Related Experiment Video
Updated: Jun 12, 2026
Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences
Published on: February 11, 2019
Upregulated expression of hITF in Crohn's disease and screening of hITF Interactant by a yeast two-hybrid system
Huaxiu Shi1, Jianlin Ren, Hongzhi Xu
1Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China.
Aims:
To study the expression of human intestinal trefoil factor (hITF) mRNA in Crohn's disease and to screen the cellular proteins that can interact with the hITF protein by a yeast two-hybrid system in order to explore the mechanism of hITF in protecting intestinal mucosa from injury.
Methods:
Seventy-eight patients underwent double-balloon enteroscopy (DBE). Expression of hITF mRNA was detected by quantitative real-time polymerase chain reaction analysis (qRT-PCR). The hITF gene was amplified by PCR and cloned into vector pDEST32. The yeast cells cotransformed with pDEST32-hITF and the human jejunal cDNA library were plated in a selective SC-Leu-Trp-His-Ura medium. The subsequent screen was performed with χ-gal detection, and true-positive clones were sequenced and analyzed with bioinformatics. Co-immunoprecipitation (Co-IP) was performed to confirm the binding of putative proteins to the hITF protein.
Results:
Thirty-nine patients were diagnosed with Crohn's disease. We found that the expression of hITF mRNA is significantly increased in Crohn's disease compared to normal controls. A total of ten colonies were selected and sequenced. Among these, six colonies were Homo sapiens zinc finger protein 193 (ZNF193), three colonies were Homo sapiens Aldo-keto reductase family 1C 1 (AKR1C1), and one colony was of an unknown gene. A reverse two-hybrid experiment and Co-IP indicated that ZNF193 and AKR1C1 might interact with hITF.
Conclusions:
The expression of hITF mRNA is increased in Crohn's disease. ZNF193 and AKR1C1 are proteins that can interact with the hITF protein by a yeast two-hybrid system and Co-IP, hITF may contribute to the mucosal repair through this interaction.
Insights
Human intestinal trefoil factor (hITF) mRNA expression increases in Crohn's disease. This study identified ZNF193 and AKR1C1 as interacting proteins, suggesting a role for hITF in mucosal repair.
Area of Science:
- Gastroenterology
- Molecular Biology
- Immunology
Background:
- Crohn's disease (CD) is a chronic inflammatory bowel disease.
- The role of human intestinal trefoil factor (hITF) in CD pathogenesis is not fully understood.
- Identifying hITF interacting proteins may elucidate its function in intestinal mucosal integrity.
Purpose of the Study:
- To quantify hITF mRNA expression in CD patients.
- To identify proteins interacting with hITF using a yeast two-hybrid system.
- To explore the mechanism of hITF in protecting the intestinal mucosa.
Main Methods:
- Quantitative real-time polymerase chain reaction (qRT-PCR) for hITF mRNA expression analysis.
- Yeast two-hybrid system screening with a human jejunal cDNA library to identify hITF interacting proteins.
- Co-immunoprecipitation (Co-IP) to validate protein interactions.
Main Results:
- hITF mRNA expression was significantly elevated in CD patients compared to controls.
- Yeast two-hybrid screening identified ZNF193 and AKR1C1 as potential hITF interacting proteins.
- Co-IP confirmed the interaction between hITF and both ZNF193 and AKR1C1.
Conclusions:
- hITF mRNA is upregulated in Crohn's disease.
- ZNF193 and AKR1C1 interact with hITF, as confirmed by yeast two-hybrid and Co-IP.
- These interactions suggest hITF may play a role in mucosal repair mechanisms in CD.
