Transcriptional repression by the Drosophila Giant protein: cis element positioning provides an alternative means of interpreting an effector gradient

Early developmental patterning of the Drosophila embryo is driven by the ac tivities of a diverse set of maternally and zygotically derived transcripti on factors, including repressors encoded by gap genes such as Kruppel, knir ps, giant and the mesoderm-specific snail. The mechanism of repression by g ap transcription factors is not well understood at a molecular level. Initi al characterization of these transcription factors suggests that they act a s short-range repressors, interfering with the activity of enhancer or prom oter elements 50 to 100 bp away, To better understand the molecular mechani sm of short-range repression, we have investigated the properties of the Gi ant gap protein. We tested the ability of endogenous Giant to repress when bound close to the transcriptional initiation site and found that Giant eff ectively represses heterologous promoter when binding sites are located at -55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression isdiminished. Rather than exhibiting a sharp 'step-fu nction' drop-off in activity, however, repression is progressively restrict ed to areas of highest Giant concentration. Less than a two-fold difference in Giant protein concentration is sufficient to determine a change in tran scriptional status of a target gene. This effect demonstrates that Giant pr otein gradients can be differentially interpreted by target promoters, depe nding on the exact location of the Giant binding sites within the gene. Thu s, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradien t. We also demonstrate that a chimeric Ga14-Giant protein lacking the basic /zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain.