What are Nucleosomes?
Chromatin, the material into which genomic DNA is packaged in eukaryotes, is a very dynamic structure. The smallest subunit of chromatin is the nucleosome, consisting of 147 base pairs of DNA wrapped around an octamer of core histone proteins. Thehistone octamer is composed of a central heterotetramer of histones H3 and H4, flanked by two heterodimers of histones H2A and H2B. Each nucleosome is separated by 10 to 60 bp of linker DNA. The resulting nucleosomal array constitutes achromatin fiber of about 10 nm in diameter. This arrangement is folded into more condensed fibers (about 30 nm) that are stabilized by binding of a linker histone (Histone H1) to each nucleosome core. Such 30 nm fibers are then condensed in vivoto form thicker interphase fibers or the most highly compacted metaphase chromosome structures. But, the role of histones and nucleosomes is not limited to the compaction of the chromatin. Epigenetic modifications such as phosphorylation, acetylation, methylation and ubiquitination at specific amino acid residues on the histone tails influence higher-order chromatin structure that regulates the nuclear processes of transcription, chromosome packaging and DNA damage repair. Many of these specific histone modifications are conserved throughout eukaryotes. While the biological significance of some histone modifications remains to be understood, some have been demonstrated to correlate very closely with specific cellular states like transcriptional activity. Transcriptionally active chromatin is referred to aseuchromatin, while transcriptionally inactive chromatin is called heterochromatin. Active Motif offers the Nucleosome Preparation Kit to enable researchers to easily isolate mononucleosomes or oligonucleosomes for use as substrates in the analysis of histone modifications, for studies of enzyme kinetics, or in screening and profiling assays. Intact nucleosomes are a better choice than peptides or recombinant histones because they provide a more physiologically relevant substrate to enable to your assay to better mimic actual cellular biology.
Mononucleosomes refers to a single monomer of the nucleosome array. Due to the number of potential binding interactions that are possible on the histone tails, histone globular domain and the nucleosomal DNA, mononucleosomes can offer a simplified substrate for chromatin analysis. Active Motif offers Nucleosome Assembly Control DNA which can be used for in vitroassembly of mononucleosomes. The 187 bp double stranded DNA can be added to your core histones for formation of a control mononucleosome with a known DNA sequence. The Nucleosome Assembly Control DNA can also be used to monitor how chromatin interacting proteins or compounds effect mononucleosome assembly kinetics.
Understanding the position of nucleosomes can help provide information about chromatin context and gene regulation. Traditional methods used to look at nucleosome positioning include nuclease digestions methods, such as MNase-Seq or DNase-Seq, which rely on the fact that a nucleosome bound to DNA will protect the DNA from enzymatic digestions. However, these techniques destroy the physical linkages between binding sites and therefore are designed to look at average distribution across a panel of remodeled nucleosomes and not to determine the status of a single DNA molecule. Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE-Seq) is a method used to enrich for nucleosome depleted DNA using formaldehyde fixation and phenol-chloroform extraction. The nucleosome depleted DNA can be sequenced to identify regions that correspond with transcriptional start sites, enhancers and promoters. While understanding nucleosome positioning has great value, the ability to study nucleosome occupancy in the context of other epigenetic modifications provides a powerful tool for researchers to use in studying the different states of chromatin and its effect on gene regulation. Active Motif is the first company to offer a method to study both nucleosome occupancy and DNA methylation within the same DNA strand. NOMe-Seq (Nucleosome Occupancy and Methylome Sequencing) is a published method that was developed by the Peter A. Jones lab which can be used for gene-specific analysis of both nucleosome occupancy and DNA methylation levels on the same DNA strand of the gene of interest. It provides a temporal relationship between nucleosomes, transcription factor binding and DNA methylation.
The assembly of genomic DNA and histones into chromatin is a fundamental process that affects a broad range of genome-dependent processes including DNA replication, DNA repair and gene expression. In general, there are ATP-dependent and ATP-independent methods for reconstituting or assembling chromatin in vitro, but only the ATP-dependent process generates an extended array of ordered nucleosomes. Active Motif's Chromatin Assembly Kit utilizes an ATP-dependent method which enables you to study your DNA sequence of interest in a native chromatin environment. The simple, easy-to-follow protocol generates assembled chromatin in hours with few manipulations, providing you with material that is ideal for downstream applications such as in vitro ChIP, transcription and histone modification assays. To study specific histone modifications within a chromatin context, Active Motif's Recombinant Histones with site and degree-specific modifications can be used as substrates for chromatin assembly.
Recombinant Histones & Nucleosomes
Recombinant nucleosomes and histones are essential tools for investigating complex functional questions about chromatin-associated proteins, epigenetic regulation, nucleosome remodeling, transcriptional regulation, replication and DNA repair. Active Motif’s Recombinant Nucleosomes save you time and effort by providing pre-assembled human nucleosomes. Our Recombinant Nucleosomes offer the advantage of providing a more physiologically relevant choice for use as substrates in in vitroassays when compared to histone proteins alone or synthetic peptides. Each recombinant nucleosome is comprised of octamers of core histone proteins (H2A, H2B, H3 and H4) bound by DNA. These intact nucleosomes are ideal substrates for in vitro studies of histone post-translational modifications, enzyme kinetics or for inhibitor screening assays. Unlabeled or biotin-labeled nucleosomes are available to give you flexibility in your experimental design. Active Motif also offers histones with site-specific methylation, acetylation or phosphorylation modifications. Histone methylation, acetylation and phosphorylation are post-translational modifications that affect nucleosome structure and therefore the ability of transcription factors to access the DNA and regulate gene expression. Click on our link to get a complete list of our Recombinant Histones and Modified Histones.