Quantitative mapping of combinatorial histone modifications

PROJECT SUMMARYNucleosomes are the repeating building blocks of chromatinmade from a histone octamer wrapped by DNADynamic regulation of histone post translational modificationsPTMsplays a vital role in controlling gene expressioncell fate specificationand other essential cellular functionsInterestinglythese PTMs form a combinatorial molecular languagei ethe histone codethat recruits specific epigenetic effector proteins to transduce downstreampatho physiological signaling cascadesConsequentlydefects in the histone PTM landscape are associated with vast human pathologiesHereEpiCypher will develop EpiTandemTMan innovative nucleosome enrichment platform to quantify combinatorial histone PTMsThe 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 combinationsQuantitative mapping of dual histone PTMs may provide novel biomarkers with disease specificityHowevercurrent assays capable of detecting combinatorial PTMs are laborious or lack precision and reproducibilityEpiCypher has recently developed the first quantitative Chromatin ImmunoPrecipitationChIPplatform termed SNAP ChIPSample Normalization and Antibody ProfilingRHGwhich uses PTM defined recombinant designer nucleosomesdNucsat a range of concentrations as DNA barcodedspike incalibration standardsHerewe will develop our DNA barcoding technology to reliably measure combinatoriallymodified nucleosomes using a tandem histone PTM reader binding domain systemReader proteins are ideal for this approach as these proteins are highly specific and naturally used to interpret the combinatorial histone codeIn Aimwe will develop DNA barcoded H K meH Kacand H K meH Kmemodified dNucs and use these reagents to optimize development of two tandem reader domain pairs for specific enrichment of dually modified mononucleosomesIn Aimwe will demonstrate the power of our EpiTandem approach by performing EpiTandem Seq to quantify the genome wide distribution of H K meH Kacand H K meH Kmemodified nucleosomes in human lung cancer cellsKey to our approach is the application of DNA barcoded singlyand combinatorially modified dNucsfrom Aimas spike in controls to aconfirm specific enrichment of dually modified nucleosomesvssingleand bquantify the distribution of these dual PTMs genome wideLastlywe will validate our EpiTandem Seq results by comparing the genome wide distribution of dually modified nucleosomes with single PTM maps generated using SNAP ChIP SeqIn Phase IIwe will expand the EpiTandem platform to incorporate additional PTM combinationse gH K meH KmeWe will also develop and validate pre clinical EpiTandem assays for next generation biomarker identification and to measure effectiveness of epigenetic targeted therapy!PROJECT NARRATIVEChromatin is regulated in part by histone post translational modificationsPTMson nucleosomesThese PTMs function in various combinations to dynamically regulate key cellular processesincluding gene expression and cell fate specificationHowevertools are lacking to quantitatively measure combinatorial histone PTMsHereEpiCypher is developing EpiTandemTMthe first platform capable of reliably mapping and quantifying combinatorial PTMs genome wideIn addition to basic research applicationsthis 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!