Abstract
Traditionally, the analysis of gene regulatory regions suffered from the caveat that it was restricted to artificial contexts (e.g. reporter constructs of limited size). With the advent of the BAC recombineering technique, genomic constructs can now be generated to test regulatory elements in their endogenous environment. The expression of the transcriptional repressor brinker (brk) is negatively regulated by Dpp signaling. Repression is mediated by small sequence motifs, the silencer elements (SEs), that are present in multiple copies in the regulatory region of brk. In this work, we manipulated the SEs in the brk locus. We precisely quantified the effects of the individual SEs on the Brk gradient in the wing disc by employing a 1D data extraction method, followed by the quantification of the data with reference to an internal control. We found that mutating the SEs results in an expansion of the brk expression domain. However, even after mutating all predicted SEs, repression could still be observed in regions of maximal Dpp levels. Thus, our data point to the presence of additional, low affinity binding sites in the brk locus.
Highlights
The Drosophila wing imaginal disc is routinely used as a model to study growth and patterning
To understand the role of the brinker silencer elements in growth and patterning of the wing we manipulated them in a genomic context, thereby changing the brinker gene’s sensitivity to Dpp signaling on the transcriptional level
Making use of genomic constructs featuring between zero and 13 functional consensus silencer elements (SEs) in their endogenous context as well as a differently labeled internal control as a reference, we developed a quantification method that allowed us to very precisely quantify the effect of single SEs or SE combinations on the Brk gradient
Summary
The Drosophila wing imaginal disc is routinely used as a model to study growth and patterning. Growth and patterning of the wing imaginal disc are regulated by gradients of morphogens. From its source Dpp spreads both into the anterior and the posterior compartment, forming a concentration gradient. Upon migration to the nucleus this complex directly activates the transcription of Dpp target genes. For most target genes this activating branch of the Dpp pathway plays only a minor role. Some target genes seem to be exclusively regulated via Brk (e.g. optomotorblind, omb; bifid) while the expression of others seems to depend on a combination of direct activation and brk repression (e.g. spalt, sal; spalt major, salm) [3,4]. The repression of brk has been termed ‘‘signal-induced repression’’ and represents an example of an interesting but poorly understood mechanism that can be found in other pathways (for review: [6])
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