Nucleosomes are basis units of DNA packaging in eukaryotic cells. A core particle (mono-nucleosome) consists of a segment of DNA called core DNA (147 bp in length) wound around a histone octamer (Fig 1.). Histone octamers are made up of 2 copies of the the core histones H2A, H2B, H3, and H4. These mono-nucleosomes are connected by linker 80bp-long DNA. H1 histone, the so-called linker histone, binds to the linker DNA close to the entry and exit of the core DNA and is involved in chromatin compaction (Fig 1.).
DNA and Histone modifications regulate gene expression
In order to regulate processes such as transcription, DNA replication and DNA repair, the chromatin structure has to be modulated in order to change the condensation level. Different enzymatic (epigenetic) modifications are correlated to either gene activation or gene silencing.
While methylation of DNA and histones causes nucleosomes to pack tightly together (thus hindering e.g. transcription factors to bind the DNA and to cause gene expression ), histone acetylation for instance cause loosening of the packing of nucleosomes (thus allowing access of factors and gene expression (Fig. 2)).
Substrates for epigenetic enzyme assays and inhibitor screenings
To measure the activity of histone modifying enzymes and screen possible inhibitors, peptides derived from specific histone sequences are often used in in vitro assays. These peptides can either be “blank” e.g. for methyltransferase reactions, or specifically modified for instance for demethylase or deacetylase reactions. Often these peptides are absolutely sufficient for screening purposes, but for some chromatin modifying enzymes, nucleosomes are a more physiologically relevant substrate than these peptides. Nucleosomes can be purified from diverse cells to serve as substrates. These nucleosomes obviously carry all modifications stemming from the parent cells. On the other hand the different core histones can be expressed recombinantly and subsequently be assembled to form histone octamers or even – by introducing core DNA – to form mono-nucleosomes. In this case the resulting mono-nucleosomes are “blank” and can be perfectly used for measuring the activity of so-called writer enzymes (enzymes which introduce modifications). Moreover, recombinant histones can be selectively modified at defined sites and used as substrates for eraser enzymes (enzyme which remove modifications)
tebu-bio has selected and cooperates with a number of partners such as Epicypher, BPS Biosciences, Anaspec and CHI Scientific supplying high quality substrates. Epicypher is even able to provide “Designer” nucleosomes, recombinant nucleosomes with single or multiple defined modifications at different histone sites (see below).
Peptide substrates
Histone derived peptides can be either “blank” (without any modifications) or modified with single or multiple modifications. Furthermore, the peptides can be fluorogenic, i.e. labeled with a fluorophore which starts to fluoresce upon modification of the peptide e.g. by a deacetylating enzyme.
There is already a huge selection of both types of peptides. To get a quick overview, have a look at this list of histone derived peptides.
Any peptide you might not find on catalog, can be offered on a customized basis – be it standard peptide sequences or peptides with modifications such as acetylations and methylations. Use the form below to get a quote – we just need the sequence, modification, amount and expected purity.
Native nucleosomes
Native nucleosomes carry all modifications and represent a highly physiological substrates for histone modifying enzymes. They can be isolated from cell lines (e.g. Hela or HEK293 cells) or for instance from chicken erythrocytes. Hela cell derived nucleosomes are available as mono-nucleosomes and poly-nucleosomes.
Here, I’ve selected a choice of different sources. To find the preparation best suited to your needs, please feel free to contact me with the form below.
- HeLa Mononucleosomes
- HeLa Polynucleosomes
- HEK293 Polynucleosomes
- Chicken Oligonucleosomes, Purified
- Native histone octamers
Recombinant nucleosomes, histones and histone polymers
If you need “blank” (free of modification) histone substrates, recombinant nucleosomes, histones, and histone dimers, tetramers and octamers are available.
- Recombinant histone, unmodified
- Recombinant histone dimers, unmodified
- Recombinant histone tetramers, unmodified
- Recombinant histone octamers, unmodified
- Recombinant nucleosomes, unmodified: Mono-nucleosomes are assembled from recombinant human histones (two each of histones H2A, H2B, H3 and H4) wrapped by 147 bp linker DNA.
- Generate your own nucleosomes from recombinant histones or polymers, with Nucleosome Assembly 601 Sequence DNA.
A few recombinant histones and histone polymers with modifications are available as well.
- Recombinant histone, modified
- Recombinant histone tetramers, modified
- Recombinant histone octamers, modified
Designer nucleosomes
As some epigenetic enzymes require highly specific substrates, Epicypher has launched a new product line – Designer Nucleosomes (dNuc). dNUCs are semi-synthetic nucleosomes incorporating specific histone post-translational modifications. These reagents represent a powerful new technology – critical in understanding chromatin biology and for the development of novel drug targets and precision therapeutics. It is known that highly specific histone modifications can be linked to certain diseases (see Table 1). dNUCs serving as substrates for the most relevant epigenetic enzymes will be added to our catalog in the coming months. If you have specific modifications in mind, which which are know to be optimal for your enzyme of interest, please let me know through the form below. Epicypher might already have this designer nucleosome in their pipeline – or might be able to produce it on a customized basis.
To get more insight into the dNuc manufacturing process Epicypher applies, you can download the white paper Not All Designer Nucleosomes are Created Equal: A Tale of Two Cysteines.
The paper compares the two currently used synthetic methods to produce dNUCs, native chemical ligation (NCL) and methyl lysine analog (MLA). The paper shows that NCL – the method used by Epicypher – yields superior nucleosome preparations.
Please feel free to leave your comments or questions in the form below!
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