Tissue formation and organ homeostasis require the precise coordination of stem cell proliferation and differentiation. Deregulation of these processes can lead to degenerative diseases or cancer. This study shows that the switch from hPSC self-renewal to differentiation is marked by the induction of specific CDKIs. In hPSCs, Activin/Nodal/TGFβ signaling maintains CDKIs in a poised state through a transcriptional complex involving SMAD2/3, NANOG, OCT4, EZH2, and SNON. During differentiation, CDKIs are activated by transcriptional complexes formed between SMAD2/3-SMYD2 and developmental regulators such as EOMES, extending the G phase of the cell cycle. This prolongation enhances SMAD2/3 transcriptional activity by blocking its linker phosphorylation, creating a positive feedback loop that drives the exit from pluripotency and stepwise cell fate specification. These findings highlight the intricate relationship between cell cycle dynamics and cell fate decisions, providing valuable insights for therapeutic applications.
Key findings from the study include:
- CDKI Regulation During Differentiation
CDKIs are largely absent in pluripotent hPSCs but are highly induced during differentiation into germ layers. Endoderm differentiation is characterized by significant G1 phase lengthening and elevated expression of CDKIs such as p15, p18, and p57. These inhibitors are induced early, preceding the appearance of lineage markers, underscoring their pivotal role in initiating differentiation.
- Epigenetic Control of Pluripotency and Differentiation
A screen of epigenetic regulators identifies EZH2 and SMYD2 as key players. EZH2 inhibition promotes differentiation by reducing pluripotency markers, while SMYD2 inhibition blocks the induction of CDKIs and endoderm markers. Chromatin immunoprecipitation analyses show that EZH2 establishes repressive marks (H3K27me3) on CDKI loci in pluripotent cells, while SMYD2 deposits activating marks (H3K4me3) during differentiation.
- SMAD2/3's Role in Maintaining and Switching Cell States
In pluripotent hPSCs, SMAD2/3 interacts with OCT4, NANOG, EZH2, and SNON to suppress CDKI expression via chromatin regulation. During differentiation, SMAD2/3 transitions to partnering with EOMES and SMYD2, leading to CDKI activation and facilitating endoderm formation. This dynamic switch is essential for the progression from self-renewal to lineage specification.
- Lineage-Specific Functions of CDKIs
Functional studies highlight the importance of CDKIs in lineage decisions. Knockdown of p15, p18, and p57 impairs endoderm and mesoderm differentiation but enhances neuroectodermal fate. Conversely, p21 knockdown boosts endoderm differentiation while suppressing neuroectoderm and mesoderm formation.
This study elucidates how CDKI expression interlinks cell cycle control and differentiation in hPSCs. SMAD2/3, in collaboration with context-specific partners (OCT4-NANOG or EOMES-SMYD2), dynamically regulates CDKI loci. These findings provide insight into the autonomous regulatory circuits of tissue formation, offering potential strategies for regenerative medicine and disease modeling. The work entitled “
SMAD2/3-SMYD2 and developmental transcription factors cooperate with cell-cycle inhibitors to guide tissue formation” was published on
Protein & Cell (published on May. 17, 2024).
DOI:
10.1093/procel/pwae031