Customized Nucleosome Assembly

By Author: Leslie Shannon
Total Articles: 3
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Chromatin structure refers to the exquisite three-dimensional architecture of DNA in a cell, which impacts gene expression by altering genome accessibility to transcription factors and other regulatory proteins. This structure is fundamental to many cellular processes, including differentiation, proliferation and survival, and aberrant regulation of chromatin is linked to multiple human diseases, such as cancer.
The base subunit of chromatin is the nucleosome, which refers to ~147 base pairs of DNA wrapped around a histone octamer. The histone octamer contains two of each core histone: H2A, H2B, H3 and H4. The tails of these histones are decorated with a diverse repertoire of post-translational modifications (PTMs), including acetylation, methylation, phosphorylation and citrullination. Unique combinations of PTMs on the histone tails regulate chromatin accessibility, both by altering the charge of the histone tails, and via interactions with various chromatin interacting proteins (i.e. transcription factors and nucleosome remodeling complexes).
In the past, histone PTMs and modifying enzymes were studied using histone ...
... tail peptides. These peptides, both modified and unmodified, were fairly easy to synthesize and inexpensive substrates for screening inhibitors, antibodies, and PTM-binding specificity.
However, recent studies have revealed that many chromatin interacting proteins and modifying enzymes require a nucleosome substrate for activity. For instance, the NSD2 methyltransferase requires a nucleosome substrate to catalyze H3K36 di-methylation [1], and the SETD8 H4K20 methyltransferase displays a 10,000-fold decrease in Km when using a nucleosome vs. histone peptide substrate [2].
To address the growing need for reliable and high-quality nucleosome substrates for epigenetics research, EpiCypher offers custom Nucleosome Assembly services. EpiCypher is expert in recombinant and semi-synthetic nucleosome assembly, and has developed an entire suite of nucleosome products that complement these services:
● Modified designer nucleosomes (dNucs): dNucs are highly pure semi-synthetic recombinant human nucleosomes, assembled with a variety of histone PTMs. Each modified histone is HPLC purified (>95% pure) and validated by mass spectrometry and western blot.

● EpiDyne chromatin remodeling substrates: EpiDyne substrates are manufactured using an elongated DNA template, and include tags or restriction enzyme sites to enable direct monitoring of nucleosome remodeling activity.

● Recombinant nucleosomes (rNucs): rNucs are pure, unmodified recombinant human nucleosomes, representing the ideal substrate for many histone modifying enzymes.

● Oncogenic nucleosomes (oncoNucs): oncoNucs are generated using histones with known cancer-associated mutations, and are useful for screening potential epigenetic inhibitors or directed therapeutics.

● Variant nucleosomes (vNucs): vNuc substrates are assembled using histone octamers containing defined variants, such as H2AZ and H2AX. These histone variants have important roles in fundamental cell processes, including the DNA damage response.

● versaNuc: versaNuc is the most recent addition to EpiCypher’s recombinant nucleosome assembly technologies. versaNuc uses a “tailless” histone H3 to generate tailless octamers. These histone octamers can be wrapped with a variety of custom DNA templates, including those with hemi- or fully-methylated sequences. Following tailless nucleosome assembly, the resulting nucleosomes are ligated to modified H3 peptides, thus generating a fully customized versaNuc. This technology can be easily expanded to create libraries of versaNucs, with varying DNA templates or H3 PTMs, thus enabling analysis of these complex chromatin substrates.
As the study of chromatin advances, there is an increasing requirement for defined and reliable tools. EpiCypher’s custom nucleosome assembly is an ideal service for high-throughput studies or exploratory analysis of understudied PTMs. For more information about EpiCypher’s recombinant nucleosome technologies, including versaNuc, visit EpiCypher.com
For more information about Chip Antibody and Recombinant Nucleosomes Please visit: EPICYPHER INC..