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McGehee, J.

Publications and source records attributed to McGehee, J..

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Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that result in either nuclear export or degradation of Dorsal when exposed to blue light. Nuclear export of Dorsal results in loss of expression for the high threshold, ventrally-expressed target gene snail (sna) but retention of the low threshold, laterally-expressed target gene short-gastrulation (sog). In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels than when Dorsal is exported from the nucleus. To elucidate how nuclear export results in loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found it reenters the nucleus even under conditions of blue-light when export is favored. The associated kinetics of being imported and exported continuously are likely responsible for loss of sna but, alternatively, can support sog. Collectively, our results show that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention. SUMMARY STATEMENTThis study shows how optogenetic tools can be used to determine how a transcription factors levels and nuclear retention impact a dynamic patterning process.

developmental biology↗

High levels of Dorsal transcription factor downregulate, not promote, snail expression by regulating enhancer action

In Drosophila embryos, genes expressed along the dorsal-ventral axis are responsive to concentration of the Dorsal (Dl) transcription factor, which varies in space; however, levels of this morphogen also build over time. Since expression of high-threshold Dl target genes such as snail (sna) is supported before Dl levels peak, it is unclear what role increasing levels have if any. Here we investigated action of two enhancers that control sna expression in embryos, demonstrating using genome editing that Dl binding sites within one enhancer located promoter proximally, sna.prox, can limit the ability of the other distally-located enhancer, sna.dis, to increase sna levels. In addition, MS2-MCP live imaging was used to study sna transcription rate in wildtype, dl heterozygote, and a background in which a photo-sensitive degron is fused to Dl (dl-BLID). The results demonstrate that, when Dl levels are high, Dl acts through sna.prox to limit the activity of sna.dis and thereby influence sna transcription rate. In contrast, when Dl levels are kept low using dl-BLID, sna.prox positively influences sna transcription rate. Collectively, our data support the view that Dls effect on gene expression changes over time, switching from promoting sna expression at low concentration to dampening sna expression at high concentration by regulating enhancer interactions. We propose this differential action of the Dl morphogen is likely supported by occupancy of this factor first to high and then low affinity binding sites over time as Dl levels rise to coordinate action of these two co-acting enhancers. Significance statementA gradient of the maternal transcription factor Dorsal is important for establishing spatial expression of target genes along the dorsal-ventral axis of Drosophila embryos. Dorsal levels are also dynamic as nuclear concentration builds in time. Surprisingly, expression of high-threshold target genes such as snail is supported before levels peak, raising the question why levels continue to build. Our data support the view that peak Dorsal levels act to preferentially support activity of one enhancer over another to effectively decrease snail expression. In addition, while the morphogen Dorsal acts early to support gene expression, later it effectively acts as a damper to limit gene expression. Our results suggest other morphogens also have effects on gene expression that change over time.

developmental biology↗