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Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor.

ABSTRACT: Self-renewal of pluripotent mouse embryonic stem (ES) cells is sustained by the cytokine leukaemia inhibitory factor (LIF) acting through the transcription factor Stat3. Several targets of Stat3 have previously been identified, most notably the reprogramming factor Klf4. However, such factors are neither required nor sufficient for the potent effect of LIF. We took advantage of Stat3 null ES cells to confirm that Stat3 mediates the self-renewal response to LIF. Through comparative transcriptome analysis intersected with genome location data, we arrived at a set of candidate transcription factor effectors. Among these, Tfcp2l1 (also known as Crtr-1) was most abundant. Constitutive expression of Tfcp2l1 at levels similar to those induced by LIF effectively substituted for LIF or Stat3 in sustaining clonal self-renewal and pluripotency. Conversely, knockdown of Tfcp2l1 profoundly compromised responsiveness to LIF. We further found that Tfcp2l1 is both necessary and sufficient to direct molecular reprogramming of post-implantation epiblast stem cells to naïve pluripotency. These results establish Tfcp2l1 as the principal bridge between LIF/Stat3 input and the transcription factor core of naïve pluripotency.

SUBMITTER: Martello G

PROVIDER: S-EPMC3791366 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor.

Martello Graziano G Bertone Paul P Smith Austin A

The EMBO journal 20130813 19

Self-renewal of pluripotent mouse embryonic stem (ES) cells is sustained by the cytokine leukaemia inhibitory factor (LIF) acting through the transcription factor Stat3. Several targets of Stat3 have previously been identified, most notably the reprogramming factor Klf4. However, such factors are neither required nor sufficient for the potent effect of LIF. We took advantage of Stat3 null ES cells to confirm that Stat3 mediates the self-renewal response to LIF. Through comparative transcriptome ...[more]

PMID: 23942233

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Project description:Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5' ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5'-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5'-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal 'enhancer' located between -3200 and -1200.

Dataset Information

Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor.

Publications

Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor.

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