Dunipace Leslie, McGehee James M, Irizarry Jihyun, Stathopoulos Angelike
Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Blvd., Pasadena, CA 91125, USA.
Genetics. 2025 Jun 4;230(2). doi: 10.1093/genetics/iyaf058.
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.
自调节反馈是一种基因产物调节自身表达的机制，可在噪声和环境变化中稳定基因活性。在黑腹果蝇中，蜗牛基因编码一种关键的转录抑制因子，该因子在胚胎早期发育过程中调节许多基因的表达，包括其自身的表达。本研究聚焦于蜗牛基因远端和近端增强子上的蜗牛蛋白占据情况，以了解自调节控制中涉及的顺式调控机制。这些增强子的协同作用导致胚胎早期发育过程中蜗牛基因表达水平受到精确限制。利用CRISPR/Cas9进行基因组编辑，我们发现单独缺失每个增强子在正常条件下与胚胎存活能力是相容的。然而，双突变体是致死的，这表明两个增强子之间存在功能相互作用。为了进一步深入了解，我们检测了固定胚胎中蜗牛基因的表达水平。我们的结果表明，蜗牛基因的负自调节依赖于近端增强子。此外，增加近端增强子中转录激活因子背腹蛋白结合位点的亲和力会损害这种自调节，这表明蜗牛蛋白在局部发挥作用以抵消背腹蛋白的输入。蜗牛自调节控制的数学模型进一步支持了我们的发现，强化了近端增强子介导负自调节反馈的观点，并暗示远端增强子参与正自调节反馈。总之，蜗牛蛋白在近端增强子上的作用对于负自调节控制至关重要，并且对于平衡远端增强子介导的激活必不可少。
