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Gtf2ird1-Dependent Mohawk Expression Regulates Mechanosensing Properties of the Tendon.

Kayama T, Mori M, Ito Y, Matsushima T, Nakamichi R, Suzuki H, Ichinose S, Saito M, Marumo K, Asahara H - Mol. Cell. Biol. (2016)

Bottom Line: In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover.Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation.Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Tokyo, Japan Department of Orthopaedic Surgery, The Jikei University School of Medicine, Tokyo, Japan.

No MeSH data available.


Functional screening for Mkx-regulatory genes. (A) A schematic of the screening system. A fragment 5 kb upstream of the transcription start site in addition to the first exon and intron, with a 3-kb region downstream of the first coding exon, was selected and cloned into a luciferase vector. This vector was cotransfected into HEK293T cells with expression vectors for a luciferase assay, which was repeated and narrowed down for a larger-scale analysis. The conservation plot was obtained using the ECR Browser (http://ecrbrowser.dcode.org/) (73). (B) First screening of 6,049 expression vectors performed in 384-well plates (n = 1). Thirty-five candidate genes with the greatest increases in luciferase activity were selected for a second screening. (C) Results of the second screening performed in 96-well plates (n = 2). Seven genes with consistent luciferase activity increases were selected for a more detailed analysis. Error bars represent standard errors of the means. (D) Results of the third screening performed in 24-well plates (n = 2). ETS2, GTF2IRD1, KIF22, HOXC11, and CCNDBP1 were found to elevate luciferase activity in the presence of an Mkx promoter. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test). (E) qRT-PCR of GTF2IRD1 transfected tenocytes confirmed GTF2IRD1 expression. GTF2IRD1 transfected cells also revealed an increase in Mkx expression in rat tenocytes. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test).
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Figure 4: Functional screening for Mkx-regulatory genes. (A) A schematic of the screening system. A fragment 5 kb upstream of the transcription start site in addition to the first exon and intron, with a 3-kb region downstream of the first coding exon, was selected and cloned into a luciferase vector. This vector was cotransfected into HEK293T cells with expression vectors for a luciferase assay, which was repeated and narrowed down for a larger-scale analysis. The conservation plot was obtained using the ECR Browser (http://ecrbrowser.dcode.org/) (73). (B) First screening of 6,049 expression vectors performed in 384-well plates (n = 1). Thirty-five candidate genes with the greatest increases in luciferase activity were selected for a second screening. (C) Results of the second screening performed in 96-well plates (n = 2). Seven genes with consistent luciferase activity increases were selected for a more detailed analysis. Error bars represent standard errors of the means. (D) Results of the third screening performed in 24-well plates (n = 2). ETS2, GTF2IRD1, KIF22, HOXC11, and CCNDBP1 were found to elevate luciferase activity in the presence of an Mkx promoter. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test). (E) qRT-PCR of GTF2IRD1 transfected tenocytes confirmed GTF2IRD1 expression. GTF2IRD1 transfected cells also revealed an increase in Mkx expression in rat tenocytes. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test).

Mentions: Currently, there are no known molecular factors linking physical forces with Mkx induction, nor have any upstream regulators of Mkx been identified. Therefore, a functional screening of upstream regulatory factors of Mkx transcription was performed with Mkx promoter-driven luciferase constructs with an MGC library of 6,049 human genes (Fig. 4A and B). Among them, 619 genes increased luciferase activity by more than 2-fold, whereas 267 genes decreased activity by more than 50%. Among the genes with increased luciferase activity from the first screening, 35 genes which activated the Mkx promoter by greater than 3-fold were analyzed for a second screening (Fig. 4C). Then, among the 32 genes which increased luciferase activity, the seven genes with the greatest luciferase activity were selected for a third screening. ETS2 (v-ets avian erythroblastosis virus E26 oncogene homolog 2) and GTF2IRD1 (general transcription factor II-I repeat domain-containing protein 1) showed the greatest relative luciferase activity increases (Fig. 4D) and were therefore selected as the prime candidate genes for further assessment with regard to Mkx promoter activity.


