Manipulating the sensitivity of signal-induced repression: quantification and consequences of altered brinker gradients.
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.
Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.
Lucia
L
Gafner
Sascha
S
Dalessi
Eliane
E
Escher
George
G
Pyrowolakis
Sven
S
Bergmann
Konrad
K
Basler
D000818:Animals; D001483:Base Sequence; D015536:Down-Regulation; D004330:Drosophila / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D029721:Drosophila Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect; D017930:Genes, Reporter; D056426:Genetic Loci; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D009154:Mutation; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics*; D015398:Signal Transduction; D037941:Silencer Elements, Transcriptional; D014921:Wings, Animal / Q000254:growth & development / Q000378:metabolism
D029721:Drosophila Proteins; D012097:Repressor Proteins; C118082:brk protein, Drosophila; C064196:dpp protein, Drosophila; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins