Author Response
Reviewer #1 (Public Review):
[...] Based on these results, the authors support a model whereby kinetic regimes are encoded in the cis-regulatory sequences of a gene instead of imposed by an evolving trans-regulatory environment.
The question asked in this manuscript is important and the eve locus represents an ideal paradigm to address it in a quantitative manner. Most of the results are correctly interpreted and well-presented. However, the main conclusion pointing towards a potential "unified theory" of burst regulation during Drosophila embryogenesis should be nuanced or cross-validated.
We thank the reviewer for their careful and insightful assessment of our manuscript. The reviewer is right in that our claims should have been more nuanced. Indeed, our proposed unified strategy only concerns even-skipped transcription under the variable conditions that exist in ectopic and endogenous eve expression regions.
Our results and those of others suggest that different developmental genes follow unified—yet different—transcriptional control strategies whereby different combinations of bursting parameters are regulated to modulate gene expression: burst frequency and amplitude for eve (Berrocal et al., 2020), and burst frequency and duration for gap genes (Zoller et al., 2018). In light of the aforementioned works, we can only claim that our results suggest a unified strategy for eve, our case of study, as we observe that eve regulatory strategies are robust to disruption of enhancers and binding sites. In the Discussion section of our revised manuscript, we will emphasize that the bursting control strategy we uncovered for eve does not necessarily apply to other genes, and speculate in more detail that genes that employ the same strategy of transcriptional bursting may be grouped in families that share a common molecular mechanism of transcription.
In addition to the lack of novelty (some results concerning the fact that koff does not change along the A/P axis/the idea of a 'unified regime' were already obtained in Berrocal et al 2020),...
Unfortunately, we believe there is a misunderstanding in terms of what we construe as novelty in our work. In our previous work (Berrocal et al., 2020), we observed that the seven stripes of even-skipped (eve) expression modulate transcriptional bursting through the same strategy—bursting frequency and amplitude are controlled to yield various levels of mRNA synthesis, while burst duration remains constant. We reproduce that result in our paper, and do not claim any novelty. However, what was unclear is whether the observed eve bursting control strategy would only exist in the wild-type stripes, whose expression—we reasoned—is under strong selection due to the dramatic phenotypic consequences of eve transcription, or if eve transcriptional bursting would follow the same strategy under trans-regulatory environments that are not under selection to deliver specific spatiotemporal dynamics of eve expression. Our results—and here lies the novelty of our work—support the second scenario, and point to a model where eve bursting strategies do not result from adaptation of eve activity to specific trans-regulatory environments. Instead, we speculate that a molecular mechanism constrains eve bursting strategy whenever and wherever the gene is active. This is something that we could not have known from our first study in (Berrocal et al., 2020) and constitutes the main novelty of our paper. To put this in other words, the novelty of our work does not rest on the fact that both burst frequency and amplitude are modulated in the endogenous eve pattern, but that this modulation remains quantitatively indistinguishable when we focus on ectopic areas of expression. We will make this point clearer in the Introduction and Discussion section of our revised manuscript.
… note i) the limited manipulation of TF environment;...
We acknowledge that additional genetic manipulations would make it possible to further test the model. However, we hope that the reviewer will agree with us that the manipulations that we did perform are sufficient to provide evidence for common bursting strategies under the diverse trans-regulatory environments present in wild-type and ectopic regions of gene expression. In the Discussion section of our revised manuscript, we will elaborate further on the kind of genetic manipulations (e.g., probing transcriptional strategies that result from swapping promoters in the context of eve-MS2 BAC; or quantifying the impact on eve transcriptional control after performing optogenetic perturbations of transcription factors and/or chromatin remodelers) that could shed further light on the currently undefined molecular mechanism that constrains eve bursting strategies, as a mean to motivate future work.
… ii) the simplicity with which bursting is analyzed (only a two-state model is considered, and not cross-validated with an alternative approach than cpHMM) and…
Based on our previous work (Lammers et al., 2020), and as described in the SI Section of the current manuscript: Inference of Bursting Parameters, we selected a three-state model (OFF, ON1, ON2) under the following rationale: transcription of even-skipped in pre-gastrulating embryos occurs after DNA replication, and promoters on both sister chromatids remain paired. Most of the time these paired loci cannot be resolved independently using conventional microscopy. As a result, when we image an MS2 spot, we are actually measuring the transcriptional dynamics of two promoters. Thus, each MS2-fluorescent spot may result from none (OFF), one (ON1) or two (ON2) sister promoters being in the active state. Following our previous work, we analyzed our data assuming the three-state model (OFF, ON1, ON2), and then, for ease of presentation, aggregated ON1 and ON2 into an effective single ON state. As for the lack of an alternative model, we chose the simplest model compatible with our data and our current understanding of transcription at the eve locus. With this in mind, we do not rule out the possibility that more complex processes—that are not captured by our model—shape MS2 fluorescence signals. For example, promoters may display more than two states of activity. However, as shown in (Lammers et al., 2020 - SI Section: G. cpHMM inference sensitivities), model selection schemes and cross-validation do not give consistent results on which model is more favorable; and for the time being, there is not a readily available alternative to HMM for inference of promoter states from MS2 signal. For example, orthogonal approaches to quantify transcriptional bursting, such as smFISH, are largely blind to temporal dynamics. As a result, we choose to entertain the simplest two-state model for each sister promoter. We appreciate these observations, as they point out the need of devoting a section in the supplemental material of our revised manuscript to clarify the motivations behind model selection.
