# Defining Gene Regulatory Elements Essential to Cancer Cell Viability

> **NIH NIH K00** · DUKE UNIVERSITY · 2020 · $84,162

## Abstract

Project Summary/Abstract:
Mono-methylation of histone 3 lysine 4 (H3K4me1) is a chromatin mark closely associated with transcriptional
enhancers and other intergenic regulatory elements; however, the functional significance of this histone
modification has yet to be demonstrated. Our lab has previously identified Trr and MLL3/4 as the major H3K4-
monomethylases in Drosophila and mammals, respectively. Recent genome-wide association studies
identified the trr human orthologues, MLL3 and MLL4, as genes frequently mutated in a wide variety of human
cancers, along with other COMPASS subunits. Previous work has established that Trr protein is necessary for
regulating enhancer function; however, the role of Trr-dependent H3K4me1 at enhancers has not been tested
directly. To address this, we have taken advantage of an embryonic recessive lethal Trr-NULL allele, trr[1], to
test the necessity of Trr-dependent methylase activity. We have found the trr[1] lethality can be rescued by
expressing a Trr transgene whose SET domain contains either a catalytic-inactive (C2398A) or a catalytic-
hyperactive (Y2383F) point mutation. As expected, western blots confirm substantial reductions of H3K4me1
in the catalytic-dead mutant, while the catalytic-hyperactive mutation shows significant increases in
H3K4me2/3. Our ChIP-seq studies verify these changes are occurring specifically at enhancer elements. Aside
from these molecular phenotypes, the two mutant fly lines show no obvious developmental, reproductive, or
behavioral differences in comparison with a control trr-WT rescue line. These observations raise three
important questions: 1) what is the function of Trr-dependent H3K4-methylation in Drosophila development, 2)
what are the non-enzymatic functions of Trr in regulating enhancer-mediated processes, and 3) how do non-
catalytic mutations found in MLL3/4 disrupt enhancer function and lead to human cancer pathogenesis? By
exploiting Drosophila melanogaster, in which MLL3/4 are represented by just one enzyme, trr, I will uncover
fundamental details regarding how these proteins function to regulate enhancer activity, and how deleterious
mutations to MLL3/4 result in cancer formation.

## Key facts

- **NIH application ID:** 10009285
- **Project number:** 5K00CA222988-04
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** Ryan Adam Rickels
- **Activity code:** K00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $84,162
- **Award type:** 5
- **Project period:** 2019-09-05 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10009285

## Citation

> US National Institutes of Health, RePORTER application 10009285, Defining Gene Regulatory Elements Essential to Cancer Cell Viability (5K00CA222988-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10009285. Licensed CC0.

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