A Few Methods for Identifying Histone Modifications


Epigenetic research, particularly work focused on chromatin structure, is shedding new light on the factors involved in gene expression and transcriptional silencing. Though the field is constantly evolving, the implications this research has for understanding disease development, diagnosis, and potential treatments is immense. Indeed, many of the epigenetic factors involved in chromatin accessibility and gene regulation are implicated in the development of human diseases, including cancer and neurodegenerative diseases.

The study of chromatin structure is often focused on factors that drive accessibility to transcription factors and other chromatin interacting proteins. For instance, Histone Modifications come in a variety of forms, including lysine methylation and acetylation, or serine phosphorylation. These post-translational modifications (PTMs) alter the compaction of chromatin, making it more or less accessible transcription factors, and thus regulate gene expression patterns. Mapping Histone Modifications is an important aspect of epigenetics research, as localization of distinct PTMs correlates with unique genomic features, such as active gene promoters, enhancers, or silenced chromatin.

There are many different protocols for profiling histone PTMs. The most common method is Chromatin Immunoprecipitation, or ChIP, which uses highly specific antibodies to enrich chromatin fragments associated with specific histone modifications. These chromatin fragments are isolated and subjected to next generation sequencing (in ChIP-seq) in order to map histone PTMs to specific locations in the genome. ChIP has been widely used in epigenetics research for over 30 years, with variations on assay readout improving the resolution over time (i.e. ChIP-microarray, ChIP-qPCR, and ChIP-seq).

Despite its widespread usage, chromatin immunoprecipitation has some drawbacks. Indeed, there have been few innovations over the past 30 years with regards to the ChIP procedure itself, and this process is still very reliant on the use of specific, validated antibodies for high-quality and accurate results. Antibodies that are nonspecific, cross-reactive, or display low binding efficiency (i.e. enrichment) will contaminate results with high background and off-target signal. All of these situations can compromise the results of a ChIP assay.

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EpiCypher offers SNAP-ChIP validated antibodies that have been extensively tested to offer high IP efficiency and low cross-reactivity. Each lot of SNAP-ChIP certified antibodies is extensively validated using defined, DNA-barcoded modified recombinant nucleosome spike-in controls to test their reliability in situ, thus delivering best-in-class performance for superior ChIP experiments.

However, new approaches are being developed that may soon surpass the widespread use of ChIP, due to their increased sensitivity and signal : noise, low sequencing depth requirements, compatibility with low cell inputs, and rapid turnaround times. These methods, including CUT&RUN (Cleavage Under Targets and Released Using Nuclease), are based on chromatin immunocleavage approaches, or ChIC. ChIC assays leverage the immuno-targeting properties of protein A and protein G to tether an enzyme, such as micrococcal nuclease (MNase) to antibody-bound chromatin loci. Following controlled activation of the MNase, cleaved chromatin fragments can be isolated for downstream sequencing or qPCR analysis.

CUT&RUN assays build on the ChIC technology by immobilizing cells or nuclei on a solid support. Cells / nuclei are permeabilized, labelled with an antibody to a unique chromatin target, and then treated with a protein A / protein G fused to MNase (pAG-MNase; similar to ChIC, above). Controlled activation of MNase with calcium results in the selective fragmentation of antibody-bound chromatin loci. The use of a solid support enables a streamlined assay protocol, including easy removal of cell debris and non-fragmented chromatin, and isolation of target chromatin fragments from assay supernatant.

As a result of these assay improvements, CUT&RUN enables analysis of low cell inputs (down to 60 cells) with high resolution and sensitivity vs. standard ChIP-seq. In addition, CUT&RUN is also compatible with extremely low sequencing depths, generating publication-quality data with only 3-5 million reads per samples (vs. 20-40 million with ChIP-seq).

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EpiCypher has launched their line of CUTANA CUT&RUN products, starting with pAG-MNase, making this remarkable technology widely available to chromatin researchers. Our pAG-MNase and optimized CUT&RUN protocol have robust activity for profiling histone modifications, as well as transcription factors, chromatin remodeling enzymes, and other chromatin associated proteins. All assays show low signal-to-noise compared with ChIP-seq, and our protocol is designed for low sequencing depths (3-5 million reads) and compatibility with benchtop sequencers (i.e. Illumina MiniSeq or MiSeq). Our streamlined protocol also enables you to go from cells to data in 2 days (vs. weeks to months with ChIP-seq), thus representing a notable advance in chromatin profiling technology.
Whether you’re in need of validated ChIP antibodies or you are waiting for the release of new and improved CUT&RUN technology, visit EpiCypher.com to keep ahead of research and to find the antibodies and enzymes required for your chromatin profiling assays.

For more information about Chip Antibody and Cut And Run Chip Seq Please visit: EPICYPHER INC..

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