… iii) the lack of comparisons with published work.
We thank the reviewer for pointing this out. In the current discussion of our manuscript, we compare our findings to recent articles that have addressed the question of the origin of bursting control strategies in Drosophila embryos (Pimmett et al., 2021; Yokoshi et al., 2022; Zoller et al., 2018). Nevertheless, we acknowledge that we failed to include references that are relevant to our study. Thus, our revised Discussion section must include recent results by (Syed et al., 2023), which showed that the disruption of Dorsal binding sites on the snail minimal distal enhancer results in decreased amplitude and duration of transcription bursts in fruit fly embryos. Additionally, we have to incorporate the study by (Hoppe et al., 2020), which reported that the Drosophila bone morphogenetic protein (BMP) gradient modulates the bursting frequency of BMP target genes. References to thorough studies of bursting control in other organisms, like Dictyostelium discoideum (Tunnacliffe et al., 2018), are due as well.
Reviewer #2 (Public Review):
The manuscript by Berrocal et al. asks if shared bursting kinetics, as observed for various developmental genes in animals, hint towards a shared molecular mechanism or result from natural selection favoring such a strategy. Transcription happens in bursts. While transcriptional output can be modulated by altering various properties of bursting, certain strategies are observed more widely. As the authors noted, recent experimental studies have found that even-skipped enhancers control transcriptional output by changing burst frequency and amplitude while burst duration remains largely constant. The authors compared the kinetics of transcriptional bursting between endogenous and ectopic gene expression patterns. It is argued that since enhancers act under different regulatory inputs in ectopically expressed genes, adaptation would lead to diverse bursting strategies as compared to endogenous gene expression patterns. To achieve this goal, the authors generated ectopic even-skipped transcription patterns in fruit fly embryos. The key finding is that bursting strategies are similar in endogenous and ectopic even-skipped expression. According to the authors, the findings favor the presence of a unified molecular mechanism shaping even-skipped bursting strategies. This is an important piece of work. Everything has been carried out in a systematic fashion. However, the key argument of the paper is not entirely convincing.
We thank the reviewer, as these comments will enable us to improve the Discussion section and overall logic of our revised manuscript. We agree that the evidence provided in this work, while systematic and carefully analyzed, cannot conclusively rule out either of the two proposed models, but just provide evidence supporting the hypothesis for a specific molecular mechanism constraining eve bursting strategies. Our experimental evidence points to valuable insights about the mechanism of eve bursting control. For instance, had we observed quantitative differences in bursting strategies between ectopic and endogenous eve domains, we would have rejected the hypothesis that a common molecular mechanism constrains eve transcriptional bursting to the observed bursting control strategy of frequency and amplitude modulation. Thus, we consider that our proposition of a common molecular mechanism underlying unified eve bursting strategies despite changing trans-regulatory environments is more solid. On the other hand, while our model suggests that this undefined bursting control strategy is not subject to selection acting on specific trans-regulatory environments, it is not trivial to completely discard selection for specific bursting control strategies given our current lack of understanding of the molecular mechanisms that shape the aforesaid strategies. Indeed, we cannot rule out the hypothesis that the observed strategies are most optimal for the expression of eve endogenous stripes according to natural selection, and that these control strategies persist in ectopic regions as an evolutionary neutral “passenger phenotype” that does not impact fitness. We recognize the need to acknowledge this last hypothesis in the updated Introduction and Discussion sections of our manuscript. Further studies will be needed to determine the mechanistic and molecular basis of *eve *bursting strategies.
Reviewer #3 (Public Review):
In this manuscript by Berrocal and coworkers, the authors do a deep dive into the transcriptional regulation of the eve gene in both an endogenous and ectopic background. The idea is that by looking at eve expression under non-native conditions, one might infer how enhancers control transcriptional bursting. The main conclusion is that eve enhancers have not evolved to have specific behaviors in the eve stripes, but rather the same rates in the telegraph model are utilized as control rates even under ectopic or 'de novo' conditions. For example, they achieve ectopic expression (outside of the canonical eve stripes) through a BAC construct where the binding sites for the TF Giant are disrupted along with one of the eve enhancers. Perhaps the most general conclusion is that burst duration is largely constant throughout at ~ 1 - 2 min. This conclusion is consistent with work in human cell lines that enhancers mostly control frequency and that burst duration is largely conserved across genes, pointing to an underlying mechanistic basis that has yet to be determined.
We thank the reviewer for the assessment of our work. Indeed, evidence from different groups (Berrocal et al., 2020; Fukaya et al., 2016; Hoppe et al., 2020; Pimmett et al., 2021; Senecal et al., 2014; Syed et al., 2023; Tunnacliffe et al., 2018; Yokoshi et al., 2022; Zoller et al., 2018) is coming together to uncover commonalities, discrepancies, and rules that constrain transcriptional bursting in Drosophila and other organisms.
