification, 5-methylcytosine or 5-Methylcytidine (5-mC), can switch a gene “on or off,” and is the most extensively studied modification among the known plant and vertebrate epigenetic marks. The 5-mC modification is also implicated in genomic imprinting and contributes to the of control gene expression. This modification occurs when DNA methyltransferases (DNMTs) catalyze the reaction of a methyl group to the fifth carbon of cytosine in a CpG dinucleotide.

Researchers recently discovered another important DNA methylation modification, the  5-hydroxymethylcytosine (5-hydroxy methylcytosine, 5-hmC) which occurs after the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of cytosine oxygenases. The5-hydroxymethylcytosinecould be involved in a pathway to demethylate DNA, as seen by the evidence that 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine.

Diagenode, the pioneer in the field of epigenetics research, facilitates DNA methylation studies with optimized assay protocols and robust products. The company provides kits, reagents, antibodies, equipment, and protocols dedicated to DNA methylation studies at the single loci or genome-wide level. The antibodies product line includes 5-hmC, 5-mC, and a Cytosine DNA Standard Pack which includes hydroxymethylated, methylated, and unmethylated DNA standards. These standards have been produced by PCR using the Diagenode’s MethylTaq DNA polymerase or5-hm dCTP (for the hydroxymethylated standard). Each standard either contains normal cytosines, 5-methylcytosines, or 5-hydroxymethylcytosines. All protocols and products are available for manual assays or for automated assays with the SX-8G IP-Star Compact automation workstation.

5-hmC  ||  5-mC  ||  5-methylcytosine  ||  5-hydroxymethylcytosine

Biotechnology has revolutionized fields like engineering, medicine, environmental research, and molecular biology. The launch of the Human Genome Project (HGP) in the United States was an important milestone for genetic engineering. The success of HGP has inspired a number of research centers and biotechnology companies worldwide to identify advanced techniques for disease diagnostics, gene analysis, gene regulation, and epigenetics.

Diagenode, the pioneer and leader in epigenetics research, has made significant contributions in the field of immunoprecipitation automation, sonication (chromatin or DNA shearing, antibody development, DNA methylation, chromatin function, and protocol optimization. The wide range of kits for chromatin immunoprecipitation developed by Diagenode and used by numerous life science labs and centers provides reproducible and robust ChIP results.

The Diagenode SX-8G IP-Star and SX-8G Compact Automated Systems represent the first innovations that brought complete automation to ChIP and DNA methylation assays. The automated ChIPdeveloped by Diagenode is ideal for improving assay consistency and for preparing precious samples analysed with Next Generation Sequencing technologies such as ChIP-Seq and epigenome mapping. This unique automation system uses a range of optimized reagents and protocols combined with a proprietary magnetic separation technology run on a high-precision workstation. Automation enables faster walk-away assays and much higher consistency in obtaining precious IP’d material.

The life sciences research market recognizes Diagenode as the leader for developingcutting-edge ChIP and DNA methylation products. Various diagnostics companies look to Diagenode for expert guidance and efficient diagnostic systems in epigenetics-based diagnostics.  Diagenode offers complete solutions for every epigenetics study.

DNA shearing  ||  epigenetics  ||  5-mC ||  5-hmC

The field of biotechnology has increasingly become more wide spread with applications in diverse areas such as agriculture, environmental research and testing, molecular biology, genetic engineering, and clinical health care.

Advances in biotechnology contributed to the success of the Human Genome Project which uncovered major findings about the genome by identifying and mapping human genes. Knowledge from the HGP facilitated research in numerous areas of clinical interest such as epigenetics to gain a deeper understanding of disease at a molecular level for potential therapeutic procedures.
 

With implications in cancer and heritable diseases, the study of epigenetics and how genes are regulated has become crucial in cancer research for understanding predisposition factors and identifying biomarkers and potential drug targets. Of specific interest in the field is the interaction between proteins and DNA and genomic modifications, which give insight into how gene expression is regulated.

Epigenetic modifications and the location of DNA binding sites on the genome for a particular protein can be determined by chromatin immunoprecipitation (ChIP), a method developed by John T. Lis and David Gilmour in 1984. Various methodologies can be used to perform ChIP such ascarrier ChIP, fast ChIP, quick and quantitative ChIP, MicroChIP, and Matrix ChIP to name a few. Continual refinement of these methodologies has advanced ChIP to new levels.

Diagenode, a pioneer in the field of epigenetic marks and epigenome studies, offers state-of-the-art products that enable high reproducibility and consistency in epigenetics research. Diagenode is committed to epigenetics research, backed by a vast worldwide network of scientists and experts working in fields like cancer research, induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC), and microRNA studies. The company offers a vast range of unique and robust products for studying epigenetic marks, unmatched by any competing alternatives. Products include antibodies, reagents, kits, and equipment for DNA or chromatin shearing, chromatin immunoprecipitation (ChIP, ChIP-Seq), methylated DNA immunoprecipitation (MeDIP, MeDIP-Seq), methyl binding domain (MethylCap, MBD), bisulfite conversion, automation ( IP-Star, SX8G Compact), and next generation sample preparation.

MeDIP  ||  5-hmC  || 5-mC