Project description: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.
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:Genomic imprinting is regulated by parental-specific epigenetic marks that differentiate between maternal and paternal chromosomes. Despite identical DNA sequence, the presence or absence of DNA methylation leads to the establishment of two distinct epigenetic states at Imprinting Control Regions (ICR). Here we combine targeted epigenome engineering to generate ectopic loci in the mouse embryonic stem cell genome that recapitulate the epigenetic properties of ICRs. We describe these ectopic ICRs as strong cis-regulatory sequences that can adopt and memorise one of two opposing epigenetic states, dependent of pre-imposed DNA methylation. This bistability is unique to ICRs and enabled us to systematically study the genetic and epigenetic determinants required for creating and maintaining the observed states. Through sequence manipulation we show that the ICR DNA sequence confers autonomy of ICRs and is required for creating epigenetic bistability. Genetic screens using DNA-methylation-sensitive reporters identify key components involved in regulating maintenance of epigenetic states. Besides DNMT1, UHRF1 and ZFP57, we identify novel factors that prevent switching between methylated and unmethylated states and validate two of these candidates, ATF7IP and ZMYM2, to be important for epigenetic memory at ICRs. In summary we show that the DNA sequence of ICRs provides the prerequisite for establishment of two distinct epigenetic states, while DNA and histone modifications ensure their stable propagation.
Project description:Genomic imprinting is regulated by parental-specific epigenetic marks that differentiate between maternal and paternal chromosomes. Despite identical DNA sequence, the presence or absence of DNA methylation leads to the establishment of two distinct epigenetic states at Imprinting Control Regions (ICR). Here we combine targeted epigenome engineering to generate ectopic loci in the mouse embryonic stem cell genome that recapitulate the epigenetic properties of ICRs. We describe these ectopic ICRs as strong cis-regulatory sequences that can adopt and memorise one of two opposing epigenetic states, dependent of pre-imposed DNA methylation. This bistability is unique to ICRs and enabled us to systematically study the genetic and epigenetic determinants required for creating and maintaining the observed states. Through sequence manipulation we show that the ICR DNA sequence confers autonomy of ICRs and is required for creating epigenetic bistability. Genetic screens using DNA-methylation-sensitive reporters identify key components involved in regulating maintenance of epigenetic states. Besides DNMT1, UHRF1 and ZFP57, we identify novel factors that prevent switching between methylated and unmethylated states and validate two of these candidates, ATF7IP and ZMYM2, to be important for epigenetic memory at ICRs. In summary we show that the DNA sequence of ICRs provides the prerequisite for establishment of two distinct epigenetic states, while DNA and histone modifications ensure their stable propagation.
Project description:Genomic imprinting is regulated by parental-specific epigenetic marks that differentiate between maternal and paternal chromosomes. Despite identical DNA sequence, the presence or absence of DNA methylation leads to the establishment of two distinct epigenetic states at Imprinting Control Regions (ICR). Here we combine targeted epigenome engineering to generate ectopic loci in the mouse embryonic stem cell genome that recapitulate the epigenetic properties of ICRs. We describe these ectopic ICRs as strong cis-regulatory sequences that can adopt and memorise one of two opposing epigenetic states, dependent of pre-imposed DNA methylation. This bistability is unique to ICRs and enabled us to systematically study the genetic and epigenetic determinants required for creating and maintaining the observed states. Through sequence manipulation we show that the ICR DNA sequence confers autonomy of ICRs and is required for creating epigenetic bistability. Genetic screens using DNA-methylation-sensitive reporters identify key components involved in regulating maintenance of epigenetic states. Besides DNMT1, UHRF1 and ZFP57, we identify novel factors that prevent switching between methylated and unmethylated states and validate two of these candidates, ATF7IP and ZMYM2, to be important for epigenetic memory at ICRs. In summary we show that the DNA sequence of ICRs provides the prerequisite for establishment of two distinct epigenetic states, while DNA and histone modifications ensure their stable propagation.
Project description:Ribosome profiling analyses showed thousands of translated open reading frames in a cell encoding noncanonical peptides. To examine their functional roles, we stably overexpressed two noncanonical peptides (uSLC35A4 and iPGRMC1) in MCF-7 cells. And then we performed RNA-seq to examine differential gene expression upon gene overexpression.
Project description:Synthetic gene circuits are often engineered by considering the host cell as an invariable 'chassis'. Circuit activation, however, may modulate host physiology, which in turn can substantially impact circuit behavior. We illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in the bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear dilution of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further experimental measurements. Our results reveal a new mechanism of generating bistability and underscore the need to account for host physiology modulation when engineering gene circuits.
Project description:Faithful maintenance of genomic imprinting is essential for mammalian development. While germline DNA methylation-dependent (canonical) imprinting is relatively stable during development, the recently discovered oocyte-derived H3K27me3-mediated noncanonical imprinting is mostly transient in early embryos with only a few genes maintain imprinted expression in the extraembryonic lineage. How these few noncanonical imprinted genes maintain their extraembryonic-specific imprinting is unknown. Here we report that maintenance of extraembryonic-specific noncanonical imprinting requires maternal allele-specific de novo DNA methylation (secondary differentially methylation regions; DMRs) at implantation. The secondary DMRs are located at the gene promoters with paternal allele-specific H3K4me3 preformed during preimplantation development. Importantly, genetic ablation of Eed and DNA methyltransferases revealed that both maternal H3K27me3 and zygotic Dnmt3a/3b are required for establishing secondary DMRs and for maintaining noncanonical imprinting. Thus, our study not only reveals the mechanism underlying maintenance of noncanonical imprinting, but also sheds light on how histone modifications in oocytes and preimplantation embryos may shape the secondary DMRs in post-implantation embryos.