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Comparative Study
, 102 (50), 18034-9

A WW Domain Protein TAZ Is a Critical Coactivator for TBX5, a Transcription Factor Implicated in Holt-Oram Syndrome

Affiliations
Comparative Study

A WW Domain Protein TAZ Is a Critical Coactivator for TBX5, a Transcription Factor Implicated in Holt-Oram Syndrome

Masao Murakami et al. Proc Natl Acad Sci U S A.

Abstract

The T-box transcription factor TBX5 plays essential roles in cardiac and limb development. Various mutations in the TBX5 gene have been identified in patients with Holt-Oram syndrome, which is characterized by congenital defects in the heart and upper extremities. In this study, we identified a WW-domain-containing transcriptional regulator TAZ as a potent TBX5 coactivator. TAZ directly associates with TBX5 and markedly stimulates TBX5-dependent promoters by interacting with the histone acetyltransferases p300 and PCAF. YAP, a TAZ-related protein with conserved functional domains, also stimulates TBX5-dependent transcription, possibly by forming a heterodimer with TAZ. TBX5 lacks a PY motif, which mediates the association of other proteins with TAZ, and interacts with TAZ through multiple domains including its carboxyl-terminal structure. Truncation mutants of TBX5 identified in patients with Holt-Oram syndrome were markedly impaired in their ability to associate with and be stimulated by TAZ. These findings reveal key roles for TAZ and YAP in the control of TBX5-dependent transcription and suggest the involvement of these coactivators in cardiac and limb development.

Figures

Fig. 1.
Fig. 1.
TAZ potently activates TBX5-dependent transcription. (A) TAZ potently activates ANF-luciferase expression in the presence of TBX5. COS-1 cells were transiently transfected with plasmids for ANF-luciferase (ANF-Luc, 100 ng) and control β-galactosidase (10 ng), TBX5 expression plasmid (10 ng), and the expression plasmids of various transcriptional coregulators (100 ng). Basal luciferase activity without expression of TBX5 and coregulators was given a value of 1. (B) TAZ activates TBX5-dependent ANF-luciferase expression in a dose-dependent manner. TBX5, 10 ng; TAZ, 5–100 ng. (C) TBEs are essential for TAZ-mediated activation of ANF-luciferase expression. (Right) Luciferase assays with the ANF-luciferase constructs (–452, –249, and –79 bp) and its mutants (muTBE). TBX5, 10 ng; TAZ, 100 ng. (Left) Structures of the ANF-luciferase constructs (–452, –249, and –79 bp) and its mutants are shown. The transcriptional initiation site is shown as +1. TBX5, 10 ng; TAZ, 100 ng. (D) TAZ activates TBX5-dependent Fgf10-luciferase (Fgf10-Luc) expression. TBX5, 50 ng; TAZ, 100 ng. (E) TAZ stimulates the activity of a luciferase reporter that contains tandem TBEs (2× TBE-Luc), but not the reporter with mutated TBEs (2× muTBE-Luc). TBX5, 10 ng; TAZ, 100 ng. In BE, COS-1 cells were transfected with the expression plasmids of TBX5 and TAZ together with luciferase and β-galactosidase plasmids. Basal activity of each luciferase reporter without TBX5 and TAZ expression was given a value of 1.
Fig. 2.
Fig. 2.
TAZ and YAP play important roles in cardiac ANF expression. (A) Structure of mouse TAZ and YAP proteins. WW, WW1, and WW2, WW domain. TAZ and YAP share WW domain(s), a coiled-coil motif, and a PDZ-binding motif. YAP contains a proline-rich domain and an SH3-binding motif. (B) YAP activates TBX5-dependent ANF-luciferase expression in a dose-dependent manner. TBX5, 10 ng; YAP, 5–200 ng. Basal luciferase activity without TBX5 and YAP expression was given a value of 1. (C) siRNA against TAZ or YAP reduced ANF-luciferase activity in cardiac myocytes. Primary neonatal mouse cardiac myocytes were cotransfected with siRNA and ANF-luciferase plasmid. siRNA, control (siRNA without known homology with mammalian sequences) (Qiagen); TAZ-site #1 and -site #2, against mouse TAZ; YAP-site #1 and -site #2, against mouse YAP. Luciferase activity with control siRNA cotransfection was given a value of 100%.
Fig. 3.
Fig. 3.
Physical interactions among TBX5, TAZ, and YAP. (A) TAZ activates UAS-luciferase reporter expression by Gal4 DNA-binding domain fused to TBX5 (DBD-TBX5). DBD or DBD-TBX5, 10 ng; TAZ, 100 ng. (B) TAZ, but not YAP, was coimmunoprecipitated with TBX5. Flag-TBX5, Myc-TAZ, and Myc-YAP were expressed in 293T cells and coimmunoprecipitation was performed by using anti-Flag antibody. Western blot analysis (IB) was performed on immunoprecipitates (IP) and total cell lysates (Input) by using anti-Myc or -Flag antibody. (C) TAZ associates with TBE DNA fragments through TBX5. Biotin-labeled TBE oligonucleotide fragments were bound to streptoavidin beads and incubated with Myc-TAZ in the presence of GST or GST-TBX5. Recovery of Myc-TAZ with streptoavidin beads was examined by Western blotting using anti-Myc antibody. (D) TAZ forms a homodimer and a heterodimer with YAP. Flag-TAZ or -YAP was immunoprecipitated by using anti-Flag antibody, and associated proteins were detected by Western blotting using anti-Myc antibody (Top). Expression of TAZ and YAP was confirmed by using anti-Myc or -Flag antibody (Middle and Bottom). (E) Coexpression of TAZ potently enhances YAP-mediated activation of TBX5-dependent transcription. COS-1 cells were cotransfected with TBX5 expression plasmid (10 ng) and different amounts of TAZ and YAP plasmids. Basal luciferase activity was given a value of 1.
Fig. 4.
Fig. 4.
TAZ associates with HAT proteins to activate transcription. (A) TAZ and YAP do not show HAT activity. In vitro HAT assays were performed by using the proteins that were expressed in 293T cells and purified by immunoprecipitation (IP). (Inset) Protein expression was confirmed by Western blot analysis. (B) TAZ physically associates with p300 and PCAF. Coimmunoprecipitation was performed as described above. (C) p300 and PCAF enhance TBX5-dependent transcription in the presence of TAZ. COS-1 cells were transfected with the expression plasmids (TBX5, 10 ng; TAZ, 50 ng; p300 or PCAF, 100 ng) and ANF-luciferase plasmid.
Fig. 5.
Fig. 5.
Effects of TAZ on various TBX5 mutants identified in HOS patients. (A) Premature-termination mutants identified in HOS patients, TBX5-R279Ter and -E316Ter, show marked reduction of TAZ-mediated transactivation. (Left) Structures of TBX5 mutants are shown. Numbers indicate the positions of the T-box and amino-/carboxyl-termini of full-length protein in the amino acid sequence. Asterisks indicate the positions of point mutations. COS-1 cells were cotransfected with expression plasmids encoding TBX5 or HOS mutants (10 ng) and/or TAZ plasmid (100 ng). Expression of TBX5 proteins and TAZ was confirmed by Western blotting. Lane numbers on the gel correspond to transfections (Right). (B) DNA-binding activity of TBX5-R279Ter and -E316Ter mutants is not affected. Electrophoretic mobility shift assay is shown. Arrowheads indicate shifted bands. (C) TBX5-R279Ter and -E316Ter show inefficient interaction with TAZ. Coimmunoprecipitation was performed as described in Fig. 3.

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