# Mapping protein signatures to single allele chromatin topologies at genomic resolution

> **NIH NIH R21** · UNIVERSITY OF ILLINOIS AT CHICAGO · 2023 · $211,378

## Abstract

Interphase chromatin is hierarchically organized in chromosome territories, active and inactive compartments, and
topologically associating domains (TADs). Gene expression is controlled by regulatory chromatin elements (enhancers
and promoters) that bind transcription factors and interact within, but not across TAD boundaries. Recent advances in
single cell biology have revealed a tremendous amount of cell-to-cell variability in both chromatin topology and protein
coverage. Even though TADs appear to delineate functional units at the population level, their boundaries only emerge as
average properties from large ensembles of cells. TAD-like structures persist even in the absence of boundaries at the
ensemble level. Similarly, single cell ChIP-seq has revealed sub-states with distinct epigenetic profiles at enhancers in a
population of stem cells. But no assay exists to link topological and functional variability by reading out protein coverage
and epigenetic signatures simultaneously from single cell chromatin traces. Here, we will leverage recent advances in
multiplexed chromatin imaging and single molecule super-resolution microscopy to fill in this gap. In Aim I, we will
characterize the tradeoff between sequence resolution and spatial precision in multiplexed chromatin imaging. We will
then use optimized conditions to map super-resolved protein signal to chromatin topologies at genomic resolution. In Aim
II, we will extend the capabilities of the assay to detect multiple protein signatures simultaneously. Such combinatorial
data on protein signatures of regulatory elements at genomic resolution is not available through any other single cell
assay. We will further characterize the performance of the assay under challenging conditions by mapping both stably
integrated, widespread histone modifications and transiently binding sequence-specific transcription factors with a sizable
unbound fraction to chromatin. Using computational clustering strategies, we will stratify the data by chromatin topology
to determine if structural variation is driven by specific protein factors such as transcription factors that orchestrate long-
range enhancer interactions. Finally, we will establish protocols and technological solutions to accelerate acquisition,
processing, and visualization of statistically meaningful datasets comprising 100s-1000s of single alleles. The resulting
datasets will provide unprecedented insight into the molecular mechanisms underlying cell-to-cell variability in chromatin
topology and serve as a powerful hypothesis generator for investigating single cell genome structure-function
relationships.

## Key facts

- **NIH application ID:** 10649096
- **Project number:** 1R21CA282715-01
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT CHICAGO
- **Principal Investigator:** Jan-Hendrik Spille
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $211,378
- **Award type:** 1
- **Project period:** 2023-04-07 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10649096

## Citation

> US National Institutes of Health, RePORTER application 10649096, Mapping protein signatures to single allele chromatin topologies at genomic resolution (1R21CA282715-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10649096. Licensed CC0.

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