Quantification of combinatorial epigenetic modifications using defined nucleosome standards

PROJECT SUMMARYNucleosomes are the fundamental repeating units of chromatincomprised of four core histone proteins that are subject to a variety of post translational modificationsPTMse glysine methylation and acetylationEpigenetic reader proteins coordinate the cellular response to these PTMs by influencing chromatin compaction and gene expression through domains that recognize specific PTMsA rapidly expanding area of research has shown that reader proteins often contain combinations of domainsi emultivalencyin order to simultaneously translate multiple PTMs that decorate a single nucleosomeReadout of thishistone codehas profound functional outcomes for cellular functionse gcell cycle regulationas well as disease statese gcancer and othersDespite increasing recognition that quantifying combinations of epigenetic marks can improve predictive validity in biomarker studies and also increase the magnitude of response to epigenetic inhibitor treatmentthe dearth of available technologies to quantify co occurring PTMs has impeded scientific discovery in this fieldA major limitation for developing assays to quantify combinatorial PTMs has been the lack of control reagents that offer sufficient resolution to detect the combinations of PTMs that are read by multivalent readersReadouts based on histone proteinsmass spectrometryhistone ELISAsrequire elaborate sample processing protocols and simply cannot detect epigenetic marks in transe gPTMs on different histone tails within a nucleosomehistone DNA interactionsetcApproaches capable of assaying PTMs at nucleosomal resolution are low throughputlaboriousand or not quantitativee gsingle nucleosome imagingre ChIPClearlydevelopment of streamlinedhigh throughputquantitative assays using defined nucleosomal calibrants is urgently needed to decipher combinatorial histone codes and accelerate epigenetic therapiesHerewe propose to developQuantiNucTMa high throughput platform of nucleosome calibrated assays to measure PTMs from biological samples with high precisionThe innovation of this proposal is the novel application of modified recombinant nucleosome calibrants to enable minimal sample processing and reliable quantification of both single and combinatorial PTMsIn Aimwe will develop a combinatorially modified nucleosome and employ it to optimize ELISAslow throughput but widely accessible applicationsas well as AlphaLISAsbead based proximity assays for high throughput applicationsThe optimized reagents and assays developed in Aimwill be used for proof of principle biological validation in Aimwhere we will apply QuantiNuc to recapitulate the unique biology of combinatorial PTMsCompletion of these aims will demonstrate feasibility for developing an expanded QuantiNuc platform in Phase II to commercialize assays that will help decipher the histone codeenable biomarker discoveryand facilitate drug development PROJECT NARRATIVE After deciphering the genetic codethe next major frontier is to understand theepigenetic codea collection of chemical changes that direct DNA function and that are emerging as highly predictive indicators of disease state and treatment responseScientific discovery in this field is impeded by a dearth of available technologies with the appropriate controls for rapidreliable quantification of combinations of epigenetic marksIn this proposalEpiCypher is developing innovativehigh throughput assays using defined controls to enable unparalleled resolution of epigenetic marks in readily accessible biological samplesCommercial development of these revolutionary assays will help to decipher the epigenetic codethereby accelerating biomarker development to diagnose and treat complex diseases