Fulcoli, Filomena Gabriella (2009) Tbx1 regulates Smad signaling. [Tesi di dottorato] (Unpublished)
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| Item Type: | Tesi di dottorato | ||||||||
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| Lingua: | English | ||||||||
| Title: | Tbx1 regulates Smad signaling. | ||||||||
| Creators: |
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| Date: | 30 March 2009 | ||||||||
| Number of Pages: | 120 | ||||||||
| Institution: | Università degli Studi di Napoli Federico II | ||||||||
| Istituzioni (extra): | CEINGE Biotecnologie Avanzate, TIGEM – Telethon Insitute of Genetics and Medicine | ||||||||
| Department: | Telethon Institute of Genetics and Medicine (TIGEM) | ||||||||
| Scuola di dottorato: | SEMM – European School of Molecular Medicine | ||||||||
| Dottorato: | PhD in Molecular Medicine (Molecular Oncology or Human Genetics) | ||||||||
| Ciclo di dottorato: | 20 | ||||||||
| Coordinatore del Corso di dottorato: |
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| Tutor: |
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| Date: | 30 March 2009 | ||||||||
| Number of Pages: | 120 | ||||||||
| Uncontrolled Keywords: | Tbx1 Smad1 | ||||||||
| Settori scientifico-disciplinari del MIUR: | Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare | ||||||||
| Additional Information: | Ciclo II/XX, Curriculum Human Genetics | ||||||||
| Date Deposited: | 10 Nov 2009 13:54 | ||||||||
| Last Modified: | 14 Jan 2015 12:18 | ||||||||
| URI: | http://www.fedoa.unina.it/id/eprint/3202 |
Abstract
To have insight into the molecular mechanisms governing the function of Tbx1, a transcription factor involved in DiGeorge syndrome (DGS) and cardiovascular development, we searched for proteins interacting with it. Using an affinity purification protocol followed by a candidate protein approach, we found that Smad1/5/8 interacted with Tbx1. To confirm this interaction, protein extracts from mouse embryos were co-immunoprecipitated with an antibody anti Tbx1 and analyzed by western-blot. Results confirmed the interaction and revealed that Smad1 is the protein directly interacting with Tbx1. Next, we tested TBX1 missense mutations found associated with a DGS phenotype but. Results indicate that one of the mutant isoforms is unable to bind Smad1. To examine the role of Tbx1 in the regulation of transcriptional responses induced by Smad1 we performed luciferase assays and the results show that Tbx1 is capable of suppressing the activity of a Smad signaling reporter in a dosage-dependent manner. We also found, using a Co-IP approach, that Tbx1 inhibits Smad signaling activity by competing with Smad4 for binding to Smad1. Using a Tbx1 mutant isoform that prevents DNA binding, we found that this suppression is not dependent upon Tbx1-DNA binding. We in addition provided evidence that Tbx1 overexpression in mice causes a phenotype similar to that caused by loss of Smad1-dependent signaling in the same tissues (including cleft lip and outflow heart defects) and downregulates Smad1 target genes such as Msx1 and 15 Msx2. In conclusion, our data demonstrate a mechanism by which Tbx1 interferes with the Bmp/Smad1 signal transduction pathway in tissue culture and during mammalian development. In addition, we provide evidence that a T-box transcription factor can have functions not directly related to or mediated by its transactivation activity.
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