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Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
Published on: January 17, 2012
Runx2-interacting genes identified by yeast two-hybrid screening of libraries generated from hypertrophic
Feifei Li1, Rui Mi2, Chuling Fan1
1Department of Pathophysiology, Anhui Medical University Hefei 230032, China.
Abstract:
Runx2, a member of the Runt domain family, is a well-known master transcription factor for osteoblast differentiation. Runx2 has also been shown to play essential roles during chondrocyte hypertrophy, an important late stage of endochondral ossification linking both bone and cartilage development. To identify the co-factors that may interact with Runx2 together to regulate this critical process, we have performed yeast two-hybrid (Y2H) screening using Runx2 as a bait to screen a cDNA library of hypertrophic chondrocytes. The bait expressing cassette was constructed by fusing Runx2 with the pGBKT7 vector containing the Gal4 DNA binding domain (BD). The Mate & Plate libraries were constructed using pGADT7-Rec and cDNAs derived from hypertrophic chondrocytes enriched limb tissues or hypertrophic MCT cells. After co-transformation of pGBKT7-Runx2 and the cDNA libraries, colonies that grew in nutrition deficient medium were selected and subjected to PCR and sequencing analysis. We successfully identified more than 30 candidate genes, including Lectin-1 (Lgals1), Col1a2, Edf1 and Timp-2. We have performed literature review and bioinformatics analysis of these genes using GenePaint. Most of them show ubiquitous expression with Lgals1 show enhanced expression in hypertrophic chondrocytes. We further performed preliminary expression analysis by quantitative PCR and detected differential expression of these candidate genes in proliferative and hypertrophic MCT cells, with Timp-2 significantly (around 3-fold) and Lgals1 moderately (around 1.5 fold) upregulated in hypertrophic MCT cells. Our results suggest that, candidate gene Timp-2 is very likely to interact with Runx2 and together to play essential function during cartilage development, and possibly its homeostasis.
Insights
Researchers identified potential co-factors interacting with Runx2, a key transcription factor in bone and cartilage development. Timp-2 was significantly upregulated, suggesting its interaction with Runx2 in chondrocyte hypertrophy and cartilage homeostasis.
Area of Science:
- Molecular Biology
- Developmental Biology
- Biochemistry
Background:
- Runx2 is a master transcription factor crucial for osteoblast differentiation.
- Runx2 also plays vital roles in chondrocyte hypertrophy, a key stage in endochondral ossification.
- Identifying Runx2 co-factors is essential for understanding cartilage development regulation.
Purpose of the Study:
- To identify novel co-factors that interact with Runx2 during chondrocyte hypertrophy.
- To elucidate the molecular mechanisms regulating cartilage development and homeostasis.
Main Methods:
- Yeast two-hybrid (Y2H) screening was employed using Runx2 as bait.
- cDNA libraries from hypertrophic chondrocytes and MCT cells were utilized.
- Candidate gene validation involved literature review, bioinformatics analysis, and quantitative PCR.
Main Results:
- Over 30 candidate genes interacting with Runx2 were identified, including Lectin-1 (Lgals1), Col1a2, Edf1, and Timp-2.
- Bioinformatics and expression analysis revealed ubiquitous expression for most candidates, with Lgals1 showing enhanced expression in hypertrophic chondrocytes.
- Quantitative PCR confirmed significant upregulation of Timp-2 (approx. 3-fold) and moderate upregulation of Lgals1 (approx. 1.5-fold) in hypertrophic MCT cells.
Conclusions:
- Timp-2 is a strong candidate for interacting with Runx2 to regulate chondrocyte hypertrophy.
- The identified co-factors, particularly Timp-2, are likely crucial for cartilage development and maintaining its homeostasis.
