Chromatin immunoprecipitation (ChIP) is one of the core methods in the study of epigenetic regulation of gene expression. Analytical methods, such as qPCR, microarray and next generation sequencing (NGS) have increased throughput, but not the reliability of ChIP datasets. Using state-of-the-art reagents, ChIP is at best semi-quantitative, and at worst unrepresentative of in vivo post-translational modification (PTM) density. To address this short-coming, EpiCypher, Inc. has partnered with Dr. Alex Ruthenburg of the University of Chicago who has pioneered a method to internally-standardize ChIP experiments for subsequent normalization and quantitation of epigenetic marks. The method, referred to here as Internally Calibrated Immunoprecipitation Sequencing, or ICeChIP-Seq, was recently licensed on an exclusive basis by EpiCypher, Inc. and relies on designer nucleosomes collaboratively developed and manufactured by EpiCypher and the Ruthenburg lab. These nucleosomes are modified with an epigenetic mark of interest and wrapped in a DNA sequence containing a unique, identifying barcode sequence. Barcoded designer nucleosomes are added to the ChIP reaction at various concentrations, containing purified chromatin and a bead-attached pull-down antibody against the epigenetic mark of interest. After immunoprecipitation, next generation sequencing data is analyzed for the number of reads detected for 1) each barcode and 2) immunoprecipitated input DNA with the resulting ratio used to compute IP enrichment quantitatively. Read number can then be normalized to input concentration for each barcoded nucleosome, providing a standard curve for quantitation of sample DNA reads. The barcoded nucleosomes serve as calibrators because they are subjected to the same sources of variability the sample chromatin experiences during the ChIP reaction, and they precisely resemble the target of the IP. Proof-of-concept development of the assay has focused on the following: synthesis of barcoded nucleosomes; analysis of their quality; development of an H3K4me3 assay; analysis algorithms; characterizing signal-to-noise ratio; minimum chromatin input demands and day-to-day variability. Given the strong data generated to date, and the urgent need for an improved ChIP-Seq assay, we have proposed a Direct to Phase II workplan toward the commercialization of this powerful method. The Phase I report provides compelling evidence that the method is a dramatic improvement over existing methods, with Phase II aims focused on manufacturing a kit for the H3K4me3 assay reagents, external validation of this assay and expanding the menu of PTM-specific ICeChIP-Seq reagents to other epigenetic marks.

Public Health Relevance

Chromatin immunoprecipitation (ChIP) is a cornerstone method to investigating epigenetic regulation of gene expression, a currently expanding area of research toward new therapeutics for cancer and other indications. ChIP, however, is a non-quantitative method, providing investigators only with information regarding the presence or absence of various protein-chromatin interactions with no data regarding abundance even on a relative basis. Currently, reproducibility issues prohibit clinical applications. The method under development here employs nucleosome-based controls, which can be added to ChIP reactions and uniquely detected using next generation sequencing methods to provide a standard curve, thereby allowing a quantitative method, referred to here as Internally Calibrated Immunoprecipitation Sequencing or ICeChIP-Seq.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
1R44HG008907-01
Application #
9045474
Study Section
Special Emphasis Panel (ZRG1-IMST-K (14))
Program Officer
Pazin, Michael J
Project Start
2015-12-15
Project End
2017-11-30
Budget Start
2015-12-15
Budget End
2016-11-30
Support Year
1
Fiscal Year
2016
Total Cost
$676,616
Indirect Cost
Name
Epicypher, Inc.
Department
Type
DUNS #
078882699
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599