Abstract
Body pattern formation during early embryogenesis of Drosophila melanogaster relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. We identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation.
Highlights
Kr (Kruppel) is an essential gene of the segmentation gene hierarchy in Drosophila melanogaster
We provide evidence that Kr exerts transcriptional repression by association with the corepressor Drosophila C terminus-binding protein (dCtBP) and by site-specific deacetylation of histones, a mechanism that involves an interaction between Kr and dSAP18
The dual mode of Kr-dependent repression might explain earlier studies showing that Kr represses eve stripe 2 expression, but not h stripe 7 expression, in a dCtBP-dependent manner [24]
Summary
Fly Genetics—The following fly stocks were used: b pr cn wx KrIf-1 (Tubingen stock collection), the balancers y,w; Sco/CyO, P(hb-lacZ) and y,w; Ly/TM3, Sb, and hs-Kr/CyO, P(hb-lacZ); h7-lacZ (for details see Ref. 24). Resins loaded with GST-Kr fusion protein were incubated with preconditioned lysate (2 h, 4 °C, constant agitation), collected by a short spin, and washed with several changes of buffer (20 mM HEPES, pH 8.0, 300 mM NaCl, 0.2% Triton X-100, 5% glycerol, 1 mM dithiothreitol, protease inhibitor mixture). g of imidazol-eluted protein was incubated with 15-l resin aliquots loaded with 15 g of either GST or GST-Kr. Incubations were carried out in 0.5 ml of binding buffer (20 mM TrisHCl, pH 7.5, 0.15 M NaCl, 0.1 mM ZnSO4, 1 mM dithiothreitol, 0.1% Triton X-100, and protease inhibitors) containing 3% bovine serum albumin as nonspecific competitor (2 h, 4 °C). The proteins were eluted (boiling in 20 l of 2ϫ Laemmli buffer) and separated by 13.5% SDS-PAGE followed by Western blotting (anti-His mouse monoclonal antibodies, DIA900; Dianova), visualized by goat anti-mouse HRP-conjugated secondary antibodies (Pierce).
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