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Updated: Jun 6, 2025
A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells
Published on: August 29, 2020
A stepwise mode of TGFβ-SMAD signaling and DNA methylation regulates naïve-to-primed pluripotency and differentiation
Bingnan Zhao1,2, Xiuwei Yu1,2, Jintong Shi3
1Department of Histoembryology, Genetics and Developmental Biology, Shanghai Key Laboratory of Reproductive Medicine, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Abstract:
The formation of transcription regulatory complexes by the association of Smad4 with Smad2 and Smad3 (Smad2/3) is crucial in the canonical TGFβ pathway. Although the central requirement of Smad4 as a common mediator is emphasized in regulating TGFβ signaling, it is not obligatory for all responses. The role of Smad2/3 independently of Smad4 remains understudied. Here, we introduce a stepwise paradigm in which Smad2/3 regulate the lineage priming and differentiation of mouse embryonic stem cells (mESCs) by collaboration with different effectors. During the naïve-to-primed transition, Smad2/3 upregulate DNA methyltransferase 3b (Dnmt3b), which establishes the proper DNA methylation patterns and, in turn, enables Smad2/3 binding to the hypomethylated centers of promoters and enhancers of epiblast marker genes. Consequently, in the absence of Smad2/3, Smad4 alone cannot initiate epiblast-specific gene transcription. When primed epiblast cells begin to differentiate, Dnmt3b becomes less actively engaged in global genome methylation, and Smad4 takes over the baton in this relay race, forming a complex with Smad2/3 to support mesendoderm induction. Thus, mESCs lacking Smad4 can undergo the priming process but struggle with the downstream differentiation. This work sheds light on the intricate mechanisms underlying TGFβ signaling and its role in cellular processes.
Insights
Smad2/3 proteins regulate mouse embryonic stem cell (mESC) differentiation by upregulating DNA methyltransferase 3b (Dnmt3b) during lineage priming. Smad4 is essential for later mesendoderm induction, highlighting a stepwise TGFβ signaling mechanism.
Area of Science:
- Developmental Biology
- Stem Cell Biology
- Molecular Biology
Background:
- The canonical transforming growth factor beta (TGFβ) pathway is critical for cellular processes, primarily mediated by Smad proteins.
- Smad4 acts as a common mediator in TGFβ signaling, but its role is not essential for all responses.
- The independent function of Smad2/3 proteins, separate from Smad4, in TGFβ signaling remains underexplored.
Purpose of the Study:
- To elucidate the distinct roles of Smad2/3 and Smad4 in mouse embryonic stem cell (mESC) differentiation.
- To investigate the stepwise mechanism of TGFβ signaling during the transition from naïve to primed pluripotency.
- To understand how Smad proteins collaborate with epigenetic modifiers like Dnmt3b.
Main Methods:
- Utilized mouse embryonic stem cells (mESCs) to study lineage priming and differentiation.
- Investigated the regulation of DNA methyltransferase 3b (Dnmt3b) by Smad2/3 during the naïve-to-primed transition.
- Analyzed the binding of Smad2/3 to hypomethylated promoters and enhancers of epiblast marker genes.
- Examined the role of Smad4 in mesendoderm induction in Smad4-deficient mESCs.
Main Results:
- Smad2/3 proteins upregulate Dnmt3b during the naïve-to-primed transition in mESCs, establishing DNA methylation patterns necessary for Smad2/3 binding to regulatory elements.
- Smad4 is not sufficient to initiate epiblast-specific gene transcription in the absence of Smad2/3.
- Smad4 forms a complex with Smad2/3 to promote mesendoderm induction during differentiation, indicating a sequential role.
- mESCs lacking Smad4 can achieve pluripotency priming but exhibit impaired downstream differentiation.
Conclusions:
- TGFβ signaling operates via a stepwise paradigm involving Smad2/3 and Smad4 in mESC differentiation.
- Smad2/3, in collaboration with Dnmt3b, drives lineage priming by establishing permissive epigenetic states.
- Smad4 is crucial for later differentiation stages, particularly mesendoderm induction, working in concert with Smad2/3.
- This study reveals a dynamic interplay between Smad proteins and epigenetic modifiers in regulating stem cell fate and development.
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