Asymmetric modification of nucleosomes for targeted drug discovery

Project SummaryNucleosomes are the fundamental units of chromatincomprised of DNA wrapped around an octamer of histone subunitsH AH BHand HChromatin regulation is achieved through the addition of reversible post translational modificationsPTMsto specific histone residuesThese PTMs orepigenetic modificationsfunction in systems to dramatically alter downstream gene expressiona hypothesis termed thehistone codeAbnormalities in the epigenetic landscape are associated with a collection of age related diseases and cellular dysfunctions including metastatic cancers and tissue degenerationDeciphering the histone code toward the development of therapeutics is thusdependent on nucleosome based reagents to model how PTM combinations alter epigenetic signalingEpiCypher is pioneering the development of recombinant nucleosomes carrying specific PTMsdesigner nucleosomesdNucsfor drug discovery applicationsdNuc based reagents howeverare limited to the production of nucleosomes carrying the same modification in a given histone subunit dimerFor exampledNucs carrying a histone Hsubunit trimethylated at lysineH K mewill have both Hsubunits modifiedRecent studies show that nearly half of histone PTMs are added asymmetrically in the genomerevealing a strikingand previously unappreciatedadditional level of PTM complexitybivalentpromoterscontaining both activeH K meH Kmeand repressiveH Kmemarksare also asymmetrically modified at the nucleosome levelThis epigenetic signature is observed in stem cellscancer cells and plays key roles in stem cell maintenancedifferentiationSignificantlyPTM symmetry exerts a pronounced effect on the activity of chromatin modifying enzymesFor instancethe activity of EZHa histone H Kspecific methyltransferase associated with several cancerse gleukemiaand adult stem cell senescenceis dramatically enhanced by asymmetric vssymmetric H K meThese findings suggest that PTM symmetry provides an additional regulatory layer to the histone codethereby altering epigenetic signaling by preferentially recruiting specific chromatin modifying proteinsCurrentlythere are no available tools to examine the function of epigenetic regulators in the context of asymmetric PTMsIn this proposalwe will optimize novel methods to generate asymmetrically modified dNucs at commercial grade andscaleAimand develop an innovative drug discovery platform that utilizes these physiological nucleosomes as biochemical substrates to recapitulate in vivo activity of chromatin regulators at H K meH Kmebivalent promotersAimIn Phase IIwe willscale up manufacturing of asymmetrically modified dNucs for our industry pharmaceutical partners anddevelop a portfolio of highthroughput assaysbiochemical and cell basedfor drug discovery and development using other diseaserelevant asymmetric PTMsincluding H KmeH Kaclung cancerand H Kmeleukemialung and pancreatic cancerProject NarrativeIt has been recently appreciated that asymmetric histone post translational modifications are widespread and play key roles in stem cell biology and tumorigenesisHoweverthe biochemical characterization of these modifications is not yet possible due to a dearth of research toolsIn this proposalEpiCypher will generate a homogenous population of asymmetrically modified nucleosomes and develop a novel drug discovery platform to examine the impact of these reagents on chromatin regulating enzymes