Rapid manufacturing of recombinant nucleosomes for high-throughput assays

DESCRIPTION provided by applicant Chromosomes are composed of genomic DNA and associated protein components that exert powerful regulatory effects The nm nucleosome represents the functional unit of chromatin comprising turns of genomic DNA bp wrapping an octamer of histone proteins Post translational modification PTM of the histones e g methylation acetylation or ubiquitination represents a central epigenetic regulatory mechanism with specific modifications recruiting effector proteins Effector proteins or more often protein complexes can include transcription factors responsible for initiating or blocking the expression of specific genes The andquot histone code hypothesisandquot asserts that certain combinations of histone PTMs on a given nucleosome exert an ensemble impact on downstream gene expression through the recruitment of various effector proteins and complexes Aberrant PTM regulation has been linked to changes in gene expression associated with specific disease states Given the link between PTMs and disease enzymes responsible for the addition and removal of certain PTMs have become the targets of drug discovery efforts with several compounds achieving marketing clearance A broad range of compelling epigenetic drug targets therefore exist including enzymes that add PTMs to histone proteins andapos writersandapos those that bind specific PTMs andapos readersandapos and those that remove PTMs andapos erasersandapos To study these interactions peptides or recombinant histones can be synthetically modified to contain PTMs but these substrates fail to replicate chromatin architecture EpiCypher tm has pioneered the production of PTM modified nucleosomes called designer nucleosomes While these methods are effective synthesis of a single new designer nucleosome can take weeks depending on the number of histone PTMs to add and the quality control steps required to ensure a pure product Condensing this process to or weeks would allow EpiCypher to produce a greater number of modified nucleosomes dramatically expanding the utility of these reagents Current screening of a given writer reader or eraser for PTM context dependent activities requires the use of either hundreds of modified peptides or a priori knowledge of the PTMs of interest to focus on a restricted handful of designer nucleosomes Rapid high throughput manufacturing of PTM carrying nucleosomes will enable EpiCypher to load each well of a well plate with unique nucleosomes enabling a dramatic increase in the throughput of these assays with substrates that more accurately represent in vivo chromatin structure Recent research advancements have developed means to rapidly synthesize proteins carrying specific PTMs using AMBER suppression technology Continuing these research efforts we will develop for the first time methods for commercial scale synthesis of designer nucleosomes carrying mono di or trimethyl PTMs This innovative advancement in designer nucleosome manufacturing will provide a transformational opportunity for drug discovery and the identification of novel therapeutic strategies PUBLIC HEALTH RELEVANCE Post translational modification PTM of histone proteins represents a central epigenetic regulatory mechanism with specific modifications regulating the recruitment of effector proteins to chromatin thereby potentiating or inhibiting downstream gene expression programs To date recombinant nucleosomes are slow to produce and modify limiting their utility in high throughput screening applications Here EpiCypher will optimize a novel means of scaling the production of PTM modified nucleosomes enabling the use of designer nucleosomes in high throughput screens for novel drug discovery and innovative assay development