Post-translational modifications on the histone constituents of nucleosomes are able to transduce changes in local chromatin states that govern the accessibility of underlying DNA, regulating processes that range from transcriptional activation to gene silencing. Yet with present technology, it is impossible to measure the absolute densities of histone modifications in a locus specific manner. Despite serving as the central experimental technique in epigenetics research, chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) suffers from a number of serious drawbacks: 1.) it is a relative measurement unthered to any external scale in a way that obviates comparison amongst experiments;and 2.) it employs antibody reagents that have differing specificity and affinity for epitopes, which are in turn variable in abundance, yet none of these factors are taken into account in present analysis. Consequently, the peaks of different histone modifications that seem to overlap on certain genomic loci cannot be meaningfully compared. To address these substantial problems, I propose a novel approach to calibrate ChIP-seq data using a panel of nucleosomes derived from recombinant and semisynthetic sources as internal standards (calChIP-seq). To that end, nucleosomes bearing a given mark will be reconstituted with a library of DNAs composed of a constant strong nucleosome positioning sequence that is flanked by a variable """"""""barcode"""""""" that represents each member?s molar concentration, then spiked into the input of a native ChIP-seq experiment. After immunoprecipitation with modification-specific antibodies followed by sequencing, the tag counts resulting from the exogenous semisynthetic nucleosome DNA series will serve as an internal-standard calibration curve for absolute quantification of mark density with the positional accuracy of ChIP-seq in a genome-wide data set. This basic scheme will be employed in a number of variations to calibrate ChIP-seq in a proof of concept form and critically examine several troublesome sources of experimental error in ChIP measurements. This proposal is centered on developing the calChIP-seq technology, although a number of potential applications that could substantially contribute to understanding how the epigenome contributes to the control of genomic information are presented. The ability to make comparisons of histone modification density on an absolute scale by calChIP-seq will be transformative for our understanding of chromatin states and enable for the first time crucial comparisons between one modification to another, one cell type to another, and from patient to another. I am in a unique position to accomplish this radical and desperately needed improvement to our field?s most important technology in that I have both expertise in making semisynthetic chromatin and experience with ChIP-sequencing experiments.
Despite its widespread use to understand the way the genome is packaged and managed, chromatin immunoprecipitation followed by sequencing (ChIP-seq) suffers from a number of serious drawbacks. We aim to remedy a number of ChIP-seq?s flaws using our unique ability to make synthetic chromatin as internal standard. Our new technology could not only revolutionize the field of epigenetics, but also reveal fundamental insights into stem cell development and the molecular mechanisms of a variety of nuclear state transitions, including diabetic glucose tolerance and cancer etiology.
|Chen, Zhonglei; Notti, Ryan Q; Ueberheide, Beatrix et al. (2018) Quantitative and Structural Assessment of Histone Methyllysine Analogue Engagement by Cognate Binding Proteins Reveals Affinity Decrements Relative to Those of Native Counterparts. Biochemistry 57:300-304|
|Grzybowski, Adrian T; Chen, Zhonglei; Ruthenburg, Alexander J (2015) Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide. Mol Cell 58:886-99|
|Chen, Zhonglei; Grzybowski, Adrian T; Ruthenburg, Alexander J (2014) Traceless semisynthesis of a set of histone 3 species bearing specific lysine methylation marks. Chembiochem 15:2071-5|