The proximal enhancer of the snail gene mediates negative autoregulatory feedback in Drosophila melanogaster
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1.
California Institute of Technology
Abstract
Autoregulatory feedback is a mechanism in which a gene product regulates its own expression, stabilizing gene activity amid noise and environmental changes. In Drosophila melanogaster, the gene snail encodes a key transcriptional repressor that regulates the expression of many genes during early embryogenesis, including its own expression. This study focuses on Snail occupancy at both distal and proximal enhancers of the snail gene to understand the cis-regulatory mechanisms involved in autoregulatory control. The coordinated action of these enhancers results in precisely constrained levels of snail expression during early embryogenesis. Using genome editing by CRISPR/Cas9, we found that deletion of each enhancer individually is compatible with embryonic viability under normal conditions. However, the double mutant is lethal, suggesting a functional interplay between the 2 enhancers. To gain further insight, we assayed snail gene expression levels in fixed embryos. Our results revealed that negative autoregulation of snail relies on the proximal enhancer. Moreover, increasing the affinity of binding sites for Dorsal, a transcriptional activator, in the proximal enhancer impaired this autoregulation, suggesting that Snail acts locally to counterbalance Dorsal's input. A mathematical model of snail autoregulatory control further supports our findings, reinforcing the view that the proximal enhancer mediates negative autoregulatory feedback, and implicating the distal enhancer in positive autoregulatory feedback. In summary, Snail's role at the proximal enhancer is pivotal for negative autoregulatory control and essential for balancing the activation mediated by the distal enhancer.
Copyright and License
© The Author(s) 2025. Published by Oxford University Press on behalf of The Genetics Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Acknowledgement
The authors are grateful to Justin Bois and Gregory Reeves for helpful discussions on the modeling approach as well as Stathopoulos lab members for comments on the manuscript. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study.
Funding
This study was supported by funding from the National Institute of Health grant R35GM118146 to A.S.
Contributions
A.S., J.I., L.D., and J.M.M. conceived the project and planned the experimental approach. A.S. directed the project. L.D. and J.I. performed all the experiments, and J.M.M. performed all the quantitative analysis of imaging data, modeling, and statistical analysis. Data were analyzed by L.D., J.M.M., J.I., and A.S. The manuscript was written by J.M.M., L.D., and A.S. with edits provided by J.I.
Data Availability
Drosophila strains and other reagents generated in this study will be available upon request from the lead contact. A github repository with the code for quantitative and statistical analyses was generated and is publicly available: https://github.com/StathopoulosLab/Dunipace_2024. Additional code used to verify image segmentation is available: https://github.com/StathopoulosLab/McGehee_2024. Any additional information required to reanalyze the data shown in this paper is available from the lead contact upon request. ChiP-Seq datasets were previously published and can be accessed using https://doi.org/10.1186/s13059-016-1057-2 GEO accession numbers: Sna: GSM1689688; toll10b_twi_1: GSM1689698; toll10b_dl_1: GSM1689690 (Koenecke et al. 2016, Edgar et al. 2002).
Supplemental material available at GENETICS online.
Code Availability
A github repository with the code for quantitative and statistical analyses was generated and is publicly available: https://github.com/StathopoulosLab/Dunipace_2024. Additional code used to verify image segmentation is available: https://github.com/StathopoulosLab/McGehee_2024.
Conflict of Interest
The author(s) declare no conflicts of interest.
Additional Information
Leslie Dunipace and James M McGehee contributed equally to this work.
Supplemental Material
Files
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Additional details
Identifiers
- PMID
- 40147870
- PMCID
- PMC12135202
Related works
- Describes
- Journal Article: 40147870 (PMID)
- Journal Article: PMC12135202 (PMCID)
Funding
- National Institutes of Health
- P40OD018537
- National Institutes of Health
- R35GM118146
Dates
- Submitted
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2025-02-19
- Accepted
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2025-03-18
- Updated
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2025-04-23Corrected and typeset
Caltech Custom Metadata
- Caltech groups
- Division of Biology and Biological Engineering (BBE)
- Publication Status
- Published