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Downregulation of the tissue-specific transcription factor Fork head by Broad-Complex mediates a stage-specific hormone response

¹ Institut für Genetik der Freien Universität Berlin, Arnimallee 7, D-14195 Berlin, Germany
² Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, 15N 2030E, Room 5200, Salt Lake City, UT 84112-5331, USA

View larger version (41K): [in a new window]   Fig. 1. BR-C protein is not detected at Sgs loci or at transposed copies of the Sgs4 regulatory region. Salivary gland polytene chromosomes were immunostained with anti-BR-C antibody SA 4256 (red) and counterstained with the DNA dye Hoechst 33258 (green). In the bottom panels of B,C, DNA and immunostaining are merged to allow proper localization of the immunofluorescent signals. (A) Staining of chromosomes of the wild-type strain Oregon R (ORN) and the BR-C null mutant npr13. The antibody specifically detects BR-C protein in the wild-type chromosomes but not in the npr13 chromosomes. (B) Sections of Oregon R wild-type chromosomes 1 (X), 2L and 3L, which contain the loci of five Sgs genes, Sgs4 (3C11), Sgs1 (25B2-3), and Sgs3, Sgs7 and Sgs8 (68C). Neither of the Sgs loci coincides the strong fluorescent signals, which are abundant on all chromosomes. (C) Staining of a wild-type fourth chromosome (ORN) and a fourth chromosome of a transformant line (T4) that carries four copies of the Sgs4 regulatory region inserted at 102 D3-5. The intensity of the immunofluorescent signal at 102 D is not changed in the presence of the insertion.

View larger version (32K): [in a new window]   Fig. 2. The levels of fkh mRNA and Fkh protein are significantly decreased in salivary glands of early prepupae. (A) SEBP2/Fkh and SEBP3 DNA-binding activities are detected in salivary gland nuclear extracts from third instar larvae (3L) but not from early prepupae (PP). Binding of equivalent amounts of extract protein to radiolabeled oligonucleotide , which contains both a SEBP2/Fkh- and a SEBP3-binding site (Lehmann and Korge, 1996), was analyzed by a mobility shift DNA-binding assay. Unlabeled competitor oligonucleotides were used to confirm the identity of the protein-DNA complexes. O5 includes wild-type SEBP3-binding site as competitor, O6 includes SEBP3-binding site mutated at two bases as competitor, and ß includes wild-type SEBP2/Fkh-binding site as competitor. Even after a tenfold longer exposure, no Fkh-DNA complex is visible in lanes loaded with prepupal extract. (B) Total RNA extracted from salivary glands of mid-third instar larvae (m3L), late-third instar larvae (l3L) and white prepupae (PP) was used as template for RT-PCR amplification of fkh and rp49 transcripts. The detection of rp49 mRNA served as an internal control for the amount and integrity of the RNA used in the reactions. Each lane contains the products of an independent RT-PCR. Reactions were performed on aliquots of separate RNA pools, each obtained from salivary glands of five animals. (C) Protein from the indicated numbers of salivary glands (SGs) of mid-third instar larvae (3L) and white prepupae (PP) was separated by SDS-PAGE, blotted onto nitrocellulose, and probed with an anti-Fkh antibody. The antibody detects two closely spaced bands. The protein in the lower band migrates with the same mobility as Fkh protein obtained by in vitro translation (IVT). The upper protein migrates only slightly more slowly and shows the same strong decline in prepupal salivary glands as the faster migrating protein. It may therefore represent a posttranslationally modified form of Fkh. The lanes were overloaded to detect Fkh in the prepupal extract, resulting in some distortion of the protein bands.

View larger version (49K): [in a new window]   Fig. 3. Sgs4 is not properly repressed in the presence of elevated levels of Fkh and in mutants of the BR-C function 2Bc. (A) Maintenance of fkh expression by heat shock induction of a Fkh-encoding transgene leads to high levels of Sgs4 mRNA in early prepupae. The expression of Sgs4 in the salivary glands of Oregon R animals (ORN) and animals of the transformant line P [hs-Fkh111] was analyzed by northern blot hybridization after a 1 hour heat shock applied 3-4 hours before dissection of the glands, or without prior heat shock. Sgs4 expression in prepupae is only maintained only in salivary glands of P[hs-Fkh111] animals after heat shock application. Equivalent results were obtained after reducing the heat shock period to 30 minutes (not shown). (B) Sgs4 expression extends into the prepupal stage in mutants of the BR-C function 2Bc. Expression of Sgs4 in males hemizygous for the 2Bc alleles 2Bc1, 2Bc2 and 2Bc4, or control males hemizygous for the balancer chromosome present in the corresponding stock, was assayed by northern blot analysis. Hybridization to rp49 was used as a control for loading and transfer. Note that, although fkh mRNA levels remain completely or nearly unchanged at pupariation in the 2Bc mutants (see Fig. 4), the amount of Sgs4 mRNA is lower in prepupae of these mutants than in prepupae expressing the ectopic Fkh. This difference is probably due to the strong heat shock induction mediated by the hsp70 promoter, leading to a high level of ectopic Fkh and enhanced Sgs4 expression. m3L and 3L, mid-third instar larvae; l3L, late-third instar larvae; PP, white prepupae.

View larger version (46K): [in a new window]   Fig. 4. Fkh is not properly downregulated at the larval-to-prepupal transition in 2Bc mutants of the BR-C. Expression of fkh in the salivary glands of three different 2Bc mutants, 2Bc1, 2Bc2 and 2Bc4, was assayed by RT-PCR as described in the legend to Fig. 2. In contrast to wild-type animals (compare Fig. 2), high levels of fkh mRNA are maintained in the salivary glands of mutant late-third instar larvae and early prepupae.

View larger version (35K): [in a new window]   Fig. 5. BR-C protein is bound to the fkh locus 98 D2-3 in salivary glands of mid- and late third instar larvae. Salivary gland polytene chromosomes of puff stage (PS) 1 and 6 were immunostained with anti-BR-C antibody SA 4256 (middle panels, red) and counterstained with Hoechst 33258 (top panels, green). In the bottom panels both stains are merged. A section of the third chromosome that encompasses the fkh locus at 98 D2-3 is shown. A strong fluorescent signal is observed at this locus when the Sgs genes are active (PS1) and after cessation of Sgs gene expression (PS 6).

View larger version (12K): [in a new window]   Fig. 6. A model for the regulatory interactions between BR-C, Fkh and the 20E receptor EcR/Usp in the control of Sgs4 expression. For details see the text.

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