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Key Tools for Chromatin Immoprecipitation (ChIP assay)

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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:

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