Barcoded nucleosomes for analyzing combinatorial epigenetic regulators

Project SummaryNucleosomes are the elementary and repeating building blocks of chromatincomprised of a histone octamer wrapped inbp DNAHistone post translational modificationsPTMsplay a dynamic role in controlling gene expression and other essential cellular functionsThese PTMs form a systems level signaling networki ethe histone codethat recruits specific epigenetic effector proteins to transduce various downstream signaling cascadesSo called `readerproteins are compelling candidates for therapeutic manipulation of gene expression in multiple diseasesincluding cancerneurodegenerationand other conditionsHoweverhigh throughput methods are not available to comprehensively evaluate the interaction between physiologically relevant PTM states and their regulatory protein partnersIn additionPTM targets are often studied on a synthetic peptide backbonea substrate that poorly models in vivo chromatin structureTo address these issuesEpiCypher is developing a barcoded nucleosome platform for high throughput PTMprotein interaction studiesToward this goalour group has developed proprietary methods for the commercial production of recombinant histones customized with a broad range of PTMstermed `designer nucleosomesor `dNucsIn Phase Iwe synthesized dNuc substrates carrying single and combinatorial PTMsto establish EpiCodeTMa powerful barcoding technology for multiplexed protein PTM interaction studiesThe innovation of this technology is the ability to screen potentially hundreds of protein nucleosomal PTM combinations in a single experimentEpiCodeTM employs uniquely modified dNucs identified by barcodes appended to their DNA assembly sequenceDNA barcoding is central to our approach as it allows multiplexed binding events to be deconvolved using qPCREpiCodeTM allows specific proteins of interest to be combined with a PTMcustomized dNuc library to identify new interactionsIn Phase IIour first goal is to generate an expanded barcoded dNuc library targeting the combinatorial impact of histone acetylation and methylationtwo highly prevalent PTM families with significant diseaserelevanceAimWe will validate our expanded barcoded dNuc library by performing multiplexed binding assays using a collection of readers that interact with PTMs represented in the libraryAimAs part of this validationwe will establish protocols compatible with next generation sequencingNGSused in place of qPCRfor streamlined deconvolution of multiplexed barcodesIn Aimwe will develop simplified workflows that utilize plate based assay designsproviding high throughput assays for major end users interested in using this platform for large discovery projectsi epharmaceutical companiesFinallyseveral external laboratory sites will validate our platform with a goal of refining protocolsto minimize characterize variabilityand demonstrating its utility as a tool to identify novel protein PTM interactionsAs a powerful new tool to decode chromatin signalingEpiCodeTM will be of broad interest to both pharmaceutical and basic researchers