Epigenetics refers to alterations in gene expression that occur independently of the underlying DNA sequence (i.e. mutations). Epigenetics research is inextricably linked to the study of chromatin architecture, as chromatin and chromatin-associated proteins are essential to the packaging and accessibility of the genome. The base subunit of chromatin is the nucleosome, which is composed of a histone octamer wrapped with DNA. Histone tails are decorated with a variety of post-translational modifications (PTMs), which impact gene expression by either 1) directly altering chromatin accessibility to transcription factors, or 2) interacting with and / or recruiting other chromatin remodeling or modifying complexes.
Chromatin structure has widespread effects on development and disease. At least 20% of all cancers contain mutations in the SWI/SNF chromatin remodeling complexes, while other cancers are known to harbor specific alterations in histone methyltransferases (i.e. a subset of leukemias contain oncogenic fusions of the H3K4 methyltransferase MLL). Thus, understanding the factors that regulate chromatin may reveal novel therapeutic targets, as well as new diagnostic / prognostic indicators of disease state.
Localization of histone PTMs and other chromatin interacting proteins on chromatin is key to elucidating their function. Indeed, several histone PTMs are associated with genomic features, such as H3K4me3 (denotes actively transcribed promoters), H3K27ac (marks enhancer) and H3K9me3 (repressed heterochromatin). The main method used to profile histone PTMs and other factors is chromatin immunoprecipitation sequencing or ChIP-seq. ChIP-seq uses factor-specific antibodies to enrich subsets of chromatin from large fragmented pools, followed by next-generation sequencing. Despite its prevalence in the field, ChIP-seq has numerous drawbacks, including high backgrounds / low resolution, high sequencing depth and cell input requirements (which can become costly), and labor-intensive protocols. As the field moves increasingly toward single-cell technologies, scientists are eager to test new methods.
The recent development of immunotethering approaches, such as CUT&Tag (Cleavage Under Targets and Tagmentation), have generated significant interest in the epigenetics field. These strategies use protein A / protein G (i.e. pAG) to “tether” an enzyme (e.g. Tn5 in the case of CUT&Tag) to antibody-marked chromatin, developing streamlined enrichment processes that generate high-resolution data with a fraction of the cell input compared to ChIP-seq.
In the case of CUT&Tag, immobilized cells are permeabilized and treated with a factor-specific antibody. Cells are then treated with a fusion of pAG and Tn5, localizing Tn5 to antibody-bound chromatin loci. Tn5 is loaded with sequencing adaptors; following activation with magnesium, Tn5 simultaneously cleaves adjacent chromatin and ligates sequencing adaptors to DNA. These adaptor-ligated fragments are then selectively amplified by PCR and used for next-generation sequencing.
The application of Tn5 combined with the solid support enables accelerated protocols and reduced backgrounds vs. ChIP-seq. CUT&Tag can be also used to profile small cell populations, down to the single cell level, and is compatible with reduced sequencing depths (3-5 million per reaction, vs. 30-40 million with ChIP-seq). The result is higher resolution data at a substantially reduced price point, making CUT&Tag a desired application in epigenetics labs.
EpiCypher is dedicated to providing the latest advances in epigenetics technologies, including immunotethering approaches. Each of our products is validated by rigorous quality control metrics, meaning that you can be confident in the accuracy and reliability of your data. We will soon be adding to our CUTANA line of immunotethering approaches, with reagents for CUT&Tag. Make sure to follow Epicypher.com for the latest news and updates on this product release.