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Noncanonical post-transcriptional feedback between microRNA and messenger RNA enables bistability and governs robust cell fate decisions

ABSTRACT: Positive feedback driven by transcriptional regulation has long been considered a key mechanism underlying cell lineage segregation during embryogenesis. Using the developing spinal cord as a paradigm, we found that canonical, transcription-driven feedback cannot explain robust lineage segregation of motor neuron subtypes marked by two cardinal factors, Hoxa5 and Hoxc8. We propose a feedback mechanism involving elementary microRNA-mRNA reaction circuits that differ from known feedback loop-like structures. Strikingly, we show that a wide range of biologically-plausible post-transcriptional regulatory parameters are sufficient to generate bistable switches, a hallmark of positive feedback. Through mathematical analysis, we explain intuitively the hidden source of this feedback. Using embryonic stem cell differentiation and mouse genetics, we corroborate that microRNA-mRNA circuits govern tissue boundaries and hysteresis upon motor neuron differentiation with respect to transient morphogen signals. Our findings reveal a previously underappreciated feedback mechanism that may have widespread functions in cell fate decisions and tissue patterning.

ORGANISM(S): Mus musculus

PROVIDER: GSE156023 | GEO | 2021/04/14

REPOSITORIES: GEO

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Project description:(I am not the first author of the paper who contributed to the experimental data, I did the modeling) Bistable switches and oscillators have long been considered key mechanisms underlying cell fate decisions and pattern formation in biology. Previous studies of these dynamical behaviors focused on regulatory networks with intuitive feedback loops. It was therefore unclear whether other common biochemical reactions can act as bistable switches or oscillators crucial for cellular and physiological dynamics. In this work, we used mass-action-based models to show that elementary production, degradation and binding reactions involving as few as two RNA species (e.g.an mRNA and a microRNA) can generate bistability and oscillation. We showed that both bistability and oscillation depend on cooperativity of two microRNA binding sites on the mRNA. We therefore termed our model the two-site mRNA-microRNA (MMI2) model. Remarkably, the network structure of the MMI2 model does not have any explicit feedback loop. We estimated that this simple reaction network is applicable to nearly half of human protein-coding genes. Using in vitro and in vivo experiments, we showed the function of a newly proposed MMI2-based switch in governing motor neuron lineage segregation in the spinal cord of mammalian embryos. Our findings reveal a previously underappreciated post-transcriptional mechanism that may have widespread functions in cell fate decisions, oscillatory cell dynamics and tissue patterning. Furthermore, our results challenge the long-standing idea of using intuitive feedback loops to explain bistability and oscillation. In addition to its significance in biology, the MMI2 model enables nontrivial mathematical analysis due to its simplicity. Using algebraic geometry and chemical reaction network theory, we obtained key conditions for bistability of the MMI2 model. These conditions include an inequality that reveals to a hidden feedback loop arising from regulated degradation. For these reasons, we expect that our model will not only provide useful insights into a wide range of problems in cell and developmental biology, but also enable new analytical approaches in systems biology and mathematical biology.

Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).

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Noncanonical post-transcriptional feedback between microRNA and messenger RNA enables bistability and governs robust cell fate decisions

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