Abstract
Numerous studies have indicated that BMP4 signaling is involved in the regulation of the early steps of development. In mouse embryonic stem cells (ESCs), BMP4 is crucial to sustain pluripotency and blocks differentiation towards neural fate. Here, through a systematic analysis of miRNAs in ESCs, we establish that BMP4 signaling regulates miR-23a, 27a and 24-2, through the recruitment of phospho-Smads at the promoter of the gene encoding this miRNA cluster. Suppression of miR-23a/b, 27a/b and 24 does not affect self-renewal or pluripotency, but induces an evident change of ESC differentiation, with a significant increase of the cells undergoing apoptosis after the transition from ESCs to epiblast stem cells (EpiSCs). BMP4 has been previously reported to cause apoptosis during ESC differentiation. By blocking BMP4 signaling, we completely prevent the apoptosis induced by suppression of the miRs. This suggests that the effects of miR suppression are the result of enhanced BMP4 signaling. This hypothesis is further supported by the observation that Smad5, the transcription factor downstream of the BMP4 receptor, is targeted by the miRNAs of the 23a and 23b clusters. Altogether, our results highlight the existence of a regulatory loop, involving Smad5 and the miR-23a clusters, that modulates the apoptotic response of ESCs to BMP4.
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Abbreviations
- ESCs:
-
embryonic stem cells
- EpiSCs:
-
epiblast stem cells
- miRs:
-
microRNAs
- UTR:
-
untraslated region
- pri-miRNA:
-
primary transcript of miRNA
- SFEBs:
-
serum-free embryoid bodies
- BMP4:
-
bone morphogenetic protein
- DCGR8:
-
DiGeorge syndrome critical region 8
- Dies1:
-
differentiation of embryonic stem cells 1
- ChIP:
-
chromatin immunoprecipitation
- qPCR:
-
quantitative PCR
- KSR:
-
knock-out serum replacement
- Id:
-
inhibitor of differentiation
- TGFβ:
-
transforming growth factor beta
- PI:
-
propidium iodide
- KD:
-
knock-down.
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Acknowledgements
This work was supported by grants from Associazione Italiana Ricerca sul Cancro and from Italian Ministry of University and Research for the projects: Futuro in Ricerca 2013 (RBFR13YZ2Y), PON 01_02782 and CESMA. A Vocca and A Gargiulo were supported by fellowships from Project STRAIN. We thank Danila Scarpa for the technical assistance and Dr Tiziana Parisi for the critical reading of the manuscript. The Sox1-GFP cells were kindly provided by Professor Austin Smith.
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Musto, A., Navarra, A., Vocca, A. et al. miR-23a, miR-24 and miR-27a protect differentiating ESCs from BMP4-induced apoptosis. Cell Death Differ 22, 1047–1057 (2015). https://doi.org/10.1038/cdd.2014.198
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DOI: https://doi.org/10.1038/cdd.2014.198
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