Gtf2ird1-Dependent Mohawk Expression Regulates Mechanosensing Properties of the Tendon.

Kayama T, Mori M, Ito Y, Matsushima T, Nakamichi R, Suzuki H, Ichinose S, Saito M, Marumo K, Asahara H - Mol. Cell. Biol. (2016)

Functional screening for Mkx-regulatory genes. (A) A schematic of the screening system. A fragment 5 kb upstream of the transcription start site in addition to the first exon and intron, with a 3-kb region downstream of the first coding exon, was selected and cloned into a luciferase vector. This vector was cotransfected into HEK293T cells with expression vectors for a luciferase assay, which was repeated and narrowed down for a larger-scale analysis. The conservation plot was obtained using the ECR Browser (http://ecrbrowser.dcode.org/) (73). (B) First screening of 6,049 expression vectors performed in 384-well plates (n = 1). Thirty-five candidate genes with the greatest increases in luciferase activity were selected for a second screening. (C) Results of the second screening performed in 96-well plates (n = 2). Seven genes with consistent luciferase activity increases were selected for a more detailed analysis. Error bars represent standard errors of the means. (D) Results of the third screening performed in 24-well plates (n = 2). ETS2, GTF2IRD1, KIF22, HOXC11, and CCNDBP1 were found to elevate luciferase activity in the presence of an Mkx promoter. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test). (E) qRT-PCR of GTF2IRD1 transfected tenocytes confirmed GTF2IRD1 expression. GTF2IRD1 transfected cells also revealed an increase in Mkx expression in rat tenocytes. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test).
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Related In: Results  -  Collection

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Figure 4: Functional screening for Mkx-regulatory genes. (A) A schematic of the screening system. A fragment 5 kb upstream of the transcription start site in addition to the first exon and intron, with a 3-kb region downstream of the first coding exon, was selected and cloned into a luciferase vector. This vector was cotransfected into HEK293T cells with expression vectors for a luciferase assay, which was repeated and narrowed down for a larger-scale analysis. The conservation plot was obtained using the ECR Browser (http://ecrbrowser.dcode.org/) (73). (B) First screening of 6,049 expression vectors performed in 384-well plates (n = 1). Thirty-five candidate genes with the greatest increases in luciferase activity were selected for a second screening. (C) Results of the second screening performed in 96-well plates (n = 2). Seven genes with consistent luciferase activity increases were selected for a more detailed analysis. Error bars represent standard errors of the means. (D) Results of the third screening performed in 24-well plates (n = 2). ETS2, GTF2IRD1, KIF22, HOXC11, and CCNDBP1 were found to elevate luciferase activity in the presence of an Mkx promoter. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test). (E) qRT-PCR of GTF2IRD1 transfected tenocytes confirmed GTF2IRD1 expression. GTF2IRD1 transfected cells also revealed an increase in Mkx expression in rat tenocytes. Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test).
Mentions: Currently, there are no known molecular factors linking physical forces with Mkx induction, nor have any upstream regulators of Mkx been identified. Therefore, a functional screening of upstream regulatory factors of Mkx transcription was performed with Mkx promoter-driven luciferase constructs with an MGC library of 6,049 human genes (Fig. 4A and B). Among them, 619 genes increased luciferase activity by more than 2-fold, whereas 267 genes decreased activity by more than 50%. Among the genes with increased luciferase activity from the first screening, 35 genes which activated the Mkx promoter by greater than 3-fold were analyzed for a second screening (Fig. 4C). Then, among the 32 genes which increased luciferase activity, the seven genes with the greatest luciferase activity were selected for a third screening. ETS2 (v-ets avian erythroblastosis virus E26 oncogene homolog 2) and GTF2IRD1 (general transcription factor II-I repeat domain-containing protein 1) showed the greatest relative luciferase activity increases (Fig. 4D) and were therefore selected as the prime candidate genes for further assessment with regard to Mkx promoter activity.

Bottom Line: In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover.Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation.Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Tokyo, Japan Department of Orthopaedic Surgery, The Jikei University School of Medicine, Tokyo, Japan.

No MeSH data available.