# Exploiting a gain-of-function SKN-1 mutant to discover novel mechanisms of transcription factor regulation

> **NIH NIH F31** · UNIVERSITY OF SOUTHERN CALIFORNIA · 2021 · $46,036

## Abstract

Project Summary:
Homeostatic control mechanisms are vital components of biological maintenance within the cell. Reactive oxygen
species (ROS) are kept under homeostatic control, in part, by “Cap’n’Collar”(CnC) transcription factors, like SKN-1
in C. elegans. A mutagenesis screen uncovered SKN-1 gain-of-function(gf) alleles that display increased resistance
to oxidative stress, but surprisingly, deregulated metabolism and shortened lifespans. These early findings suggest
a link between uncontrolled regulation of cytoprotective transcription factors and disease pathology. Regulation of
SKN-1 has been documented primarily through genetic studies. This has left many mechanistic details of SKN-1
regulation unstudied. This proposal leverages the opportunity to examine a constitutively active transcription factor to
characterize aberrant regulation normally applied in the wild type condition. This proposal will CRISPR/cas9-
generated GFP tagged variants of SKN-1 and SKN-1gf at the endogenous locus, which avoids problems with multi-
copy transgenic arrays used in previous studies. In Aim 1, I will utilize ChIP-seq to assess target selection as well as
a Biacore surface plasmon resonance (SPR) studies to define binding affinity and dissociation constants, which based
on our RNAseq studies are predicted to be different across target genes. Preliminarily ChIP-qPCR experiments have
been performed for AIM1 and have revealed that SKN-1gf occupies target gene promoters more than wildtype. We
will expand this approach to a genome-wide assessment and begin with these established targets for our binding
studies. In Aim 2, I will examine the interplay of phosphorylation and OGlcNAcylation post-translational modifications
on SKN-1 activity and target selection. Phosphorylation on SKN-1 is critical for its regulation as well as
OGlcNAcylation. A preliminary assessment of phosphorylation stoichiometry has been performed via PhosTag SDS
PAGE, where an increase in phosphorylation on SKN-1gf compared to wildtype was observed. It is unclear what
kinases are causing this shift, but my combined genetic and biochemical approach will define these important
regulators. Taken together these biochemical assessments will fill a critical knowledge gap for SKN-1 activity and
target gene selection, which is a topic of great interest to the study of transcription factor regulation.

## Key facts

- **NIH application ID:** 10142226
- **Project number:** 5F31GM137587-02
- **Recipient organization:** UNIVERSITY OF SOUTHERN CALIFORNIA
- **Principal Investigator:** Christian David Turner
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 5
- **Project period:** 2020-04-03 → 2023-04-02

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10142226

## Citation

> US National Institutes of Health, RePORTER application 10142226, Exploiting a gain-of-function SKN-1 mutant to discover novel mechanisms of transcription factor regulation (5F31GM137587-02). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10142226. Licensed CC0.

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