Characterization of the TBX5 binding site and analysis of mutations that cause Holt-Oram syndrome

Abstract

Holt-Oram syndrome is caused by mutations in TBX5, a member of the T-box gene family. In order to identify DNA sequences to which the TBX5 protein binds, we have performed an in vitro binding site selection assay. We have identified an 8 bp core sequence that is part of the Brachyury consensus-binding site. We show that TBX5 binds to the full palindromic Brachyury binding site and to the half-palindrome, whereas Brachyury does not bind to the TBX5 site. Amino acids 1-237 of TBX5 are required for DNA binding. Analysis of the effects of specific substitution mutations that arise in Holt-Oram patients indicates that G80R and R237Q eliminate binding to the target site. DNA database analysis reveals that target sites are present in the upstream regions of several cardiac-expressed genes including cardiac alpha actin, atrial natriuretic factor, cardiac myosin heavy chain alpha, cardiac myosin heavy chain beta, myosin light chain 1A, myosin light chain 1V and Nkx2.5. Cell transfection studies demonstrate that TBX5 activates the transcription of an atrial natriuretic factor reporter construct and this effect is significantly reduced by deletion of the TBX5 binding site.

The NK-2 class homeodomain factor CEH-51 and the T-box factor TBX-35 have overlapping function inC. elegansmesoderm development

“…4C), which matches 7/8 sites of the Brachyury half-site TCACACCT (matches underlined) (Kispert and Herrmann, 1993). Hence, it is likely that TBX-35 binds DNA as a monomer, similar to mouse Tbx20 and Tbx5 (Ghosh et al, 2001;Macindoe et al, 2009;Stennard et al, 2003). A competitor oligonucleotide containing two of the candidate sites competed the shifts, whereas a competitor with both sites mutated did not (Fig.…”

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

“…Several reports have described that HOS-associated Tbx5 mutant proteins have deficient transcriptional regulatory activity in vitro (Ghosh et al, 2001;Hiroi et al, 2001;Fan et al, 2003a,b;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.…”

XTbx1 is a transcriptional activator involved in head and pharyngeal arch development in Xenopus laevis

“…As yet, no consensus binding site has been determined for Tbx1 proteins. However, site selection experiments for T-box family members have invariably identified the core motif TCACACCT (Conlon et al, 2001;Ghosh et al, 2001;Wilson and Conlon, 2002). The palindromic arrangement of this core motif was first identified as the preferred binding site for the prototypical T-box protein, Brachyury (Kispert and Herrmann, 1993) and has been demonstrated to bind to human TBX1A by electrophoretic mobility shift assay (Sinha et al, 2000).…”