Development of novel spike-in controls for quantitative chromatin-associated protein profiling

PROJECT SUMMARYAlterations in chromatin regulators are associated with diverse human pathologiesThe ability to quantitatively assess these factors in healthy and diseased cells is essential to accelerate the development of therapeutics targeting epigenetic regulationa growing area of studywith many candidates already in clinical trialsHoweverChIP Seqthe most widely used approach to map the genomic location of Chromatin Associated ProteinsChAPsis often limited by poor resolutionsensitivityand reliabilityDrSteven Henikoff s group recently developed CUTandamp RUNCleavage Under Targets and Released Using Nucleasea new mapping approach with vastly improved assay performance vsChIP SeqCUTandamp RUN uses ChAP targeting antibodies to locally tether protein A micrococcal nucleasepA MNaseto chromatin in intact nucleifollowed by controlled MNase activation to cleave nearby DNASequencing of the subsequently released DNA fragments yields precise target localization profiles using fractionsvsChIP Seqof the required cellular inputfold lessand sequencing depthandgtfold lessThe efficiency of this method could now enable pre clinical applications in a high throughput formatsuch as quantifying the genome wide effects of epigenetic therapeuticsHoweverdelivering on such promise will require the development of quantitative spike insIn this Fast Track SBIR proposalEpiCypheris partnering with DrKami Ahmad of the Henikoff lab to develop quantitative spike in controls for ChAPs using CUTandamp RUNChAP CUTandamp RUNEpiCypher has recently developed the application of DNA barcoded recombinant designer nucleosomesdNucsas quantitative spike in controls for histone post translational modificationPTMChIP studiesi eSNAP ChIPHoweverthere are no tools to normalize mapping data for ChAPswhich make up the largest segment of the ChIP Seq marketThe innovation of this project is the engineering of DNA barcoded dNucs that contain eithera ChAP epitopeora Short Peptide TagSPTe gFLAGfused to the N terminus of histone HThese can then be used to capture ChAPor SPT specific antibodiesboth commonly used for ChAP mapping studiesin a CUTandamp RUN workflowIn AimPhase Iwe will develop a set of DNA barcoded dNuc spike ins for quantitative analysis of ChAPse gCTCFtranscription factorand BRDchromatin interactorPhase I will be successfully completed when we use these dNucs in CUTandamp RUN for quantitative sample normalizationIn AimPhase IIwe will expandscale manufacturing of ChAP CUTandamp RUN spike in control panels and apply these reagents to establish robust workflows for quantitative sample normalization genomewideIn Aimwe will develop and externally validate ChAP CUTandamp RUN beta kitsWe envision ChAP CUTandamp RUN will become one of the most widely used assays in the epigenetics fieldgiven the vast gain in assay metrics vsChIP Seqwith the potential to open new markets for the routine analysis of limitedi epreciousclinical samples PROJECT NARRATIVESpike in controls are needed for all genome wide analyses as theyiare essential for normalizing samples to enable cross sample comparisonsand iican be used as internal controls to monitor assay variatione gtechnical variabilityEpiCypherhas recently developed the application of DNA barcoded semi synthetic designer nucleosomesdNucsas qualitativequantitative spike in controls for histone post translational modificationPTMChromatin ImmunoPrecipitationSNAP ChIPThere are no equivalent tools for chromatin associated proteinsChAPseven though ChAPs comprise the majority of the rapidly growing ChIP Seq marketTo fill this needEpiCypher is developing novel spike in controls for quantitativehigh resolution mapping of ChAPs