# The role of protein-protein interaction motifs in coordinating the DNA binding and regulatory specificity of Hox proteins

> **NIH NIH F32** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2020 · $17,327

## Abstract

Project Summary: Transcription factors perform a key role in regulating gene expression, but our ability to
interpret regulatory DNA by identifying transcription factor binding sites or by predicting regulatory function is
lacking. One generally unappreciated contributing factor is that each transcription factor has the potential to
interact with cofactors, sometimes in structurally distinct ways, thus adding to the ability of transcription factor
complexes to bind distinct DNA sequences. Perhaps the best illustration of this idea is the Hox family of
proteins, which pattern the anterior/posterior axis in all metazoans. When bound to the cofactor Extradenticle
(Exd), Hox proteins exhibit latent differences in DNA-binding specificity necessary to carry out their unique
activities. In addition, they exhibit paralogue specific binding by recognizing low-affinity DNA-binding sites,
illustrating a fundamental tradeoff between DNA binding specificity and affinity. In this proposal, I will extend
these observations by studying the role of protein-protein interaction (PPI) motifs that mediate the interaction
between Hox proteins and their cofactors. The physical interaction between Hox and Exd, mediated by a
canonical motif present in all Hox proteins, has been particularly-well studied. However, some Hox proteins
contain additional motifs. These motifs may promote alternate Hox-Exd-DNA conformations, a potential
mechanism underlying paralogue-specific activity. To test this hypothesis, combinations of Exd interaction
motifs will be mutated in the endogenous loci of three Hox genes: Ultrabithorax (Ubx), abdominal-A (abd-A)
and Abdominal-B (Abd-B). ChIP-seq (Chromatin ImmunoPrecipitation) will be used to assess differences in in
vivo DNA-binding profiles to identify motif-redundant and motif-specific binding sites. Next, differences in DNA-
binding affinity between mutant Hox-Exd complexes will be determined by utilizing a version of SELEX-seq
(Systematic Evolution of Ligands by Exponential Enrichment) modified to capture low affinity DNA-binding.
Comparing the ChIP-seq and SELEX-seq datasets will generate a list of motif-specific sites driven to bind only
by the interaction between Hox and Exd, and a list of binding events caused by other mechanisms. The
second aim of this study is to evaluate the role of Exd interaction motifs in regulating the transcriptional
potential of Hox proteins as Hox proteins can both activate and repress genes expression. To address this
question, RNA-seq, ATAC-seq and epigenetic mark specific ChIP-seq will be performed on tissues that
express Hox proteins with mutant Exd interaction motifs. The third aim of this study is to study how different
isoforms of Homothorax (Hth), another Hox cofactor that is required for the nuclear localization of Exd, regulate
Hox activity. Studying the mechanisms guiding Hox paralogue specificity and regulatory activity is important
because of (1) the deep conservation of Hox genes in higher orga...

## Key facts

- **NIH application ID:** 10228347
- **Project number:** 3F32GM125329-02S1
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** William Joseph Glassford
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $17,327
- **Award type:** 3
- **Project period:** 2018-09-01 → 2020-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10228347

## Citation

> US National Institutes of Health, RePORTER application 10228347, The role of protein-protein interaction motifs in coordinating the DNA binding and regulatory specificity of Hox proteins (3F32GM125329-02S1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10228347. Licensed CC0.

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