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Eomesodermin requires transforming growth factor-beta/activin signaling and binds Smad2 to activate mesodermal genes.

ABSTRACT: The T-box gene Eomesodermin (Eomes) is required for early embryonic mesoderm differentiation in mouse, frog (Xenopus laevis), and zebrafish, is important in late cardiac development in Xenopus, and for CD8+ T effector cell function in mouse. Eomes can ectopically activate many mesodermal genes. However, the mechanism by which Eomes activates transcription of these genes is poorly understood. We report that Eomes protein interacts with Smad2 and is capable of working in a non-cell autonomous manner via transfer of Eomes protein between adjacent embryonic cells. Blocking of Eomes protein transfer using a farnesylated red fluorescent protein (CherryF) also prevents Eomes nuclear accumulation. Transfer of Eomes protein between cells is mediated by the Eomes carboxyl terminus (456-692). A carbohydrate binding domain within the Eomes carboxyl-terminal region is sufficient for transfer and important for gene activation. We propose a novel mechanism by which Eomes helps effect a cellular response to a morphogen gradient.

SUBMITTER: Picozzi P

PROVIDER: S-EPMC2629102 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Eomesodermin requires transforming growth factor-beta/activin signaling and binds Smad2 to activate mesodermal genes.

Picozzi Paola P Wang Fengxiang F Cronk Kevin K Ryan Kenneth K

The Journal of biological chemistry 20081126 4

The T-box gene Eomesodermin (Eomes) is required for early embryonic mesoderm differentiation in mouse, frog (Xenopus laevis), and zebrafish, is important in late cardiac development in Xenopus, and for CD8+ T effector cell function in mouse. Eomes can ectopically activate many mesodermal genes. However, the mechanism by which Eomes activates transcription of these genes is poorly understood. We report that Eomes protein interacts with Smad2 and is capable of working in a non-cell autonomous mann ...[more]

PMID: 19036723

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Project description:BACKGROUND:The simplicity of Transforming Growth Factor ß (TGF?) signaling pathway, linear and non-amplified, hardly sustains its variety of responses. This is often justified by the complex regulation showed by Smad proteins, TGF? signaling intracellular transducers, object of post-translational modifications that modulate TGF?-dependent transcription. Protein acetylation is emerging as a compelling mechanism affecting the activities of significant transcription factors, including p53, FOXO or NF-kB. Smad proteins might be controlled by this mechanism, implying that accessory factors capable of altering Smads-transcriptional complexes acetylation status and hence regulate TGF? responses remain to be identified. Understanding this interaction may help in the assessment of TGF? signaling outcomes, extending from healthy physiology to pathological conditions and cancer. METHODS:A two-hybrid chimera interacting system allowed to identify Sirt1, a NAD+ dependent type III histone deacetylase, as a novel Smad2 interactor. Several well stablished cellular models were applied to characterize this interaction by means of co-immunoprecipitation of tagged proteins and immuno-fluorescence staining. The occurrence of the interaction at Smad2 driven transcriptomic complexes was studied by means of DNA-pull-down and chromatin immunoprecipitation (ChIP), while its effects were assessed by protein over-expression and siRNA applied into a TGF?-dependent reporter gene assay. RESULTS:The interaction was confirmed and observed to be enhanced upon Smad2 acetylation, a known feature of active and nuclear Smad2. However, Sirt1 did not play a major role in Smad2 deacetylation. Anti-Sirt1 ChIP showed increased recovery of promoter regions corresponding to Smad2-driven genes after TGF?-stimulation, while its occurrence at Smad2-dependent transcriptomic complexes on DNA was found to effectively modulate gene expression. CONCLUSIONS:Sirt1 presence on Smad2-driven TGF?-dependent regulatory elements was detected and found to increase after TGF? treatment. Moreover, Sirt1 overexpression resulted in a decrease of the activity of a Smad2-driven TGF?-dependent reporter gene, while Sirt1 interference increased its activity. This would confirm the relevance of the discovered Sirt1-Smad2 interaction for the regulation of TGF?-dependent gene transcription.

Dataset Information

Eomesodermin requires transforming growth factor-beta/activin signaling and binds Smad2 to activate mesodermal genes.

Publications

Eomesodermin requires transforming growth factor-beta/activin signaling and binds Smad2 to activate mesodermal genes.

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