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Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

ABSTRACT: Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

SUBMITTER: Wilson MZ

PROVIDER: S-EPMC5591080 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

Wilson Maxwell Z MZ Ravindran Pavithran T PT Lim Wendell A WA Toettcher Jared E JE

Molecular cell 20170817 5

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes ...[more]

PMID: 28826673

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Project description:In prior work we developed an optogenetic system for delivering highly precise, time-varying inputs to Ras, termed OptoSOS (Toettcher et al., 2013). This system relies on a membrane-targeted photoswitchable protein (Phy-CAAX) and a cytoplasmic Ras activator (PIF-SOScat) whose localization to the membrane can be controlled with light. In this system, Phy/PIF heterodimerization can be triggered on and off by exposure to 650 and 750 nm light, respectively. We found that this system could be used to deliver highly precise levels and dynamics of Ras/Erk signaling both in vitro and in vivo. Here, we aimed to globally assess the transcriptional response to light-activated Ras and compare it to that induced by growth factor stimulation. We stimulated NIH3T3 OptoSOS cells with either constant activating red light or PDGF and measured transcriptional responses by RNAseq. Total mRNA was collected after 0, 30, 60 and 120 minutes and used to track the dynamics of transcript abundance in both conditions. Genes were defined as upregulated if they satisfied two criteria: (i) induced at least three-fold over unstimulated cells, and (ii) induced at least two consecutive timepoints. By these criteria we detected 118 genes that were upregulated within 2 h by either PDGF or light stimulation, a comparable number of Ras-responsive genes to that found in previous studies. We found that both PDGF and light induced nearly identical profiles of gene expression, with 100/118 genes induced by PDGF and 110/118 induced by light. At each time point we found excellent agreement between the levels of gene induction in response to both stimuli. This agreement also extended to response dynamics. where hierarchical clustering revealed three classes of dynamic response: an early response peaking within 30 min, an intermediate response peaking at ~1 h, and a late response where gene expression gradually increased over the full 2 h timecourse. In all three classes, we found that light and PDGF led to highly similar expression changes over time. We thus concluded that sole stimulation of the Ras/Erk pathway by light was sufficient to recapitulate at least the first two hours of the PDGF-induced transcriptional response.

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Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

Publications

Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

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