PMC

Among more than 20 TBX5 mutations, Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I represent missense mutations identified in patients with Holt-Oram syndrome. TBX5 consists of 9 exons with exons 3–7 encoding the T-domain responsible for DNA binding. Note that two mutations (Q49K and I54T) are in the N terminus, four mutations (G80R, G169R, R237Q, and R237W) are within the T-box domain, and one mutation (S252I) is in the C terminus.

A, WT, cell transfected with the FLAG-tagged wild type TBX5 construct; Q49K, I54T, G80R, R237Q, R237W, G169R, and S252I represent cells over-expressing various mutant TBX5 proteins. Transfected cells were immunostained with anti-FLAG (green) for TBX5 and the nucleus was stained with DAPI (blue). No detectable immunofluorescence staining was observed in non-transfected cells. Note that wild type TBX5 is completely localized into the nucleus, whereas TBX5 proteins with various missense mutations are distributed in both the nucleus and cytoplasm. B, percentage of nuclear versus cytoplasm distribution of TBX5 for wild type (WT) and mutant (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I). The data were based on the images in panel A.

The wild type and mutant TBX5 proteins were synthesized in vitro using the TNT-coupled transcription/translation system. The TBX5-binding site is a synthetic double-stranded DNA fragment corresponding to the region from −257 to −242 bp upstream from the ANF transcriptional start site (5′-aataTCACACCTgtac-3′). A, EMSA. Lane “−,” no TBX5 protein; WT, wild type TBX5; Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I, TBX5 with individual mis-sense mutations. B, a silver-stained protein gel showing the approximately equal level of synthesis of wild type and various mutant TBX5 proteins used in EMSA. Lane 1, no TBX5 expression plasmid DNA; lane 2, wild type TBX5; lanes 3–9 represent Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I mutant TBX5, respectively. C, EMSA with various amounts of wild type TBX5 and mutants G80R and R237Q. D, the amount of the TBX5-DNA complex in panel C was plotted against TBX5 concentrations. The fitted slope for specific binding of wild type TBX5 (WT) and mutants G80R and R237Q is 79.5, 19.5, and 11.7, respectively.

A, HeLa cells were transiently transfected with expression plasmids for His-TBX5 and NKX2.5-HA. Total cell lysates containing His-tagged TBX5 and/or HA-tagged NKX2.5 were incubated with Ni-NTA beads, separated by 12% SDS-PAGE, and analyzed by Western blot with an anti-His antibody (upper panel) or with an anti-HA monoclonal antibody (middle panel). Proteins bound to Ni-NTA beads were washed with washing buffer, eluted, and fractionated by 12% SDS-PAGE and analyzed by Western blot with anti-mouse HA for NKX2.5. Lane 1, wild type (WT) TBX5 without co-transfection of NKX2.5; lane 2, wild type TBX5 with co-transfection of NKX2.5; lanes 2–9, TBX5 with Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I co-transfected with NKX2.5-HA, respectively. B, GST-NKX2.5 fusion protein and in vitro-translated His-tagged TBX5 were incubated with glutathione-Sepharose 4B beads, and proteins bound to the beads were washed with PBS, eluted, fractionated by 12% SDS-PAGE, and analyzed by Western blot with an anti-His antibody for detecting TBX5. Approximately equal amounts of wild type and various mutant TBX5 proteins were used in the GST pull-down assay as shown in the legend to .

A, the reporter gene used for transcriptional activation assay. The promoter region, from −270 to +1 bp upstream from the transcriptional start site, of ANF was fused to the luciferase gene (LUC). B, transcriptional activation assay for all seven missense mutations of TBX5 in the absence (left block) and presence (right block) of NKX2.5. Transcriptional activity is shown as relative luciferase activity on the y-axis. The transcriptional activity for the vector only was set arbitrarily to 1. WT, wild type. Note that all missense mutations of TBX5 abolished synergistic transcription activation of the ANF promoter between TBX5 and NKX2.5. C, Western blot analysis to determine whether mutant TBX5 were successfully expressed in transfected COS-7 cells. BE, purified wild type TBX5 control from a bacterial over-expression system; WT, lane with lysate from COS-7 cells containing expressed FLAG-tagged wild type TBX5; Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I, lanes with lysates from COS-7 cells containing expressed TBX5 proteins with corresponding mutations.