Interplay between distinct cis-regulatory modules mediates combinatorial regulation by Runt and other pair-rule transcription factors during Drosophila segmentation

The relatively simple combinatorial rules responsible for establishing the initial metameric expression of sloppy-paired-1 ( slp1) in two cell wide stripes in the posterior half of each parasegment in the Drosophila embryo make this system an attractive model for investigating the mechanism of regulation by Runt and other pair-rule transcription factors. I analyzed various slp1-lacZ reporter genes in order to identify cis-regulatory sequences responsible for this early pattern. This work identifies two distinct elements, a proximal early stripe element (PESE) and a distal early stripe element (DESE) located from -3.8 kb to -1.8 kb and from -8.7 kb to -6.6 kb upstream of the slp1 promoter, respectively. The distal element expresses both odd and even-numbered stripes with inappropriate expression in the anterior half of the odd-numbered parasegments due to an inability to respond to repression by Even-skipped (Eve). In contrast, the proximal element expresses only even-numbered stripes and mediates repression by Eve as well as by the combination of Runt and Fushi-tarazu (Ftz). A minimal PESE element retains Eve-dependent repression, but is expressed throughout the even-numbered parasegments due to the loss of repression by Runt and Ftz. Importantly, a composite reporter gene containing both cis-elements faithfully recapitulates slp1 regulation in a manner beyond what is expected from combining their individual patterns. I propose a model whereby the metameric expression of slp1 is achieved through Runt dependent regulation of interactions between the DESE and PESE enhancers and the slp1 promoter. The role of DNA-binding by Runt in slp1 regulation was further investigated by examining expression of DESE-lacZ reporters containing mutations in different Runt binding sites. These results indicate these sites are important for repression of DESE-lacZ but are not essential for activation. Complementary studies using a DNA-binding defective form of Runt confirm the importance of DNA-binding for slp1 repression and also reveal a role for DNA-binding by Runt in activation of slp1 and the DESE-lacZ reporter. This work reveals new insights into Runt dependent regulation and opens the door for future studies on the mechanisms underlying the developmental regulation of gene expression.