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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.


GTF2IRD1 enhances Mkx promoter activity. (A) Schematic for Mkx-luciferase deletion constructs. A region 5′ upstream of Mkx ATG and a 3-kb downstream region were deleted as shown. TSS, transcription start site. (B) Mkx deletion constructs were cotransfected into HEK293T cells with GTF2IRD1 for luciferase assays. Luciferase activity was severely reduced when the Mkx upstream region was shortened to within 666 bp upstream of the transcription start site. Error bars represent standard errors of the means (*, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student's t test). (C) Luciferase assay with shorter constructs with the TK promoter narrows down the GTF2IRD1 binding region to 329 bp (Mkx-del 1). On closer inspection of the Mkx-del 1 sequence, a 146-bp region was highly conserved in mammals. Luciferase assay of this 146-bp region (Mkx-del 4) did not show a decrease in luciferase activity compared to that in the Mkx-del 1 construct, thus indicating that this 146-bp region is sufficient for the binding of GTF2IRD1. *, P < 0.05; **, P < 0.01, two-tailed Student's t test. (D) A section within the 146-bp Mkx-del 4 deletion construct described in panel C reveals a previously described GATTA motif and CATTT, a GATTA-like-containing sequence. A 68-bp region containing these motifs was deleted from the Mkx-luc 5 reporter vector for subsequent analysis. (E) Luciferase activity was reduced in the reporter vector with the 68-bp deletion to confirm the importance of the motif-containing deleted region. Error bars represent standard errors of the means (*, P < 0.05, two-tailed Student's t test).
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Figure 6: GTF2IRD1 enhances Mkx promoter activity. (A) Schematic for Mkx-luciferase deletion constructs. A region 5′ upstream of Mkx ATG and a 3-kb downstream region were deleted as shown. TSS, transcription start site. (B) Mkx deletion constructs were cotransfected into HEK293T cells with GTF2IRD1 for luciferase assays. Luciferase activity was severely reduced when the Mkx upstream region was shortened to within 666 bp upstream of the transcription start site. Error bars represent standard errors of the means (*, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student's t test). (C) Luciferase assay with shorter constructs with the TK promoter narrows down the GTF2IRD1 binding region to 329 bp (Mkx-del 1). On closer inspection of the Mkx-del 1 sequence, a 146-bp region was highly conserved in mammals. Luciferase assay of this 146-bp region (Mkx-del 4) did not show a decrease in luciferase activity compared to that in the Mkx-del 1 construct, thus indicating that this 146-bp region is sufficient for the binding of GTF2IRD1. *, P < 0.05; **, P < 0.01, two-tailed Student's t test. (D) A section within the 146-bp Mkx-del 4 deletion construct described in panel C reveals a previously described GATTA motif and CATTT, a GATTA-like-containing sequence. A 68-bp region containing these motifs was deleted from the Mkx-luc 5 reporter vector for subsequent analysis. (E) Luciferase activity was reduced in the reporter vector with the 68-bp deletion to confirm the importance of the motif-containing deleted region. Error bars represent standard errors of the means (*, P < 0.05, two-tailed Student's t test).

Mentions: Having identified GTF2IRD1 as the candidate transcription factor that enhances Mkx promoter activity in an Mkx-Luc vector containing a 7-kb region upstream and 3-kb region downstream of the first coding exon (exon 2), we generated deletion constructs to narrow down the Mkx promoter region that Gtf2ird1 regulates (Fig. 6A). Luciferase assay of the six deletion constructs revealed low luciferase activity in empty-vector constructs and in the construct containing the 5′ untranslated region (UTR) (Fig. 6B, luc 6). Luciferase activity increased significantly when the deletion constructs were increased to contain a 666-bp region upstream of the transcription start site, suggesting that the critical Mkx-regulating region of Gtf2ird1 exists between bp +264 and −666 upstream of exon 1 (Fig. 6B, luc 5).


