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Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein.

Böhm J, Heinritz W, Craig A, Vujic M, Ekman-Joelsson BM, Kohlhase J, Froster U - BMC Med. Genet. (2008)

Bottom Line: Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript.In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS.The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Humangenetik und Anthropologie, Universität Freiburg, Freiburg, Germany. Johann.Boehm@titus.u-strasbg.fr

ABSTRACT

Background: Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS.

Methods: The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF.

Results: The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter.

Conclusion: The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.

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Functional effect of the TBX5 c.1333delC mutation on transcriptional regulation. COS-7 cells were transiently co-transfected either with mutant or wild type TBX5 expression constructs and a reporter encoding the luciferase gene under the control of the ANF promoter. Values were calculated by normalizing against Renilla reniformis luciferase activity and compared to the empty FLAG vector (column 1). Wild type TBX5-FLAG and TBX5-GFP effectively induced expression of the reporter gene (columns 2 and 3), whereas cells transfected with rising amounts of the mutant TBX5 construct demonstrated entire abrogation of reporter gene activation compared to wild type TBX5 (column 4, 5, 6).
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Figure 4: Functional effect of the TBX5 c.1333delC mutation on transcriptional regulation. COS-7 cells were transiently co-transfected either with mutant or wild type TBX5 expression constructs and a reporter encoding the luciferase gene under the control of the ANF promoter. Values were calculated by normalizing against Renilla reniformis luciferase activity and compared to the empty FLAG vector (column 1). Wild type TBX5-FLAG and TBX5-GFP effectively induced expression of the reporter gene (columns 2 and 3), whereas cells transfected with rising amounts of the mutant TBX5 construct demonstrated entire abrogation of reporter gene activation compared to wild type TBX5 (column 4, 5, 6).

Mentions: An important functional domain of TBX5 is the T-box motif involved in DNA-binding and protein-protein interactions [10,11]. The majority of the described TBX5 mutations result in preterminal stop codons. The functional effect of the c.1333delC mutation – leading to an elongated protein – on transcriptional regulation was assessed using a luciferase assay. COS-7 cells were transiently co-transfected with mutant/wild type TBX5 expression constructs (pFLAG-N3) and an ANF-luciferase construct as reporter. The upstream region of the ANF (atrial natriuretic factor gene) promoter contains a TBX5 binding site [21]. The luciferase activity was normalized in reference to the Renilla reniformis luciferase activity, used as an internal control. The constructs were analyzed by immunoblot for appropriate expression (data not shown). Reporter gene activation significantly increased in cells transfected with wild type TBX5 compared to cells transfected with the empty expression vector. Transfection of the TBX5-GFP construct lead to the same result, indicating that neither the FLAG-tag nor the GFP-tag (as used in the localization studies) have a detrimental effect on the protein function. The luciferase activity of the mutated TBX5-FLAG construct was strongly reduced to a value comparable to cells transfected with the empty pFLAG-N3 vector. An increased amount of transfected TBX5mut plasmid (300 ng, 450 ng, 600 ng) did not induce luciferase activity, ruling out a dosage effect (Fig. 4). Under the experimental conditions of cell culture mediated luciferase assays, the mutation c.1333delC abrogates activation of the ANF promoter by TBX5.


Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein.

Böhm J, Heinritz W, Craig A, Vujic M, Ekman-Joelsson BM, Kohlhase J, Froster U - BMC Med. Genet. (2008)

Functional effect of the TBX5 c.1333delC mutation on transcriptional regulation. COS-7 cells were transiently co-transfected either with mutant or wild type TBX5 expression constructs and a reporter encoding the luciferase gene under the control of the ANF promoter. Values were calculated by normalizing against Renilla reniformis luciferase activity and compared to the empty FLAG vector (column 1). Wild type TBX5-FLAG and TBX5-GFP effectively induced expression of the reporter gene (columns 2 and 3), whereas cells transfected with rising amounts of the mutant TBX5 construct demonstrated entire abrogation of reporter gene activation compared to wild type TBX5 (column 4, 5, 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567295&req=5

Figure 4: Functional effect of the TBX5 c.1333delC mutation on transcriptional regulation. COS-7 cells were transiently co-transfected either with mutant or wild type TBX5 expression constructs and a reporter encoding the luciferase gene under the control of the ANF promoter. Values were calculated by normalizing against Renilla reniformis luciferase activity and compared to the empty FLAG vector (column 1). Wild type TBX5-FLAG and TBX5-GFP effectively induced expression of the reporter gene (columns 2 and 3), whereas cells transfected with rising amounts of the mutant TBX5 construct demonstrated entire abrogation of reporter gene activation compared to wild type TBX5 (column 4, 5, 6).
Mentions: An important functional domain of TBX5 is the T-box motif involved in DNA-binding and protein-protein interactions [10,11]. The majority of the described TBX5 mutations result in preterminal stop codons. The functional effect of the c.1333delC mutation – leading to an elongated protein – on transcriptional regulation was assessed using a luciferase assay. COS-7 cells were transiently co-transfected with mutant/wild type TBX5 expression constructs (pFLAG-N3) and an ANF-luciferase construct as reporter. The upstream region of the ANF (atrial natriuretic factor gene) promoter contains a TBX5 binding site [21]. The luciferase activity was normalized in reference to the Renilla reniformis luciferase activity, used as an internal control. The constructs were analyzed by immunoblot for appropriate expression (data not shown). Reporter gene activation significantly increased in cells transfected with wild type TBX5 compared to cells transfected with the empty expression vector. Transfection of the TBX5-GFP construct lead to the same result, indicating that neither the FLAG-tag nor the GFP-tag (as used in the localization studies) have a detrimental effect on the protein function. The luciferase activity of the mutated TBX5-FLAG construct was strongly reduced to a value comparable to cells transfected with the empty pFLAG-N3 vector. An increased amount of transfected TBX5mut plasmid (300 ng, 450 ng, 600 ng) did not induce luciferase activity, ruling out a dosage effect (Fig. 4). Under the experimental conditions of cell culture mediated luciferase assays, the mutation c.1333delC abrogates activation of the ANF promoter by TBX5.

Bottom Line: Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript.In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS.The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Humangenetik und Anthropologie, Universität Freiburg, Freiburg, Germany. Johann.Boehm@titus.u-strasbg.fr

ABSTRACT

Background: Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS.

Methods: The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF.

Results: The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter.

Conclusion: The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.

Show MeSH
Related in: MedlinePlus