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Updated: Dec 14, 2025
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Published on: May 30, 2012
Ddx56 maintains proliferation of mouse embryonic stem cells via ribosome assembly and interaction with the Oct4/Sox2
Jingwen Wang1, Jiahui Liu1, Miaoman Ye1
1MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
Background:
Embryonic stem cells (ESCs) are important source of clinical stem cells for therapy, so dissecting the functional gene regulatory network involved in their self-renewal and proliferation is an urgent task. We previously reported that Ddx56 interacts with the core transcriptional factor Oct4 by mass spectrometry analysis in ESCs. However, the exact function of Ddx56 in ESCs remains unclear.
Methods:
We investigated the role of Ddx56 in mouse ESCs (mESCs) through both gain- and loss-of-function strategies. The effect of Ddx56 on mESCs was determined based on morphological changes, involvement in the network of pluripotency markers (Nanog, Oct4, Sox2), and altered lineage marker expression. In addition, the role of Ddx56 in mESCs was evaluated by polysome fractionation, qRT-PCR, and co-immunoprecipitation (co-IP). Finally, RNA sequencing was applied to explore potential network regulation by Ddx56 in mESCs.
Result:
We found that Ddx56 participated in ribosome assembly, as knockout or RNAi knockdown of Ddx56 led to ribosome dysfunction and cell lethality. Surprisingly, exogenous expression of C-terminal domain truncated Ddx56 (Ddx56 ΔC-ter) did not affect ribosome assembly, but decreased mESC proliferation by downregulation of proliferation-related genes and cell cycle changing. In terms of mechanism, Ddx56 interacted with the Oct4 and Sox2 complex by binding to Sox2, whereas Ddx56 ΔC-ter showed weaker interaction with Sox2 and led to retardation of mESC proliferation.
Conclusions:
Ddx56 maintains ESC proliferation by conventional regulation of ribosome assembly and interaction with the Oct4 and Sox2 complex.
Insights
The study reveals that Ddx56 is crucial for embryonic stem cell (ESC) self-renewal and proliferation. It maintains ESCs by regulating ribosome assembly and interacting with key pluripotency factors Oct4 and Sox2.
Area of Science:
- Stem cell biology
- Molecular and Cellular Biology
- Gene Regulation
Background:
- Embryonic stem cells (ESCs) are vital for regenerative medicine, necessitating understanding their self-renewal and proliferation mechanisms.
- The interaction between Ddx56 and Oct4 in ESCs was previously identified but their functional relationship remained elusive.
Purpose of the Study:
- To elucidate the specific functions of Ddx56 in mouse embryonic stem cells (mESCs).
- To investigate the molecular mechanisms underlying Ddx56's role in ESC self-renewal, proliferation, and pluripotency maintenance.
Main Methods:
- Utilized gain- and loss-of-function strategies (knockout, RNAi, exogenous expression) in mESCs.
- Assessed effects on cell morphology, pluripotency markers (Oct4, Sox2, Nanog), lineage markers, and cell cycle.
- Employed polysome fractionation, qRT-PCR, co-immunoprecipitation (co-IP), and RNA sequencing to analyze Ddx56 function and interactions.
Main Results:
- Ddx56 is essential for ribosome biogenesis; its depletion causes ribosome dysfunction and cell death.
- A truncated Ddx56 (Ddx56 ΔC-ter) variant impaired mESC proliferation by downregulating proliferation genes and altering cell cycle progression.
- Ddx56 directly interacts with the Oct4-Sox2 complex, specifically binding Sox2, which is critical for maintaining mESC proliferation.
Conclusions:
- Ddx56 plays a dual role in maintaining ESC proliferation.
- It ensures ESC self-renewal through ribosome assembly and by facilitating the interaction within the Oct4-Sox2 pluripotency network.
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