# High-throughput, untargeted approaches to identify and define the functions of transcription factors regulating key life cycle transitions in Giardia

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2024 · $196,545

## Abstract

Giardia lamblia is a widespread protozoan parasite of humans and animals causing significant diarrheal disease
worldwide. Giardia cysts are transmitted between hosts through feces. Once ingested, cysts transform into
trophozoites that colonize the duodenal epithelium – a region of varied nutrients, redox stress, and immune
responses. Unknown external cues in the gut trigger trophozoites to encyst, and then cysts are passed to new
hosts. Giardia is also reported to have a quasi-meiotic stage, termed diplomixis, that occurs in late encystation
and is characterized by fusing and exchange of DNA between cyst nuclei. Due to a historical lack of molecular
genetic tools, the pathogenesis and basic biology of Giardia is grossly understudied. Developmental transitions
and environmental responses are commonly mediated by transcription factors (TFs) – modular proteins that often
contain DNA binding domains (DBDs). However, only 36 TFs with DBDs are predicted in the Giardia genome
as compared to about 200 predicted in similar sized eukaryotic genomes. Over 500 proteins have more general
motifs such as basic leucine-zipper domains, and these can also function as TFs. Only a handful of Giardia TFs
have been studied in any detail; all are associated with early to mid-encystation. Basal TFs, redox responsive TFs,
late encystation/diplomixis TFs, or cell cycle related TFs have not yet been identified. Our lack of knowledge of
the identities and functions of Giardia TFs has severely limited our understanding of genetic regulatory
networks throughout the Giardia life cycle, particularly those associated with cell division and pathogenesis.
To address these deficits, we will use an unbiased, high-throughput, genome-wide yeast one-hybrid (Y1H) screen
to identify additional DNA binding proteins likely representing candidate TFs (Aim 1). In many systems, such
screens are commonly used to define genome-wide protein-DNA interactions. Using robotic screening of arrayed
“prey” libraries, we will screen a total of nine promoter “baits” (three constitutive loci; three redox stress-
associated loci, and three encystation loci) against three “prey” cDNA libraries (constitutive; redox stress; late
encystation/diplomixis) for a total of 27 (3 x 3 x 3) combinations. Both predicted DBD proteins and candidate TFs
identified in the Y1H screen will be additionally prioritized by CRISPRi mediated knockdown, with phenotypic
screening for defects in growth, mid-encystation and genetic marker exchange in cysts (diplomixis), and redox
stress. Knockdowns of candidate TFs with strong defects will be further characterized using promoter-luciferase
assays and a novel dCas-TF luciferase fusion assay to evaluate transcriptional activation or repression (Aim 2).
Overall, this project will define and prioritize Giardia TFs as groundwork for future interrogation of Giardia
regulatory networks throughout the life cycle.

## Key facts

- **NIH application ID:** 10878886
- **Project number:** 5R21AI178544-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Scott C Dawson
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $196,545
- **Award type:** 5
- **Project period:** 2023-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10878886

## Citation

> US National Institutes of Health, RePORTER application 10878886, High-throughput, untargeted approaches to identify and define the functions of transcription factors regulating key life cycle transitions in Giardia (5R21AI178544-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10878886. Licensed CC0.

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