# Quantitative mapping of combinatorial histone modifications

> **NIH NIH R44** · EPICYPHER, INC. · 2021 · $1,024,752

## Abstract

PROJECT SUMMARY
 Nucleosomes are the repeating building blocks of chromatin, composed of a histone octamer wrapped
with DNA. Histone tails are decorated with a variety of post-translational modifications (PTMs), which together
form a combinatorial molecular language (i.e. “the histone code”) that regulates gene expression and other
physiological processes. Defects in this complex landscape are associated with vast human pathologies, most
notably cancer, and histone PTMs are rapidly emerging diagnostic / prognostic indicators. For instance,
H3K4me3 + H3K27me3 “bivalent” promoters are frequent targets of DNA hypermethylation in cancer, resulting
in drastically altered expression of important developmental regulators. Reliable quantification of dual (i.e.
combinatorial) PTMs may provide novel access to new biomarkers or drug targets with disease specificity, a
major limitation of single PTM biomarkers to date. However, tools to study dual PTMs in vivo are lacking.
 Here, EpiCypher is developing EpiTandem™ Sensors, a first-in-class technology that uses combinations
of chromatin reader domains as next-generation affinity reagents to directly detect dual PTMs. These breakout
tools leverage the enhanced avidity of multivalent interactions for combinatorial PTMs, a key mechanism
displayed by dual chromatin reader domains in vivo. A central innovation of this project is the application of
EpiCypher’s recombinant designer nucleosomes (dNuc) technology to characterize EpiTandem Sensor binding
specificity against singly- and combinatorially-modified nucleosome substrates. dNucs faithfully replicate the
endogenous three-dimensional nucleosome structure, which is crucial to accurately define cooperative,
multivalent chromatin interactions. We will apply our validated EpiTandem Sensors to CUT&RUN, an ultra-
sensitive chromatin profiling method that generates high quality mapping data with significantly lower input and
sequencing requirements vs. ChIP-seq. We will develop optimized CUT&RUN protocols for EpiTandem Sensors,
demonstrating their utility to interrogate combinatorial PTMs genome-wide. In Phase I, we developed two
EpiTandem Sensors and utilized dNucs (singly- and combinatorially-modified) to characterize and validate their
exquisite binding specificity for dual PTMs (up to 90-fold vs. single PTMs). We then applied an EpiTandem
Sensor and DNA-barcoded dNuc spike-ins to CUT&RUN, providing strong proof-of-concept for genomic
mapping applications. In Phase II, we will develop a collection of five EpiTandem Sensors and complementary
dNuc spike-ins (Aim 1), and rigorously validate them in CUT&RUN assays using a range of cell types, sample
processing methods, and inputs, including drug treatment time course experiments to highlight their value in
clinical research projects (Aim 2). In Aim 3 we will optimize commercial-scale manufacturing of the five Sensors
and dNuc panels, assemble EpiTandem beta kits, and launch in-house CUT&RUN assay services for dual PTM
...

## Key facts

- **NIH application ID:** 10324501
- **Project number:** 2R44HG010595-02
- **Recipient organization:** EPICYPHER, INC.
- **Principal Investigator:** JONATHAN MICHAEL BURG
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $1,024,752
- **Award type:** 2
- **Project period:** 2019-06-12 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10324501

## Citation

> US National Institutes of Health, RePORTER application 10324501, Quantitative mapping of combinatorial histone modifications (2R44HG010595-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10324501. Licensed CC0.

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