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First published online March 7, 2005
doi: 10.1242/10.1242/dev.01737

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Direct regulation of knot gene expression by Ultrabithorax and the evolution of cis-regulatory elements in Drosophila

University of Wisconsin-Madison, Howard Hughes Medical Institute, 1525 Linden Drive, Madison, WI 53706, USA

View larger version (94K): [in a new window]   Fig. 4. UBX binds a cluster of sites important for repression of knot in the haltere. (A) knMel701-1991 sequence as in Fig. 3A, with UBX TAAT core sequences now indicated in brown. Extent of the knMel701-1835 construct, which eliminates a cluster of core UBX sites, is indicated in blue. UBX sites identified by DNaseI footprinting are numbered. (B) Reporter expression directed by knMel701-1835 is observed in both the wing and the haltere. Because the posterior compartment of the haltere is smaller than the anterior compartment, expression of knot at the compartment boundary is shifted with respect to the center of the disc. (C,D) DNaseI footprinting of knMel1599-1991 with purified UBX homeodomain. Lane 1, no UBX protein; lanes 2-6, 3.3-90.0 ng UBX protein; lane 7, G+A sequencing ladder. Four sites protected by UBX are observed. The sequence of UBX site 1 (C) and UBX sites 2-4 (D) are indicated next to the footprints; TAAT core sequences within the each footprint are highlighted.

View larger version (50K): [in a new window]   Fig. 1. The knot gene is a candidate UBX-regulated target gene. (A) Knot protein (red) is expressed at the anteroposterior compartment boundary in the developing wing of Drosophila melanogaster, but is absent from the haltere (inset). Engrailed protein (green) indicates the extent of the posterior compartment, which is smaller in the haltere than in the wing. (B) The distribution of Knot protein in D. pseudoobscura is identical to that in D. melanogaster. (C) In mitotic clones that overexpress UBX protein (blue), Knot is cell-autonomously repressed. (C) Courtesy of Ron Galant.

View larger version (45K): [in a new window]   Fig. 2. Localization of a wing-specific regulatory element of knot. (A) Genomic region upstream of the knot gene. The first exon of knot is shown in blue. Exons of neighboring genes are shown in gray. Regions 10-15 kb upstream of the transcription start site were tested for cis-regulatory activity. Regions directing expression in the wing are indicated in blue and regions lacking activity are indicated in black. (B) VISTA plot (Couronne et al., 2003) of knMel1-2330 fragment aligned with sequence from D. pseudoobscura and D. virilis. A window size of 50 bp was used, and regions that are greater than 70% identical are indicated in orange. Shown below are the constructs generated on the basis of the distribution of sequence conservation among Drosophila. (C) lacZ reporter expression in the wing imaginal disc under the control of the knMel701-1991 fragment. No expression is observed in the haltere imaginal disc (inset). (D) Wing imaginal disc of individual carrying knMel701-1991 fragment stained for Knot protein (red) and lacZ (green). The yellow stripe indicates complete overlap of the two patterns.

View larger version (27K): [in a new window]   Fig. 3. A single Cubitus interruptus (Ci) binding site is crucial for activation of knot in the wing. (A) The locations of potential Ci-binding sites (seven out of nine residues or greater match consensus) are indicated in blue above the construct. Blocks of conserved sequence between D. melanogaster and D. pseudoobscura are indicated in red. A single nucleotide was altered individually in each of three conserved Ci-binding sites (indicated by a green or red `X'). Mutation of Ci1047 and Ci1341 sites has no effect (data not shown), whereas mutation of site Ci1680 (B) almost completely eliminates reporter gene expression.

View larger version (93K): [in a new window]   Fig. 5. UBX-binding sites and an additional site contribute to repression of knot in the haltere. (A) Mutation of UBX site 1 alone in knMel701-1991 (knMel701-1991UBX1KO) does not cause any de-repression of reporter gene expression in the haltere. (B) Mutation of 10 UBX-binding sites in knMel701-1991 (knMel701-1991KO) causes partial de-repression of reporter gene expression in the haltere. (C) knMel701-1878, which contains 43 nucleotides not present in knMel701-1835 but no additional UBX-binding sites, directs weak reporter gene expression in the haltere, indicating that repression is partially independent of UBX. (D) Point mutation of knMel1834-1837 (knMel701-1991mut) results in partial de-repression. (E) Mutation of both knMel1834-1837 and the UBX-binding sites (knMel701-1991KOmut) results in full de-repression in the haltere.

View larger version (77K): [in a new window]   Fig. 6. Functional UBX binding sites are not conserved within Drosophila (A) Schematic of a 1.9 kb fragment of D. pseudoobscura DNA, indicating the position of putative Ci-binding sites in blue and UBX TAAT core sequences in brown. This fragment (knPse1-1935) and a derivative that truncates 300 nucleotides from the 3' end (knPse1-1643) were cloned from D. pseudoobscura and injected into D. melanogaster. (B) Alignment of knot enhancer sequences from five Drosophila species: D. melanogaster, D. mauritiana, D. biarmipes, D. malerkotliana and D. pseudoobscura. (Above) Aligned block 1, which contains the footprinted UBX binding sites (red), the functional Ci binding site (blue) and the mutated site at the boundary of the knMel701-1835 construct (purple). (Below) Aligned block 2, with conserved TAAT sequences indicated in red. Arrows in aligned block 2 indicate the knMel2499-2722 sequence added to knMel701-1835 to generate the knMelcomposite construct.

View larger version (133K): [in a new window]   Fig. 7. D. melanogaster possesses a second UBX-responsive knot regulatory element that is conserved with D. pseudoobscura. (A) knPse1-1935 is expressed in the wing, but not in the haltere. (B) knPse1-1643, which eliminates the conserved block of TAAT core sequences, is expressed in both the wing and the haltere. (C) Addition of 222 nucleotides (knMel2499-2722) of D. melanogaster sequence to the de-repressed knMel701-1835 construct (to generate knMelcomposite) is sufficient to restore repression, indicating that this conserved region is a functional repressor sequence in both D. melanogaster and D. pseudoobscura. (D) Mutation of UBX sites alone in knMelcomposite (knMelcompositeKO) is sufficient for full de-repression, suggesting that UBX does not require the function of a collaborator at this repressor element. (E) The knot wing regulatory region. The crucial Ci binding site is indicated in dark blue, the UBX repressor element novel to the D. melanogaster lineage is indicated in light blue, the conserved UBX repressor element is indicated in green and the collaborating repressor site is indicated in pink.