Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 23, Issue 23, Pages -Publisher
MDPI
DOI: 10.3390/ijms232315242
Keywords
dCas9; CAPTURE; Nanog; pluripotency; reprogramming
Funding
- JSPS KAKENHI
- Takeda Science Foundation [JP19H03203, JP19K22945, JP19K07343, JP21H02678]
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This study identified and characterized proteins associated with the Nanog promoter in mouse embryonic stem cells (mESCs) using unbiased methods. The results revealed the importance of these proteins, including RNA-binding proteins (RBPs), in the maintenance of pluripotency and the reprogramming of somatic cells. The findings highlight the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.
Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.
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