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

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.

SUBMITTER: Li F

PROVIDER: S-EPMC5209497 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Li Feifei F Mi Rui R Fan Chuling C Zhang Ping P Zhu Ting T Wang Qian Q Lu Yaojuan Y Gu Junxia J Zheng Qiping Q

American journal of translational research 20161215 12

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 ...[more]

PMID: 28078017

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Project description:BackgroundTheileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.MethodsThe cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid) for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP) and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING.ResultsTwo host proteins, CRBN (Bos taurus cereblon transcript variant X2) and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit) were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs.ConclusionsThis study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are involved in many cellular processes, such as ubiquitylation regulating, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. The interaction with CRBN and Ppp4C indicate that TaCP possibly is involved in regulating signaling pathways and cell proliferation, which is helpful for understanding the interaction between T. annulata and host cells.

Dataset Information

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

Publications

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

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