UCSF

Transcription factors (TFs) are important messengers in the information cascade necessary for cells to respond to their environment. In both Saccharomyces cerevisiae and mammalian cells, the limited set of TFs must use a variety of strategies to convey complex information about the cellular environment to the many genes that they regulate downstream. These strategies include TF identity, concentration, and combinations of TFs. A subset of TFs also modulate their subcellular localization with distinct dynamic patterns in response to environmental stimuli; this dynamic information may also be used to regulate downstream genes.

Previous studies have attempted to elucidate the effect of spatiotemporal dynamics on downstream genes by using environmental inputs to induce localization changes. Using these stimuli, these studies have shown that TF spatiotemporal dynamics likely transmit information which is then decoded by downstream genes. An important limitation of these works is that the inputs used modulate not only TF spatiotemporal dynamics, but also other factors, like TF concentration and activation of other TFs and transcriptional regulators. In this study, we develop an optogenetic tool, termed CLASP, which sidesteps these limitations to precisely quantify the effect of TF spatiotemporal dynamics on downstream genes.

Specifically, we used CLASP in S. cerevisiae to control the localization of multiple TFs, and find that canonical downstream genes activate more efficiently in response to constant TF nuclear localization than to short pulses of TF localization. In contrast to these data, we focus on Crz1, a pulsatile TF, and find that many of its downstream genes activate more efficiently in response to short, pulsed TF inputs. We then use computational modeling to interrogate the promoter architecture which could yield either outcome, and find that simple promoters respond similarly to many of the downstream genes of Crz1, with a higher output in response to pulsed TF inputs. Surprisingly, a more complex model is needed to explain the phenotype seen for one Crz1 gene and the genes profiled for other TFs, where the gene is more efficiently induced by constant inputs.

In the second study, we extend CLASP to mammalian cells, and show that it is also capable of regulating localization of multiple TFs across multiple cell lines. We focus on a single TF, RelA, in mouse fibroblasts, and perform RNA-seq to measure the effect of its spatiotemporal dynamics on all genes. Additionally, we measure the effect of TNF-alpha, a common environmental stimulus used to modulate RelA spatiotemporal dynamics. First, we find that TNF-alpha regulates many genes, even when RelA is not expressed in cells, and that this regulation is extremely different from that caused by RelA-CLASP. Secondly, we find that RelA-CLASP activates many downstream genes, and that these genes can respond differently to pulsed and constant inputs. Critically, this is a novel demonstration of the ability of RelA to regulate downstream genes using only translocation, without post-translational modifications or activation of other transcriptional regulators. Using a simple model of gene expression to simulate a subset of genes in response to constant inputs, we qualitatively predict the response to pulsed inputs for some genes. However, more complex models are needed to explain others.

Together, these studies provide direct demonstration across eukaryotes of the importance of spatiotemporal TF dynamics in regulating gene expression. Additionally, these studies demonstrate an integrated approach of engineering novel tools, measuring dynamic gene expression, and modeling of promoter activation to elucidate how genes respond to complex inputs.