Runx2-interacting genes identified by yeast two-hybrid screening of libraries generated from hypertrophic chondrocytes.

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.