This work investigates the role of DNA-binding by Runt in regulating the sloppy-paired-1 (slp1) gene, and in particular two distinct cis-regulatory elements that mediate regulation by Runt and other pair-rule transcription factors during Drosophila segmentation. We find that a DNA-binding defective form of Runt is ineffective at repressing both the distal (DESE) and proximal (PESE) early stripe elements of slp1 and is also compromised for DESE-dependent activation. The function of Runt-binding sites in DESE is further investigated using site-specific transgenesis and quantitative imaging techniques. When DESE is tested as an autonomous enhancer, mutagenesis of the Runt sites results in a clear loss of Runt-dependent repression but has little to no effect on Runt-dependent activation. Notably, mutagenesis of these same sites in the context of a reporter gene construct that also contains the PESE enhancer results in a significant reduction of DESE-dependent activation as well as the loss of repression observed for the autonomous mutant DESE enhancer. These results provide strong evidence that DNA-binding by Runt directly contributes to the regulatory interplay of interactions between these two enhancers in the early embryo.

这项工作研究了 Runt 与 DNA 结合在调节sloppy-paired-1 ( slp1 ) 基因中的作用，特别是在果蝇分割过程中介导 Runt 和其他配对规则转录因子调节的两个不同的顺式调节元件。我们发现 Runt 的 DNA 结合缺陷形式在抑制 slp1 的远端 (DESE) 和近端 (PESE) 早期条纹元素方面无效并且还因 DESE 依赖性激活而受到损害。使用位点特异性转基因和定量成像技术进一步研究 DESE 中 Runt 结合位点的功能。当 DESE 作为自主增强子进行测试时，Runt 位点的诱变导致 Runt 依赖性抑制明显丧失，但对 Runt 依赖性激活几乎没有影响。值得注意的是，在也包含 PESE 增强子的报告基因构建体的背景下，这些相同位点的诱变导致 DESE 依赖性激活显着降低，以及对自主突变 DESE 增强子观察到的抑制丧失。这些结果提供了强有力的证据，证明 Runt 的 DNA 结合直接有助于早期胚胎中这两种增强子之间相互作用的调节相互作用。