# Biochemical and Computational Analysis of Notch signal transduction

> **NIH NIH R01** · CINCINNATI CHILDRENS HOSP MED CTR · 2020 · $468,961

## Abstract

Abstract
Transcription factors (TFs) activate transcription through a variety of mechanisms, including opening of
chromatin by displacing linker histones or repositioning of nucleosomes, promoting looping between
regulatory elements, and triggering or enhancing maintenance of RNA polymerase activity. We will
address the open question of why few enhancers are selected by a given TF from a multitude of
available sites, with a focus on Notch/RBPj. Our previous data indicate that target selection reflects
strength (defined here as the ultimate number of intracellular domain of Notch (NICD) molecules
reaching the nucleus, which integrates ligand-mediated release and nuclear translocation but is
agnostic to differences between Notch and Notch2), and duration (half life of NICD/RBPjk/MAML/DNA
complexes, which integrates cooperativity and stability). In this proposal, we leverage novel
experimental and computational tools to explore the distinct steps used by the Notch transcriptional
complex to select and regulate mammalian target genes. To enable investigation of this question in
high resolution, we developed Split DamID (SpDamID, a protein complementation version of DamID(14))
to specifically interrogate target selection by multi-member transcription complexes or factors binding
simultaneously near each other in only 100-1000 cells. Adenine methylation at GAmTC occurs only
when two halves complement each other on the same chromosome and is blind to complex size, a
source of artifacts in ChIP(15). Motif enrichment analyses of NICD bound peaks identified Runx as a
frequent collaborator, binding within 100bp of RBPj sites(2). Co-binding of NICD and Runx1 was
confirmed by Runx1D/NICDAM SpDamID(2). In combination with other tools, SpDamID enables
investigation of the following questions: What is the biochemical basis for Runx1/NICD
collaboration? What genomic features distinguish Notch-dependent RBPj peaks from other
RBPj sites? What is the mechanism NICD utilizes to increase target gene expression
frequency?

## Key facts

- **NIH application ID:** 9853793
- **Project number:** 5R01GM055479-21
- **Recipient organization:** CINCINNATI CHILDRENS HOSP MED CTR
- **Principal Investigator:** RAPHAEL KOPAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $468,961
- **Award type:** 5
- **Project period:** 1996-08-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9853793

## Citation

> US National Institutes of Health, RePORTER application 9853793, Biochemical and Computational Analysis of Notch signal transduction (5R01GM055479-21). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9853793. Licensed CC0.

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