Epigenetics research is transforming how researchers study complex biological processes, particularly those related to gene expression. Chromatin structure directly impacts the accessibility of transcription factors to target loci, and thus has important roles in cellular development, proliferation, and survival. In addition, there is extensive research on epigenetic factors that are correlated with and/or contribute to the development of human disease. Epigenetics research is inextricably linked to the development of multiple diseases, including cancer and Alzheimer’s disease.
As indicated above, epigenetics research is largely focused on chromatin structure, which refers to the exquisite packaging of DNA in the cell. The base subunit of chromatin is the nucleosome, which refers to ~147 base pairs of DNA wrapped around a histone octamer core. This “beads-on-a-string” structure is then further modified and folded, creating complex chromatin profiles that regulate multiple downstream processes in the cell, and are integral to proper gene expression.
The histone octamer core of nucleosomes is composed of 4 different histone proteins (2 of each: H2A, H2B, H3, and H4. The tails of histone proteins are decorated with a variety of modifications, or post-translational modifications (PTMs), including methylation, phosphorylation, and acetylation. Different modifications have distinct impacts on chromatin architecture; for instance, acetylation increases accessibility of chromatin to transcription factors and other interacting proteins, and in general is associated with gene activation. Specific histone PTMs have also been found to denote genomic function. Trimethylation on histone H3 Lysine (H3K4me3) is associated with active transcription start sites, while trimethylation of H3K27 denotes gene repression. Thus, profiling the location of histone PTMs may help annotate the genome as well as identify underlying changes in chromatin structure that lead to deregulated gene expression in disease.
Accurate and sensitive chromatin profiling of histone PTMs, such as H3K4me3, is essential to understanding the role of chromatin structure in complex processes. The most common assay used to map histone PTMs is chromatin immunoprecipitation combined with next-generation sequencing, or ChIP-seq. In ChIP, scientists use a histone PTM-specific antibody to enrich chromatin containing the target PTM from a large pool of fragmented chromatin. DNA is subsequently isolated from the isolated chromatin fragments, and prepared for sequencing.
One of the most important things to consider when developing a protocol for ChIP is to make sure your assay utilizes highly specific antibodies. Indeed, an antibody must be highly specific to a particular target in order to gain accurate results from testing, and to limit contaminating signals from off-target marks. Recent studies, including those from EpiCypher1, have found that a significant number of antibodies to histone PTMs (i.e. H3K4me3) display cross-reactivity to related PTMs, and have resulted in erroneous biological conclusions. This is largely due to the methods used to validate these antibodies, such as modified histone peptide arrays, which lack the context of the endogenous nucleosome structure and fail to replicate experimental conditions in ChIP-seq.
EpiCypher has developed SNAP-ChIP (Sample Normalization and Antibody Profiling for ChIP) technology to address this unmet need in the field. SNAP-ChIP uses panels of recombinant modified nucleosomes (i.e. spike-ins) to identify highly specific antibodies within the context of a ChIP-seq experiment. For best-in-class performance ChIP antibodies, EpiCypher now offers a set of highly validated SNAP-ChIP certified antibodies, such as their H4K8Ac antibody and H3K4me3 Antibody. Each SNAP-ChIP certified antibody, including their H3K4me3 antibody, has been extensively tested in situ using SNAP-ChIP spike-ins to validate the antibodies for high on-target specificity and enrichment and low cross-reactivity. Their H3K4me3 antibody, for example, is a monoclonal antibody which exhibits no cross-reactivity with H3K4me1, H3K4me2 or any other methyl-lysine states represented in EpiCypher’s SNAP-ChIP K-MetStat panel.
To learn more about EpiCypher’s SNAP-ChIP spike-in controls for antibody validation or to see a complete list of their SNAP-ChIP certified antibodies s, make sure to visitEpiCypher.com.
1.Shah et al., 2019. “Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies.” Mol Cell 72(1):162-177. PMID: 30244833.