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

Holt-Oram syndrome is caused by mutations in TBX5, a member of the T-box ge ne family. In order to identify DNA sequences to which the TBX5 protein bin ds, we have performed an in vitro binding site selection assay. We have ide ntified an 8 bp core sequence that is part of the Brachyury consensus-bindi ng 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 TBX 5 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 pat ients indicates that G80R and R237Q eliminate binding to the target site. D NA database analysis reveals that target sites are present in the upstream regions of several card iac-expressed genes including cardiac a actin, atri al natriuretic factor, cardiac myosin heavy chain a, cardiac myosin heavy c hain beta, myosin light chain 1A, myosin light chain 1V and Nkx2.5. Cell tr ansfection studies demonstrate that TBX5 activates the transcription of an atria[ natriuretic factor reporter construct and this effect is significant ly reduced by deletion of the TBX5 binding site.