# Administrative Supplement for R01-GM079428: Mechanisms of homeodomain transcriptional specificity

> **NIH NIH R01** · CINCINNATI CHILDRENS HOSP MED CTR · 2021 · $59,635

## Abstract

PROJECT ABSTRACT
Homeodomain (HD) proteins comprise a large family of transcription factors (TFs) that regulate numerous aspects of animal
development. For example, members of the Hox-like (HoxL) and Nkx-like (NKL) HD proteins regulate processes ranging
from patterning of the anterior-posterior axis (A-P) of the embryo to specifying individual cell fates within different organ
systems. Intriguingly, the HoxL and NKL proteins have highly similar HDs that bind largely overlapping AT-rich DNA
sequences in vitro. These findings provide a classic TF specificity paradox: How do TFs with highly similar in vitro DNA
binding activities achieve sufficient in vivo specificity to ensure the accurate regulation of genetic programs in different cell
types? To address this paradox, my lab is focused on defining how HD TFs achieve in vivo specificity by forming
cooperative TF complexes on cis-regulatory modules. Our preliminary and published data reveal that members of the HoxL
and NKL TFs differ in their ability to form homo- and heterodimer TF complexes on DNA. For instance, we unexpectedly
found that the Gsx/Ind TFs, which specify neuronal cell fates in animals from flies to mammals, differentially regulate gene
expression when bound to DNA as monomers versus homodimers. In contrast, the Abdominal-A (Abd-A) Hox TF, which
specifies distinct cell fates in the Drosophila abdomen, does not bind DNA as a homodimer, but instead cooperatively binds
DNA with three other HD proteins: Extradenticle (Exd), Homothorax (Hth), and Engrailed (En). These data support the
hypothesis that HD TFs achieve target and regulatory specificity by binding distinct combinations of AT-rich DNA sites as
monomers, cooperative homodimers, or cooperative heterodimers. To test this hypothesis, we propose two aims: In Aim1,
we propose to determine how HD monomer versus homodimer binding impacts target gene binding and regulation. To
achieve this goal, we will (1) systematically define which HoxL and NKL HDs cooperatively bind DNA as homodimers;
(2) assess the regulatory potential of each HD on monomer vs dimer sites in cell culture assays; and (3) define the mechanism
and function of Ind homodimer formation on Drosophila neuroblast gene expression using structural biology and transgenic
reporter, CUT&RUN, and RNA-seq assays. In Aim2, we propose to define how the choice of Hox heterodimer partner
impacts the DNA binding and regulatory specificity of the Abd-A Hox TF. To achieve this goal, we will (1) define the DNA
motifs and molecular domains required for cooperative Abd-A/Hth and Abd-A/En complexes; (2) test the role of Abd-A
heterodimerization domains in gene activation and repression assays in the Drosophila embryo; (3) define the in vivo
binding motifs and target genes regulated by Abd-A with a focus on identifying heterodimer binding events using
CUT&RUN and RNA-seq assays. Since the TFs and biological processes studied are highly conserved between flies and
mammals, we are optimistic ou...

## Key facts

- **NIH application ID:** 10402451
- **Project number:** 3R01GM079428-11S1
- **Recipient organization:** CINCINNATI CHILDRENS HOSP MED CTR
- **Principal Investigator:** BRIAN GEBELEIN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $59,635
- **Award type:** 3
- **Project period:** 2008-04-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10402451

## Citation

> US National Institutes of Health, RePORTER application 10402451, Administrative Supplement for R01-GM079428: Mechanisms of homeodomain transcriptional specificity (3R01GM079428-11S1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10402451. Licensed CC0.

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