Dataset Information

Temporal Control of the TGF-? Signaling Network by Mouse ESC MicroRNA Targets of Different Affinities.

ABSTRACT: Although microRNAs (miRNAs) function in the control of embryonic stem cell (ESC) pluripotency, a systems-level understanding is still being developed. Through the analysis of progressive Argonaute (Ago)-miRNA depletion and rescue, including stable Ago knockout mouse ESCs, we uncover transforming growth factor beta (TGF-?) pathway activation as a direct and early response to ESC miRNA reduction. Mechanistically, we link the derepression of weaker miRNA targets, including TGF-? receptor 1 (Tgfbr1), to the sensitive TGF-? pathway activation. In contrast, stronger miRNA targets impart a more robust repression, which dampens concurrent transcriptional activation. We verify such dampened induction for TGF-? antagonist Lefty. We find that TGF-? pathway activation contributes to the G1 cell-cycle accumulation of miRNA-deficient ESCs. We propose that miRNA target affinity is a determinant of the temporal response to miRNA changes, which enables the coordination of gene network responses.

SUBMITTER: Kelly TJ

PROVIDER: S-EPMC6939994 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Temporal Control of the TGF-β Signaling Network by Mouse ESC MicroRNA Targets of Different Affinities.

Kelly Timothy J TJ Brümmer Anneke A Hooshdaran Nima N Tariveranmoshabad Mito M Zamudio Jesse R JR

Cell reports 20191101 9

Although microRNAs (miRNAs) function in the control of embryonic stem cell (ESC) pluripotency, a systems-level understanding is still being developed. Through the analysis of progressive Argonaute (Ago)-miRNA depletion and rescue, including stable Ago knockout mouse ESCs, we uncover transforming growth factor beta (TGF-β) pathway activation as a direct and early response to ESC miRNA reduction. Mechanistically, we link the derepression of weaker miRNA targets, including TGF-β receptor 1 (Tgfbr1) ...[more]

PMID: 31775039

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Project description:BackgroundREST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network.MethodsEmploying biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature.ResultsWe show that Rest+/- and Rest-/- AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/- and Rest-/- AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest-/- AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST's role in ESC pluripotency.ConclusionsREST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.

Project description:It is well known that microRNAs play a very important role in regulating reprogramming, pluripotency and cell fate decisions. Porcine induced pluripotent stem cells (piPSCs) are now available for studying the pluripotent regulation network in pigs. Two types of piPSCs have been derived from human and mouse embryonic stem cell (ESC) culture conditions: hpiPSCs and mpiPSCs, respectively. The hpiPSCs were morphologically similar to human ESCs, and the mpiPSCs resembled mouse ESCs. However, our current understanding of the role of microRNAs in the development of piPSCs is still very limited. Here, we performed small RNA sequencing to profile the miRNA expression in porcine fibroblasts (pEFs), hpiPSCs and mpiPSCs. There were 22 differential expressed (DE) miRNAs down-regulated in both types of piPSCs compared with pEFs, such as ssc-miR-145-5p and ssc-miR-98. There were 27 DE miRNAs up-regulated in both types of piPSCs compared with pEFs. Among these up-regulated DE miRNAs in piPSCs, ssc-miR-217, ssc-miR-216, ssc-miR-142-5p, ssc-miR-182, ssc-miR-183 and ssc-miR-96-5p have much higher expression levels in mpiPSCs, while ssc-miR-106a, ssc-miR-363, ssc-miR-146b, ssc-miR-195, ssc-miR-497, ssc-miR-935 and ssc-miR-20b highly expressed in hpiPSCs. Quantitative stem-loop RT-PCR was performed to confirm selected DE miRNAs expression levels. The results were consistent with small RNA sequencing. Different expression patterns were observed for key miRNA clusters, such as the miR-17-92 cluster, the let-7 family, the miR-106a-363 cluster and the miR-182-183 cluster, in the mpiPSCs and hpiPSCs. Novel miRNAs were also predicted in this study, including a putative porcine miR-302 cluster: ssc_38503, ssc_38503 and ssc_38501 (which resemble human miR-302a and miR-302b) found in both types of piPSCs. The miR-106a-363 cluster and putative miR-302 cluster increased the reprogramming efficiency of pEFs. The study revealed significant differences in the miRNA signatures of hpiPSCs and mpiPSCs under different pluripotent states that were derived from different culture conditions. These differentially expressed miRNAs may play important roles in pluripotent regulation in pigs, and this information will facilitate the understanding of the mechanism of pluripotency in pigs.

Dataset Information

Temporal Control of the TGF-? Signaling Network by Mouse ESC MicroRNA Targets of Different Affinities.

Publications

Temporal Control of the TGF-β Signaling Network by Mouse ESC MicroRNA Targets of Different Affinities.

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