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Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. Here, we investigate how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein, despite expressing both proteins with pulsatile dynamics. A rapid and sustained twofold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a twofold shift in the ratio. A biophysical model based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how twofold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The biochemical regulatory network downstream of receptor tyrosine kinase (RTK) signaling is controlled by two opposing ETS family members: the transcriptional activator Pointed (Pnt) and the transcriptional repressor Yan. A bistable switch model has been invoked to explain how pathway activation can drive differentiation by shifting the system from a high-Yan/low-Pnt activity state to a low-Yan/high-Pnt activity state. Although the model explains yan and pnt loss-of-function phenotypes in several different cell types, how Yan and Pointed protein expression dynamics contribute to these and other developmental transitions remains poorly understood. Toward this goal we have used a functional GFP-tagged Pnt transgene (Pnt-GFP) to perform a comparative study of Yan and Pnt protein expression throughout Drosophila development. Consistent with the prevailing model of the Pnt-Yan network, we found numerous instances where Pnt-GFP and Yan adopt a mutually exclusive pattern of expression. However we also observed many examples of co-expression. While some co-expression occurred in cells where RTK signaling is presumed low, other co-expression occurred in cells with high RTK signaling. The instances of co-expressed Yan and Pnt-GFP in tissues with high RTK signaling cannot be explained by the current model, and thus they provide important contexts for future investigation of how context-specific differences in RTK signaling, network topology, or responsiveness to other signaling inputs, affect the transcriptional response., (© 2013 Published by Elsevier Inc.)

Gene expression has to withstand stochastic, environmental, and genomic perturbations. For example, in the latter case, 0.5%-1% of the human genome is typically variable between any two unrelated individuals. Such diversity might create problematic variability in the activity of gene regulatory networks and, ultimately, in cell behaviors. Using multigenerational selection experiments, we find that for the Drosophila proneural network, the effect of genomic diversity is dampened by miR-9a-mediated regulation of senseless expression. Reducing miR-9a regulation of the Senseless transcription factor frees the genomic landscape to exert greater phenotypic influence. Whole-genome sequencing identified genomic loci that potentially exert such effects. A larger set of sequence variants, including variants within proneural network genes, exhibits these characteristics when miR-9a concentration is reduced. These findings reveal that microRNA-target interactions may be a key mechanism by which the impact of genomic diversity on cell behavior is dampened., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In Drosophila, Notch and Egfr signaling regulate the determination of many cell types, and yet how these common signals generate cell-specific transcription is not well understood. In the compound eye, prospero (pros) is transcribed specifically in R7 photoreceptors and cone cells. We show that the transcription of pros is activated by two visual-specific transcription selectors, Glass and Sine Oculis, that bind to an enhancer and promote its activation. Together with the pre-patterning transcription factor Lozenge, these factors work in a highly combinatorial manner, such that cells missing any one factor transcribe pros only weakly, if at all. However, the factors are not sufficient to activate the enhancer because of an additional requirement for both Notch and Egfr signals. The loss of Notch signaling produces a ;salt and pepper' effect, with some cells expressing near-normal levels and others expressing no detectable pros at all; thus, the signaling loss does not produce a uniformly reduced level of transcription activity in cells. This suggests a probabilistic mechanism, in which Notch signals influence the likelihood that the enhancer is inactive or fully active in any given cell. The activity level, therefore, is dictated by the proper combination of highly cooperative selector and pre-pattern factors present in the cell.

Lola is a transcription repressor that regulates axon guidance in the developing embryonic nervous system of Drosophila. Here, we show that Lola regulates two binary cell fate decisions guided by Notch inductive signaling in the developing eye: the R3-R4 and the R7-cone cell fate choices. Lola is required cell-autonomously in R3 for its specification, and Lola transforms R4 into R3 if overexpressed. Lola also promotes R7 fate at the expense of cone cell fate. Lola antagonizes Notch-dependent gene expression and Notch-dependent fate transformation. Expression analysis shows that Lola is constitutively present in all photoreceptors and cone cells. We propose that when a precursor cell receives a weak Notch inductive signal, it is not sufficiently strong to overcome the constitutive repression of target gene transcription by Lola. A precursor that receives a strong Notch inductive signal expresses target genes despite a constitutive repression by Lola. The predicted consequence of this mechanism is to sharpen a cell's responsiveness to Notch signaling by creating a threshold.

Yan is an ETS-domain transcription factor responsible for maintaining Drosophila eye cells in a multipotent state. Yan is at the core of a regulatory network that determines the time and place in which cells transit from multipotency to one of several differentiated lineages. Using a fluorescent reporter for Yan expression, we observed a biphasic distribution of Yan in multipotent cells, with a rapid inductive phase and slow decay phase. Transitions to various differentiated states occurred over the course of this dynamic process, suggesting that Yan expression level does not strongly determine cell potential. Consistent with this conclusion, perturbing Yan expression by varying gene dosage had no effect on cell fate transitions. However, we observed that as cells transited to differentiation, Yan expression became highly heterogeneous and this heterogeneity was transient. Signals received via the EGF Receptor were necessary for the transience in Yan noise since genetic loss caused sustained noise. Since these signals are essential for eye cells to differentiate, we suggest that dynamic heterogeneity of Yan is a necessary element of the transition process, and cell states are stabilized through noise reduction. [ABSTRACT FROM AUTHOR]

ETS family transcription factors are evolutionarily conserved downstream effectors of Ras/MAPK signaling with critical roles in development and cancer. In Drosophila, the ETS repressor Yan regulates cell proliferation and differentiation in a variety of tissues; however, the mechanisms of Yan-mediated repression are not well understood and only a few direct target genes have been identified. Yan, like its human ortholog TEL1, self-associates through an N-terminal sterile α-motif (SAM), leading to speculation that Yan/TEL1 polymers may spread along chromatin to form large repressive domains. To test this hypothesis, we created a monomeric form of Yan by recombineering a point mutation that blocks SAM-mediated self-association into the yan genomic locus and compared its genome-wide chromatin occupancy profile to that of endogenous wild-type Yan. Consistent with the spreading model predictions, wild-type Yan-bound regions span multiple kilobases. Extended occupancy patterns appear most prominent at genes encoding crucial developmental regulators and signaling molecules and are highly conserved between Drosophila melanogaster and D. virilis, suggesting functional relevance. Surprisingly, although occupancy is reduced, the Yan monomer still makes extensive multikilobase contacts with chromatin, with an overall pattern similar to that of wild-type Yan. Despite its near-normal chromatin recruitment, the repressive function of the Yan monomer is significantly impaired, as evidenced by elevated target gene expression and failure to rescue a yan null mutation. Together our data argue that SAM-mediated polymerization contributes to the functional output of the active Yan repressive complexes that assemble across extended stretches of chromatin, but does not directly mediate recruitment to DNA or chromatin spreading. [ABSTRACT FROM AUTHOR]