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 enhances Mkx promoter activity. (A) Schematic for Mkx-luciferase deletion constructs. A region 5′ upstream of Mkx ATG and a 3-kb downstream region were deleted as shown. TSS, transcription start site. (B) Mkx deletion constructs were cotransfected into HEK293T cells with GTF2IRD1 for luciferase assays. Luciferase activity was severely reduced when the Mkx upstream region was shortened to within 666 bp upstream of the transcription start site. Error bars represent standard errors of the means (*, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student's t test). (C) Luciferase assay with shorter constructs with the TK promoter narrows down the GTF2IRD1 binding region to 329 bp (Mkx-del 1). On closer inspection of the Mkx-del 1 sequence, a 146-bp region was highly conserved in mammals. Luciferase assay of this 146-bp region (Mkx-del 4) did not show a decrease in luciferase activity compared to that in the Mkx-del 1 construct, thus indicating that this 146-bp region is sufficient for the binding of GTF2IRD1. *, P < 0.05; **, P < 0.01, two-tailed Student's t test. (D) A section within the 146-bp Mkx-del 4 deletion construct described in panel C reveals a previously described GATTA motif and CATTT, a GATTA-like-containing sequence. A 68-bp region containing these motifs was deleted from the Mkx-luc 5 reporter vector for subsequent analysis. (E) Luciferase activity was reduced in the reporter vector with the 68-bp deletion to confirm the importance of the motif-containing deleted region. Error bars represent standard errors of the means (*, P < 0.05, two-tailed Student's t test).
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Figure 6: GTF2IRD1 enhances Mkx promoter activity. (A) Schematic for Mkx-luciferase deletion constructs. A region 5′ upstream of Mkx ATG and a 3-kb downstream region were deleted as shown. TSS, transcription start site. (B) Mkx deletion constructs were cotransfected into HEK293T cells with GTF2IRD1 for luciferase assays. Luciferase activity was severely reduced when the Mkx upstream region was shortened to within 666 bp upstream of the transcription start site. Error bars represent standard errors of the means (*, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student's t test). (C) Luciferase assay with shorter constructs with the TK promoter narrows down the GTF2IRD1 binding region to 329 bp (Mkx-del 1). On closer inspection of the Mkx-del 1 sequence, a 146-bp region was highly conserved in mammals. Luciferase assay of this 146-bp region (Mkx-del 4) did not show a decrease in luciferase activity compared to that in the Mkx-del 1 construct, thus indicating that this 146-bp region is sufficient for the binding of GTF2IRD1. *, P < 0.05; **, P < 0.01, two-tailed Student's t test. (D) A section within the 146-bp Mkx-del 4 deletion construct described in panel C reveals a previously described GATTA motif and CATTT, a GATTA-like-containing sequence. A 68-bp region containing these motifs was deleted from the Mkx-luc 5 reporter vector for subsequent analysis. (E) Luciferase activity was reduced in the reporter vector with the 68-bp deletion to confirm the importance of the motif-containing deleted region. Error bars represent standard errors of the means (*, P < 0.05, two-tailed Student's t test).
Mentions: Having identified GTF2IRD1 as the candidate transcription factor that enhances Mkx promoter activity in an Mkx-Luc vector containing a 7-kb region upstream and 3-kb region downstream of the first coding exon (exon 2), we generated deletion constructs to narrow down the Mkx promoter region that Gtf2ird1 regulates (Fig. 6A). Luciferase assay of the six deletion constructs revealed low luciferase activity in empty-vector constructs and in the construct containing the 5′ untranslated region (UTR) (Fig. 6B, luc 6). Luciferase activity increased significantly when the deletion constructs were increased to contain a 666-bp region upstream of the transcription start site, suggesting that the critical Mkx-regulating region of Gtf2ird1 exists between bp +264 and −666 upstream of exon 1 (Fig. 6B, luc 5).

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.