Nucleosomes are the repeating building blocks of chromatin, made from a histone octamer wrapped by DNA. Dynamic regulation of histone post-translational modifications (PTMs) plays a vital role in controlling gene expression, cell-fate specification, and other essential cellular functions. Interestingly, these PTMs form a combinatorial molecular language (i.e. the histone code) that recruits specific epigenetic effector proteins to transduce downstream (patho)physiological signaling cascades. Consequently, defects in the histone PTM landscape are associated with vast human pathologies. Here, EpiCypher will develop EpiTandemTM, an innovative nucleosome enrichment platform to quantify combinatorial histone PTMs. The innovation of this approach is the use of recombinant tandem reader domains and barcoded modified recombinant nucleosomes to map the genomic location and quantify the relative abundance of specific histone PTM combinations. Quantitative mapping of dual histone PTMs may provide novel biomarkers with disease specificity. However, current assays capable of detecting combinatorial PTMs are laborious or lack precision and reproducibility. EpiCypher has recently developed the first quantitative Chromatin ImmunoPrecipitation (ChIP) platform termed SNAP-ChIP (Sample Normalization and Antibody Profiling; R44HG008907), which uses PTM-defined recombinant designer nucleosomes (dNucs) at a range of concentrations as DNA-barcoded ?spike in? calibration standards. Here, we will develop our DNA-barcoding technology to reliably measure combinatorially- modified nucleosomes using a tandem histone PTM reader binding domain system. Reader proteins are ideal for this approach as these proteins are highly specific and naturally used to interpret the combinatorial histone code.
In Aim 1, we will develop DNA-barcoded H3K4me3/H4K16ac- and H3K9me3/H3K36me2- modified dNucs and use these reagents to optimize development of two tandem reader domain pairs for specific enrichment of dually-modified mononucleosomes.
In Aim 2, we will demonstrate the power of our EpiTandem approach by performing EpiTandem-Seq to quantify the genome-wide distribution of H3K4me3/H4K16ac- and H3K9me3/H3K36me2-modified nucleosomes in human lung cancer cells. Key to our approach is the application of DNA-barcoded singly- and combinatorially-modified dNucs (from Aim 1) as spike in controls to a) confirm specific enrichment of dually modified nucleosomes (vs. single) and b) quantify the distribution of these dual PTMs genome-wide. Lastly, we will validate our EpiTandem-Seq results by comparing the genome-wide distribution of dually-modified nucleosomes with single PTM maps generated using SNAP-ChIP-Seq. In Phase II, we will expand the EpiTandem platform to incorporate additional PTM combinations (e.g. H3K4me3/H3K27me3). We will also develop and validate pre-clinical EpiTandem assays for next-generation biomarker identification and to measure effectiveness of epigenetic-targeted therapy. !
Chromatin is regulated in part by histone post-translational modifications (PTMs) on nucleosomes. These PTMs function in various combinations to dynamically regulate key cellular processes, including gene expression and cell fate specification. However, tools are lacking to quantitatively measure combinatorial histone PTMs. Here, EpiCypher is developing EpiTandemTM, the first platform capable of reliably mapping and quantifying combinatorial PTMs genome-wide. In addition to basic research applications, this technology will propel epigenetic drug development by providing access to validated dual PTM biomarkers that remain challenging to analyze using current state of the art tools. !
