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First published online 19 April 2006
doi: 10.1242/dev.02373

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The TAGteam DNA motif controls the timing of Drosophila pre-blastoderm transcription

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204, USA.

View larger version (57K): [in a new window]   Fig. 1. Sex-determination genes are among the handful of Drosophila genes known to be transcribed prior to nuclear cycle 14, the cellular blastoderm stage. Pre-cycle 14 (pre-CB) genes whose precise time of onset of transcription is known to within one nuclear cycle are ordered with respect to that parameter. The biological function of their pre-CB expression is also indicated. Pre-CB genes for which less precise information on timing is available include outstretched (Harrison et al., 1998), huckebein (Bronner et al., 1994), runt (Klingler and Gergen, 1993), twist (Thisse et al., 1988), brinker (Jazwinska et al., 1999) and short gastrulation (Markstein et al., 2002). Scale bar: 100 mm. a(Pritchard and Schubiger, 1996); b(Erickson and Cline, 1993); c(Barbash and Cline, 1995); d(Schejter and Wieschaus, 1993); e(Tautz et al., 1987); f(Ibnsouda et al., 1993); g(Eldon and Pirrotta, 1991; Kraut and Levine, 1991); h(St Johnston and Gelbart, 1987); i(Ingham et al., 1985); j(Coulter et al., 1990); k(Anderson and Lengyel, 1979; Lamb and Laird, 1976).

View larger version (48K): [in a new window]   Fig. 2. Identification of the TAGteam group of heptanucleotide sequences based on their relative over-representation upstream of pre-cellular-blastoderm genes. (A,B) Probability that the observed difference between pre-CB and post-CB genes with regard to the number of heptamer sites found within a 500-bp window at the specified distance from the transcription (txn) start site (+1) is the result of chance. The baseline cutoff is P=0.01 (-log[0.01]=2). (C,D) Comparison of the same pre-CB and post-CB genes with respect to the observed number of TAGteam sequences within 500 bp upstream of their transcription start sites versus the number expected based on %GC for the set. Gene sets are defined in Materials and methods (melanogaster: n=25 pre-CB versus 78 post-CB; pseudoobscura orthologs: n=24 pre-CB versus 70 post-CB).

View larger version (85K): [in a new window]   Fig. 3. Conservation of TAGteam sites in pre-cellular blastoderm genes is variable. (A) Alignment of bnk promoter sequences for Drosophila melanogaster (mel), yakuba (yak), ananassae (ana), pseudoobscura (pse), willistoni (will), mojavensis (moj), hydei (hyd), virilis (vir) and Zaprionus tuberculatus (Zap). Grey shading indicates at least 50% identity among nucleotides at sites present in at least two species. The TATA site is boxed in blue, while TAGteam sites are boxed in red. An arrow denotes the transcription start site. (B) Phylogenetic relationships, estimated divergence times (Kwiatowski et al., 1994; Powell and DeSalle, 1995; Russo et al., 1995), and number of TAGteam sites in bnk for the nine species. Parentheses indicate values from species for which less DNA sequence was available. (C) Schematic of a five-species alignment for the promoter-proximal upstream 400 bp of five pre-CB genes, and for TAGteam-rich enhancer elements further upstream in two cases (distances not to scale). Circles represent CAGGTAG sites, squares tAGGTAG and diamonds CAGGcAG. Overlapping symbols indicate overlapping motifs. In the rare event that a TAGteam site appeared to be replaced by its reverse complement, that complement was diagrammed as a separate site.

View larger version (23K): [in a new window]   Fig. 4. CAGGcAG contributes to pre-cellular blastoderm scute function. (A) Schematic of the sc X-chromosome signal element (XSE) transgene. TAGteam sequences in the promoter region are indicated by colored symbols. (B) XSE activity of the indicated transgene based on its ability to rescue scnull mutant females. Viability of scM6/scM6 females was measured relative to that of their scM6/+ sisters (n>140 per line) from the cross scM6w/Binsinscy X scM6w/Y; P{sisB w+mC}/+ at 25°C. a Values from Wrischnik et al. (Wrischnik et al., 2003). Extant representative lines from the previous study were tested in parallel with the new transgenes to confirm that the comparison was valid.

View larger version (56K): [in a new window]   Fig. 5. TAGteam sequences affect SxlPe responsiveness. (A) Schematic of wild-type and TAGteam-mutant versions of the 1.4-kb SxlPe-lacZ reporter transgene. (B-E) Representative samples of ß-galactosidase antibody-stained 3- to 5-hour embryos (25°C) carrying the indicated transgene. (F-I) Distribution of ß-galactosidase antibody staining intensities among stage 8-11 embryos carrying the indicated transgene (n>2400 total embryos per construct, with four independent lines sampled for each). If all females and only females express SxlPe, 50% of the embryos should stain. Scale bar: 250 µm.

View larger version (78K): [in a new window]   Fig. 6. TAGteam sequences affect the timing and penetrance of transcription from SxlPe. (A,C) In situ hybridization to lacZ RNA from a cycle 14 embryo carrying, respectively, the wild-type or the mutTAGteam 1.4-kb SxlPe-lacZ reporter transgene described in Fig. 5. (B,D) Nuclear dots of lacZ RNA hybridization in a cycle 13 embryo of the genotype as in A or C, respectively, co-stained for DNA (DAPI) to reveal nuclei. (E) Expression measured as the average number of dot-containing nuclei per presumed female embryo (n30 embryos per cycle per transgene). Arrows indicate onset of transcription (first cycle 5% expressing nuclei). Dot analysis was performed on a single wild-type and TAGteam mutant transgene line, each determined to be representative based upon ß-galactosidase staining. Scale bars: A,C, 50 µm; B,D, 20 µm.

View larger version (71K): [in a new window]   Fig. 7. TAGteam sites in the zen ventral repression element (VRE) help activate transcription. (A) Schematic of transgenes showing the segments of the VRE that were used to drive an eve-lacZ reporter. (B-G) lacZ RNA in situ hybridization to cycle 14 embryos carrying the 600VRE (B), mut600VRE (C), 250VRE (D), 111VRE (E), mut111VRE (F), or (2X)111VRE (G) transgene. (H) Nuclear RNA hybridization dots in a cycle 7 embryo carrying the (2X)111VRE transgene, co-stained with DAPI for DNA to reveal nuclei. The arrow indicates a dot-containing nucleus, which is magnified in the inset. (I) Expression measured as the average percentage of dot-containing nuclei per embryo. Transcription onset is defined as the first cycle 5%. Six lines were examined for the mut111VRE construct, three for all others. For each nuclear cycle, 18 embryos were assayed for the 250VRE construct, and 37 for all others. Scale bars: B-G, 50 µm; H-J, 20 µm.