Limits...
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.


Gtf2ird1 modifies chromatin at the Mkx promoter. (A) ChIP-qPCR analysis of the GTF2IRD1 binding site with anti-Gtf2ird1 antibody using primers designed upstream of the Mkx TSS revealed enrichment of DNA at bp −600 upstream (not drawn to scale). Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test performed for results between primers designed at bp −600 and primers designed at the H1foo intron selected as a control). (B) ChIP-qPCR with histones and Pol II at the putative GTF2IRD1 binding site. Anti-H3K4me3, anti-H4ac, and anti-Pol II antibodies were used to assess active histone modification, which confirmed enrichment at the GTF2IRD1 binding site identified from the experiment shown in panel A. Error bars represent standard errors of the means (*, P < 0.05; ***, P < 0.001, by a two-tailed Student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836271&req=5

Figure 7: Gtf2ird1 modifies chromatin at the Mkx promoter. (A) ChIP-qPCR analysis of the GTF2IRD1 binding site with anti-Gtf2ird1 antibody using primers designed upstream of the Mkx TSS revealed enrichment of DNA at bp −600 upstream (not drawn to scale). Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test performed for results between primers designed at bp −600 and primers designed at the H1foo intron selected as a control). (B) ChIP-qPCR with histones and Pol II at the putative GTF2IRD1 binding site. Anti-H3K4me3, anti-H4ac, and anti-Pol II antibodies were used to assess active histone modification, which confirmed enrichment at the GTF2IRD1 binding site identified from the experiment shown in panel A. Error bars represent standard errors of the means (*, P < 0.05; ***, P < 0.001, by a two-tailed Student's t test).

Mentions: Having identified the promoter region that Gtf2ird1 acts upon, chromatin immunoprecipitation (ChIP) analysis was performed to assess whether Gtf2ird1 binds to the Mkx promoter. The results demonstrate a 2.5- to 5-fold increase of immunoprecipitate in the primer designed around a region 600 bp upstream compared to the amount with the surrounding primers and a negative control (Fig. 7A). This region, which showed the greatest enrichment, was consistent with the finding from the luciferase assay. Furthermore, ChIP-quantitative PCR (qPCR) with H3K4me3, H4ac, and Pol II antibodies revealed enrichment compared with levels with the negative control, demonstrating chromatin modification at this location (Fig. 7B).


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)

Gtf2ird1 modifies chromatin at the Mkx promoter. (A) ChIP-qPCR analysis of the GTF2IRD1 binding site with anti-Gtf2ird1 antibody using primers designed upstream of the Mkx TSS revealed enrichment of DNA at bp −600 upstream (not drawn to scale). Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test performed for results between primers designed at bp −600 and primers designed at the H1foo intron selected as a control). (B) ChIP-qPCR with histones and Pol II at the putative GTF2IRD1 binding site. Anti-H3K4me3, anti-H4ac, and anti-Pol II antibodies were used to assess active histone modification, which confirmed enrichment at the GTF2IRD1 binding site identified from the experiment shown in panel A. Error bars represent standard errors of the means (*, P < 0.05; ***, P < 0.001, by a two-tailed Student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836271&req=5

Figure 7: Gtf2ird1 modifies chromatin at the Mkx promoter. (A) ChIP-qPCR analysis of the GTF2IRD1 binding site with anti-Gtf2ird1 antibody using primers designed upstream of the Mkx TSS revealed enrichment of DNA at bp −600 upstream (not drawn to scale). Error bars represent standard errors of the means (**, P < 0.01, two-tailed Student's t test performed for results between primers designed at bp −600 and primers designed at the H1foo intron selected as a control). (B) ChIP-qPCR with histones and Pol II at the putative GTF2IRD1 binding site. Anti-H3K4me3, anti-H4ac, and anti-Pol II antibodies were used to assess active histone modification, which confirmed enrichment at the GTF2IRD1 binding site identified from the experiment shown in panel A. Error bars represent standard errors of the means (*, P < 0.05; ***, P < 0.001, by a two-tailed Student's t test).
Mentions: Having identified the promoter region that Gtf2ird1 acts upon, chromatin immunoprecipitation (ChIP) analysis was performed to assess whether Gtf2ird1 binds to the Mkx promoter. The results demonstrate a 2.5- to 5-fold increase of immunoprecipitate in the primer designed around a region 600 bp upstream compared to the amount with the surrounding primers and a negative control (Fig. 7A). This region, which showed the greatest enrichment, was consistent with the finding from the luciferase assay. Furthermore, ChIP-quantitative PCR (qPCR) with H3K4me3, H4ac, and Pol II antibodies revealed enrichment compared with levels with the negative control, demonstrating chromatin modification at this location (Fig. 7B).

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.