Chromatin dynamics involved in gene regulation
DNA in cells is wrapped around proteins called histones forming structures known as nucleosomes. These nucleosomes can fold into higher ordered structures, concealing DNA within them. This reduces accessibility to the DNA and adds another layer to the regulation of gene expression. This is a new target for drug discovery.
Opening of these structures to expose the DNA is influenced, among other factors, by gene expression levels. The deregulation of gene expression levels could be responsible for many diseases possibly because proteins in charge of the burial of DNA in the nucleosomes are themselves deregulated. Studying these potential changes in gene expression without altering the underlying DNA sequence is known as Epigenetics, an actively expanding research field.
There is a growing interest in manipulating chromatin dynamics for applications in cell signalling, drug discovery and chromatin research. Progress in this area requires the identification of design rules for the chromatin system.
Native nucleosome substrates
EpiCypher has pioneered the manufacture of nucleosomes for next generation epigenetic research and drug discovery, producing the highest quality nucleosomes available.
They now offer a rapidly expanding portfolio of fully defined and homogeneous recombinant nucleosomes incorporating different histone and DNA modifications, site mutations, or histone variants and chromatin remodeling assay substrate. Epicypher is even able to provide “designer” nucleosomes (dNuc), recombinant nucleosomes with single or multiple defined modifications at different histone sites.
To produce dNuc nucleosomes with native modification, Epicipher uses the best method, which is Native Chemical Ligation (NCL). NCL results in the scar-less incorporation of native methyllysine as shown on the illustration below. The classic methyl-lysine analog leads to different structures than the native ones.
To complete the native nucleosomes tool box, you can also find highly purifed nucleosomes isolated from Chicken and HeLa.
EpiCypher has also developed standardized criteria, such as modified histone synthesis/purification level, the proper integration of post-translational modifications (PTMs) or octamer and nucleosome assembly, as minimal requirements to validate these reagents for use in chromatin research and drug discovery applications. Indeed, all EpiCypher’s modified histones are validated by analytical HPLC and high-resolution mass spectrometry, as undesired species could alter the histone structure and impact electrostatic or hydrophobic interactions, therefore compromising nucleosome assembly.
Barcoded nucleosome spike-in for chromatin IP
The latest EpiCypher product is the K-MetStat™ panel, a Sample Normalization & Antibody Profiling for Chromatin Immunoprecipitation (SNAP-ChIP) for histone lysine methylation. SNAP-ChIP uses DNA barcoded designer nucleosomes as next-generation spike-ins for ChIP. For the first time, users can monitor antibody specificity and evaluate technical variability within a ChIP experiment, setting SNAP-ChIP apart from any other spike-in controls currently available on the market.
Convenient screening substrates for chromatin remodeling modulators
Chromatin remodeling enyzmes are involved in human diseases because they modulate the access of the DNA and so they have a major role in gene expression and genome repair. Unfortunately, the enzymes are challenging to study. Indeed, their subtrates, the nucleosomes, have a complex and dynamic structure. Therefore, EpiCypher has developped two kinds of nucleosome substrates for in vitro functional screening. They are called EpiDyne nucleosome substrates. There is an EpiDyne REA Nucleosome Remodeling Substrate for restriction enzyme assays. It’s a nucleosome including a DNA target motif (in purple on the picture below). The target site can be a restriction enzyme recognition site or a DAM methylation sequence.
The other option is an innovation. It’s a fluorescent version for HTS platforms, including FRET, fluorescence polarization and AlphaScreen Assays. The principle of this EpiDyne FRET Substrate is illustrated below.
Nucleosomes – the key to epigenetic drug discovery and cell signalling studies
Research shows that nucleosomes are the optimal substrates to characterize many chromatin regulators. These reagents represent powerful tools, critical in understanding chromatin biology for the discovery of new drug targets resistant to DNA or histone peptide-based approaches and the development of effective therapeutic agents like exogenous nucleosome-binding molecules (eNBMs). Recombinant nucleosomes are truly the next step towards epigenetic drug discovery but also cell signalling studies.
Your toolbox for chromatin dynamics studies
Here’s an overview of just some of EpiCypher’s products available in Europe here: