Functional Analysis of TBX5 Missense Mutations Associated with Holt-Oram Syndrome

Abstract

TBX5 is a T-box transcription factor that plays a critical role in organogenesis. Seven missense mutations in TBX5 have been identified in patients with Holt-Oram syndrome characterized by congenital heart defects and upper limb abnormalities. However, the functional significance and molecular pathogenic mechanisms of these mutations are not clear. In this study we describe functional defects in DNA binding, transcriptional activity, protein-protein interaction, and cellular localization of mutant TBX5 with these missense mutations (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I). Mutations G80R, R237Q, and R237W represent a group of mutations that dramatically reduce DNA-binding activity of TBX5, leading to reduced transcription activation by TBX5 and the loss of synergy in transcriptional activation between TBX5 and NKX2.5. The second group of mutations includes Q49K, I54T, G169R, and S252I, which have no or moderate effect on DNA-binding activity and the function of transcription activation of TBX5 but cause the complete loss of synergistic transcription activity between TBX5 and NKX2.5. All seven missense mutations greatly reduced the interaction of TBX5 with NKX2.5 in vivo and in vitro. Immunofluorescent staining showed that wild type TBX5 was localized completely into the nucleus, but mutants were localized in both nucleus and cytoplasm. These results demonstrate that all seven missense mutations studied here are functional mutations with a spectrum of defects ranging from decreases in DNA-binding activity and transcriptional activation to the dramatic reduction of interaction between TBX5 and NKX2.5, and loss of synergy in transcriptional activation between these two proteins, as well as impairment in the nuclear localization of TBX5. These defects are likely central to the pathogenesis of Holt-Oram syndrome.

TBX5 intragenic duplication: a family with an atypical Holt–Oram syndrome phenotype

“…The normal cellular localisation of the TBX5 protein is in the nucleus in cardiomyocytes. 28 Fan et al 29,30 analysed various missense mutations and in frame deletions and described their possible pathogenetic mechanisms as either: (a) abnormal cellular localisation of mutant TBX5 protein or (b) altered interactions with cardiac or limb specific co-factors (eg, NKX2.5, GATA4, Cx40 and SALL4). They showed increased concentrations within the cytoplasm of TBX5 mutant proteins from various missense mutations and one in-frame deletion, providing evidence of altered nuclear localisation.…”

Microarray analysis of Tbx5‐induced genes expressed in the developing heart

“…The induction of prCAD by Tbx5 and their overlapping embryonic expression patterns are consistent with Tbx5 regulation of prCAD during the development of the eye and heart. HOS-associated mutations affect transcriptional function of Tbx5 by disrupting DNA binding, preventing association with other transcription factors, or by eliminating functional domains (Fan et al, 2003b;Plageman and Yutzey, 2004). For example, the missense mutation Tbx5(R237Q) inhibits DNA binding and association with Nkx2.5, whereas the nonsense mutation Tbx5(R279ter) prematurely stops translation and causes Tbx5 to lack a transcriptional activation domain (Fan et al, 2003b;Plageman and Yutzey, 2004).…”

“…HOS-associated mutations affect transcriptional function of Tbx5 by disrupting DNA binding, preventing association with other transcription factors, or by eliminating functional domains (Fan et al, 2003b;Plageman and Yutzey, 2004). For example, the missense mutation Tbx5(R237Q) inhibits DNA binding and association with Nkx2.5, whereas the nonsense mutation Tbx5(R279ter) prematurely stops translation and causes Tbx5 to lack a transcriptional activation domain (Fan et al, 2003b;Plageman and Yutzey, 2004). Previously, we demonstrated that these HOS-associated mutations decrease the ability of Tbx5 to activate the nppa promoter in vitro (Plageman and Yutzey, 2004).…”

“…To determine whether these mutations compromise the ability of Tbx5 to induce genes identified in the mircroarray, adenoviruses were generated expressing the HOS-associated mutant alleles Tbx5(R237Q) and Tbx5(R279ter) and used to infect 1H cells. The R237Q mutation affects Tbx5 transcriptional activity by disrupting DNA binding and inhibiting its association with Nkx2.5 (Fan et al, 2003b). The R279ter mutation is missing a strong activation domain due to a premature stop codon and cannot activate transcription in vitro (Plageman and Yutzey, 2004).…”

Epidemiology and Etiology of Congenital Heart Diseases

“…TBX5 was found expressed in embryonic human heart and limb. Mutations in this gene have been associated with Holt-Oram syndrome (Fan et al, 2003), which is characterized by skeletal malformations of the upper extremities and CHD, most commonly secundum ASD but also VSD and TOF (Basson et al, 1999;Faria et al, 2008;Li et al, 1997;Xin et al, 2009).  GATA4, GATA binding protein 4; is related to zinc finger transcription factors.…”