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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.

Manipulating the Sensitivity of Signal-Induced Repression: Quantification and Consequences of Altered Brinker Gradients