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A balanced Oct4 interactome is crucial for maintaining pluripotency.

ABSTRACT: Oct4 collaborates primarily with other transcriptional factors or coregulators to maintain pluripotency. However, how Oct4 exerts its function is still unclear. Here, we show that the Oct4 linker interface mediates competing yet balanced Oct4 protein interactions that are crucial for maintaining pluripotency. Oct4 linker mutant embryonic stem cells (ESCs) show decreased expression of self-renewal genes and increased expression of differentiation genes, resulting in impaired ESC self-renewal and early embryonic development. The linker mutation interrupts the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 is decreased. In contrast, interactions between Oct4 and Cbx1, Ctr9, and Cdc73 are increased, disrupting the epigenetic state of ESCs. Control of the expression level of Klf5, Cbx1, or Cdc73 rebalances the Oct4 interactome and rescues the pluripotency of linker mutant ESCs, indicating that such factors interact with Oct4 competitively. Thus, we provide previously unidentified molecular insights into how Oct4 maintains pluripotency.

SUBMITTER: Han D

PROVIDER: S-EPMC8849292 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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A balanced Oct4 interactome is crucial for maintaining pluripotency.

Han Dong D Wu Guangming G Chen Rui R Drexler Hannes C A HCA MacCarthy Caitlin M CM Kim Kee-Pyo KP Adachi Kenjiro K Gerovska Daniela D Mavrommatis Lampros L Bedzhov Ivan I Araúzo-Bravo Marcos J MJ Schöler Hans R HR

Science advances 20220216 7

Oct4 collaborates primarily with other transcriptional factors or coregulators to maintain pluripotency. However, how Oct4 exerts its function is still unclear. Here, we show that the Oct4 linker interface mediates competing yet balanced Oct4 protein interactions that are crucial for maintaining pluripotency. Oct4 linker mutant embryonic stem cells (ESCs) show decreased expression of self-renewal genes and increased expression of differentiation genes, resulting in impaired ESC self-renewal and ...[more]

PMID: 35171666

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Project description:Oct4 is considered a master transcription factor for pluripotent cell self-renewal and embryo development. It primarily collaborates with other transcriptional factors or coregulators to maintain pluripotency. However, it is still unclear how Oct4 interacts with its partners. Here, we show that the Oct4 linker interface mediates competing and balanced Oct4 protein interactions which are crucial for maintaining pluripotency. Linker mutant ESCs maintain the key pluripotency genes expression, but show decreased expression of self-renewal genes and increased expression of differentiation genes which result in impaired ESCs self-renewal and early embryonic lethality. Linker mutation dose not affect Oct4 genomic binding and transactivation potential, but breaks the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 was decreased, but Oct4-Cbx1, Ctr9, Cdc73 interactions were increased which disrupt the epigenetic state of ESCs. Overexpression of Klf5 or knockdown Cbx1, Cdc73 rescue the cellular phenotype of linker mutant ESCs by rebalancing Oct4 interactome indicating that different partners interact with Oct4 competitively. Thus, by showing how Oct4 interacts with different partners, we provide novel molecular insights to explain how Oct4 contributes to the maintenance of pluripotency. RNA samples to be analyzed by qRT-PCR and microarrays were prepared using RNeasy Mini Kit (QIAGEN) with on-column DNA digestion. Complementary DNA for qRT-PCR was synthesized with the M-MLV Reverse Transcriptase (Affymetrix). Transcript levels were determined using ABI PRISM Sequence Detection System 7900 and gene expression was normalized to the housekeeping gene Gapdh. 300 ng of total RNA per sample was used as input using a linear amplification protocol (Ambion) which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer’s instructions. After washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina). The bead intensities were mapped to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model (Irizarry et al., 2003). Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in MATLAB. Hierarchical clusters of genes and samples were performed with the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method.

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A balanced Oct4 interactome is crucial for maintaining pluripotency.

Publications

A balanced Oct4 interactome is crucial for maintaining pluripotency.

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