Dataset Information

Endogenous OCT4 interactome in hESC

ABSTRACT: In two widely-cited OCT4-protein interactome papers, Ding et al. (Ding et al., 2012) used murine ESC nuclear extract to identify OCT4 interactome in nucleus and Pardo et al. (Pardo et al., 2010) used whole cell lysate to identify OCT4 interactome in murine ESCs. In both papers, Benzonase, a genetically engineered endonuclease was employed to fragment genome DNA which significantly improved the enrichment efficiency of nuclear TFs. Since our focus was on cytosolic pool of OCT4, we used mild conditions for cell lysis and modified the Pardo’s method by omitting the addition of any nucleases to the lysis buffer. Such conditions not only retained the protein/RNA interactions, but also enabled precipitation of the nuclear TFs with genome DNA, and hence a relative enrichment of the cytosolic proteins and other soluble nuclear proteins (such as splicing factors) in the supernatant, and therefore allowed us to minimize the interference of the TF roles of OCT4 and to focus more on its novel RBP roles.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Bo Kang

LAB HEAD: Ying-Jie Wang

PROVIDER: PXD033840 | Pride | 2025-05-06

REPOSITORIES: Pride

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Publications

OCT4 translationally promotes AKT signaling as an RNA-binding protein in stressed pluripotent stem cells.

Chen Wenjie W Chen Xinyu X Chen Cheng C She Shiqi S Li Xia X Shan Lina L Zhang Xiaobing X Dan Songsong S Wang Yisha Y Zhou Yan-Wen YW Cao Qingyi Q Wang Wenxin W Hu Jianwen J Wei Yaxun Y Xue Yaqiang Y Zhang Yi Y Zhang Songying S Wang Ying-Jie YJ Kang Bo B

Stem cell research & therapy 20250223 1

<h4>Background</h4>Despite numerous studies addressing the molecular mechanisms by which pluripotent stem cells (PSCs) maintain self-renewal and pluripotency under normal culture conditions, the fundamental question of how PSCs manage to survive stressful conditions remains largely unresolved. Post-transcriptional/translational regulation emerges to be vital for PSCs, but how PSCs coordinate and balance their survival and differentiation at translational level under extrinsic and intrinsic stres ...[more]

PMID: 39988663

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Project description:Oct4 is an essential regulator of embryonic stem (ES) cell pluripotency in vivo and in vitro, as well as a key mediator of the reprogramming of somatic cells to form induced pluriopotent stem (iPS) cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homologue of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported ES cell self-renewal in vitro at lower concentrations than required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF-dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes. Two independent clones from each cell line were used as biological replications. Cy3-CTP-labelled sample targets were prepared from 2.5 ?g of total RNA using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP-labelled reference target was produced from 2.5 ?g of Stratagene Universal Mouse Reference RNA. Purified target RNA were hybridized to the NIA Mouse 44K Microarray v3.0 (whole genome 60-mer oligo arrays, Agilent Technology, design ID 015087) (Carter et al., 2005) according to the manufacturer's protocol (Two-Colour Microarray-Based Gene Expression Analysis Protocol, Version 5.0.1). Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% PMT for the Cy3 channel and 10% PMT for the Cy5 channel with a scan resolution of 5µm. Data was analyzed using NIA Array Analysis (Sharov et al., 2005) using standard statistical conditions (FDR<0.05, 2-fold expression levels change) to unveil genes with changes in expression levels between the samples and controls.

			Action	DRS
	Control_IP.raw	Raw
	Control_IP_txt.rar	Other
	OCT4_IP.raw	Raw
	OCT4_IP_txt.rar	Other
	TAP_OCT4_IP.raw	Raw