Epigenetics describes the phenomenon in which phenotypic differences are observed without alterations to the underlying genome sequence, and is largely regulated by changes in gene expression patterns. Epigenetics and gene expression directly relate to chromatin structure, the three-dimensional compaction of DNA in the nucleus. The fundamental unit of chromatin is the nucleosome, composed of a histone octamer wrapped with DNA. In order for gene expression to occur, DNA must be accessible to transcriptional machinery. Tightly wound chromatin (i.e. heterochromatin) inhibits gene expression since transcription factors and other chromatin interacting proteins cannot access the DNA. On the other hand, an open chromatin structure promotes gene expression by allowing transcription factors and other complexes to bind DNA. Changes in chromatin structure typically occur through modification of amino acid residues on histone tails.
Posttranslational modifications alter the chromatin landscape
Post-translational modification (PTM) of histone tails is one of the main factors influencing chromatin structure. Examples of these PTMs include acetylation, methylation, and phosphorylation. PTMs can alter chromatin structure both directly, by impacting DNA accessibility, or indirectly, by interacting with other proteins or chromatin remodeling complexes.
Acetylation typically occurs on lysine residues of histone tails and promotes a relaxed chromatin state, supporting transcription factor binding and gene expression. For example, the histone acetyltransferase CBP/p300 facilitates acetylation of the lysine 27 on histone H3 (H3K27ac). This PTM promotes gene expression by relaxing chromatin structure. CBP/p300 also directly binds acetylated histones (via its conserved bromodomain), and promotes recruitment of transcriptional activators. H3K27ac is enriched at enhancers and regulates genes involved in development as well as in disease, such as Alzheimer’s disease and hematological cancers. However, this is just one model of how epigenetics is linked to disease. Other examples include cell differentiation, proliferation and survival. Thus, studying the epigenetic regulation of gene expression is paramount to our understanding of human disease.
ChIP-seq requires highly specific antibodies
Chromatin immunoprecipitation (ChIP) is one of the main ways to study histone PTMs, such as H3K27ac. In this example ChIP experiment, a H3K27ac antibody conjugated to beads is added to a sample of fragmented chromatin. The H3K27ac antibody binds to chromatin carrying the H3K27ac PTM and is immunoprecipitated. Afterwards, the enriched DNA fragments are purified, sequenced and analyzed (ChIP-seq). This provides a genome-wide map of H3K27ac localization on DNA.
One of the main problems when carrying out a ChIP experiment is that antibodies often exhibit significant cross-reactivity to related PTMs. This can lead to errors in data interpretation and incorrect assignment of histone PTM function. Thus, the use of validated antibodies for ChIP-seq ensures accurate results by reducing cross-reactivity and contamination from off-target PTMs. Using a validated, highly specific antibody for ChIP-seq also results in more reliable sequencing data.
EpiCypher’s novel SNAP-ChIP process validates antibodies for ChIP-seq
EpiCypher recently launched the rigorous Sample Normalization and Antibody Profiling for ChIP (SNAP-ChIP) technique for validating anti-histone PTM antibodies for ChIP-seq assays. SNAP-ChIP uses panels of DNA-barcoded recombinant designer nucleosomes (dNucs) that carry distinct histone PTMs as spike-in controls for ChIP-seq experiments. These spike-ins enable in-application testing of antibody specificity and enrichment against a defined nucleosome substrates, providing unparalleled control over a ChIP assay. Further, spiking them into samples allows for direct comparisons between ChIP experiments by normalizing to the amount of on-target dNuc recovered from each sample.
EpiCypher offers a large selection of extensively tested SNAP-ChIP Certified antibodies, including their highly specific H3K27ac antibody, that exhibit high IP efficiency, high specificity, and low cross-reactivity. Therefore, experiments utilizing SNAP-ChIP Certified antibodies are more reliable and replicable. For more information about how SNAP-ChIP uses spike-in controls for highly specific antibody validation, visit EpiCypher’s SNAP-ChIP website.