Abstract
Krüppel (Kr), a member of the gap class of Drosophila segmentation genes, encodes a DNA binding zinc finger-type transcription factor. In addition to its segmentation function at the blastoderm stage, Krüppel also plays a critical role in organ formation during later stages of embryogenesis. To systematically identify in vivo target genes of Krüppel, we isolated DNA fragments from the Krüppel-associated portion of chromatin and used them to find and map Krüppel-dependent cis-acting regulatory sites in the Drosophila genome. We show that Krüppel binding sites are not enriched in Krüppel-associated chromatin and that the clustering of Krüppel binding sites, as found in the cis-acting elements of Krüppel-dependent segmentation genes used for in silico searches of Krüppel target genes, is not a prerequisite for the in vivo binding of Krüppel to its regulatory elements. Results obtained with the newly identified target gene ken and barbie (ken) indicate that Krüppel represses transcription and thereby restricts the spatial expression pattern of ken during blastoderm and gastrulation.
Highlights
The Drosophila segmentation gene Kruppel (Kr)1 participates in the subdivision of the embryo into increasingly smaller segment equivalents along the anterior-posterior axis [1, 2]
A few Kruppel-regulated genes have been identified on the basis of altered gene expression patterns in Kr mutant embryos, by in vitro studies showing that Kruppel binds to the respective cis-acting control elements [14, 27, 28], and by genetic modifier screens involving the dominant Kr mutation Irregular facets (If) [29, 30]
We present an initial screen in which we identified 82 putative Kruppel target DNA fragments of which more than half were examined with respect to enrichment in Kruppel-associated chromatin and Kruppel binding properties in vitro
Summary
Fly Strains and Construction of Transgenes Expressing Tagged Kruppel—Flies were cultured under standard conditions using Oregon R as a wild type strain. Homozygous Kr mutant embryos were identified by the absence of lacZ activity in the y,w; Sco/Cyo, P[hb-lacZ] Krl/SM5 strain. Hs-Kr/CyO, P[hb-lacZ] strain [13] was used to induce ectopic Kr espression in response to heat-shock treatment. The double-tagged FX(10.7)-Kr-2F transgene included 10.7 kb of the Kr upstream region, 1.9 kb of the transcribed region, and 1.45 kb of downstream DNA. It was generated by fusing five separate DNA fragments. They were separately amplified from Drosophila Oregon R DNA by PCR (Stratagene, La Jolla, CA) using the primers listed in Supplemental Table 1 (for their location see Fig. 1). White flies were transformed [33] with the 14-kb-long FX(10.7)-Kr-2F gene inserted into the pP(CaSpeR-4) vector [59]
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