Unknown,Transcriptomics,Genomics,Proteomics

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ChIP Seq of Oct4, Nanog, H3K27me3 in Oct4+/+ and Oct4 +/- mES cells

ABSTRACT: Embryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centred on the transcription factors Oct4 and Nanog. ESCs fluctuate between states of high and low Nanog expression that direct efficient or inefficient self-renewal. To date, robust self-renewing ESC states have only been attained by chemical inhibition of signalling pathways or enforced transgene expression. Here we show that ESCs expressing a reduced range of Oct4 concentrations, typified by Oct4 heterozygous ESCs exhibit stable robust pluripotency. Despite this reduced Oct4 concentration range, this state is characterised by increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency and delayed differentiation kinetics. In this state, ESCs exhibit increased wnt expression, enhanced LIF-sensitivity, non-responsiveness to FGF signalling and can clonally maintain pluripotency without BMP but remain dependent upon LIF. Robust pluripotency is destabilised either by alteration of the Oct4 level or by removal of LIF. Our findings suggest that robust pluripotency originates from cells with a reduced Oct4 protein concentration and that the wild-type Oct4 range enables effective differentiation.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: Jonathan Goke

PROVIDER: E-MTAB-1617 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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ChIP Seq of Oct4, Nanog, H3K27me3 in Oct4+/+ and Oct4 +/- mES cells

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