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Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming.

ABSTRACT: Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.

SUBMITTER: Ruetz T

PROVIDER: S-EPMC5732323 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming.

Ruetz Tyson T Pfisterer Ulrich U Di Stefano Bruno B Ashmore James J Beniazza Meryam M Tian Tian V TV Kaemena Daniel F DF Tosti Luca L Tan Wenfang W Manning Jonathan R JR Chantzoura Eleni E Ottosson Daniella Rylander DR Collombet Samuel S Johnsson Anna A Cohen Erez E Yusa Kosuke K Linnarsson Sten S Graf Thomas T Parmar Malin M Kaji Keisuke K

Cell stem cell 20171122 6

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) ...[more]

PMID: 29174331

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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

Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming.

Publications

Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming.

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