QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Galant, R. Articles by Carroll, S. B. Search for Related Content PubMed PubMed Citation Articles by Galant, R. Articles by Carroll, S. B.

Hox repression of a target gene: extradenticle-independent, additive action through multiple monomer binding sites

Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, Wisconsin, 53706, USA

View larger version (61K): [in a new window]   Fig. 1. Ubx cell-autonomously regulates several target genes in the wing. (A-C) Confocal photomicrographs of third instar wing discs that express various potential targets of Ubx regulation (green) and bear clones of cells ectopically expressing Ubx (purple). (A) The vgQ enhancer is cell autonomously repressed by Ubx in clones close to the DV boundary. (A'-A''') Close-up views of the area boxed in A showing (A') ß-galactosidase expression driven by the vgQ enhancer and expression of Ubx together, (A'') Ubx alone, and (A''') vgQ enhancer-driven reporter gene expression alone. (B) Ubx cell-autonomously represses Kn on the AP boundary. (B'-B''') High-magnification views of the area boxed in B showing the expression of (B') Ubx and Kn together, (B'') Ubx alone, and (B''') Kn alone. (C) Sal is cell-autonomously repressed by Ubx. (C'-C''') Close-up views of the area boxed in C showing the expression of (C') Ubx and Sal together, and (C'') Ubx and (C''') Sal alone. In each panel, ventral is to the top and anterior is to the left.

View larger version (37K): [in a new window]   Fig. 2. Ubx binds to seven sites in a flight appendage-specific cis-regulatory element of sal. (A) DNase I footprinting of the sal 328 cis-regulatory element reveals three sites protected by Ubx homeodomain and is representative of footprinting of the entire sal 1.1 element. A G+A sequencing ladder is shown in the first lane, and DNase I digestions incubated with increasing concentrations of Ubx homeodomain from 0 to 90 ng, in three-fold increments, are shown in subsequent lanes. Sites numbered 5-7 are schematized to the right of the lanes and are represented by boxes; their orientation is indicated by the arrows. (B) A list of the sequences of the seven sites bound by Ubx in DNaseI footprinting assay showing 14 base pairs centered on the TAAT core sequence (highlighted in red). The numbers indicate the position of the sites within the 1.1 kb sal element. Below each Ubx binding site is the altered sequence (mut) that was introduced in the mutant sal elements to abolish the ability of Ubx to bind specifically to the site. The altered base pairs are highlighted in blue. (C) A schematic representation of the sal elements. The blue circles indicate Sd binding sites identified by Guss et al. (Guss et al., 2001), and red circles represent the seven Ubx binding sites identified by footprinting. We note that the sal 1.1 element contains other TAAT sites that were not footprinted by Ubx. (D) Gel shifts of oligos containing a Ubx consensus binding site (lanes 1-5), Ubx binding site 2 from the sal 1.1 element (lanes 6-10) or its mutant variant (lanes 11-15) using Ubx homeodomain protein indicate that the mutant variant of site 2 exhibits an approximate ten-fold decrease in affinity for Ubx. The open triangle indicates increasing concentrations of Ubx homeodomain, ranging from 0 to 30 ng in three-fold increments. The black arrowheads indicate the lane for each oligo in which binding of Ubx is closest to half-maximal.

View larger version (91K): [in a new window]   Fig. 3. (A-H) The sal flight appendage-specific cis-regulatory element is directly repressed in the haltere by Ubx. Nomarski photomicrographs of third larval instar wing (A,C,E,G) and haltere (B,D,F,H) imaginal disks assayed for ß-galactosidase activity driven by various elements in transgenic animals carrying reporter constructs. In these panels, anterior is to the left and ventral is to the top. (A) The sal 1.1 element drives reporter activity in the wing field straddling AP boundary. (B) No sal 1.1 element driven reporter activity is seen in the haltere (arrowhead). (C) Mutation of all seven Ubx binding sites in the sal 1.1 u1-7 element does not alter the pattern of reporter activity in the wing. Therefore, the abilities of trans-activating factors required to activate the sal 1.1 element have not been affected by the mutations. (D) Mutating all seven Ubx binding sites in the sal 1.1 u1-7 element results its dramatic derepression in the haltere, as indicated by its ability to drive strong ß-galactosidase activity in a pattern very similar to that in the wing. (E) The sal 328 element drives wing-specific reporter activity in a pattern complementary to that of the sal 1.1 element. (F) No reporter activity driven by this element is present in the haltere. (G) The mutant sal 328 u5-7 element in which the three Ubx binding sites were abolished drives ß-galactosidase activity in the wing in a pattern largely similar to that driven by the wild-type sal 328 element. We note that reporter activity driven by the mutant element is expanded towards the AP boundary compared to that driven the wild-type one. This may have occurred because the Ubx binding site mutations in the sal 328 u5-7 element also affected binding sites for other transcription factors that regulate it. (H) The mutant sal 328 u5-7 element drives reporter activity very strongly in the haltere.

View larger version (123K): [in a new window]   Fig. 4. Multiple Ubx binding sites are necessary for complete repression of a sal flight appendage cis-regulatory element and individual sites are sufficient to mediate its partial repression in the haltere. (A-F) Nomarski photomicrographs of third instar wing (left) and haltere (right) imaginal discs assayed for ß-galactosidase activity driven by various Ubx binding site mutant sal 328 elements to test the necessity (A-C) and sufficiency (D-F) of individual Ubx binding sites. Schematic representations of the mutant sal 328 elements are displayed at the bottom of each panel. Binding sites for Ubx and Sd are indicated as in Fig. 2C, and mutant binding sites are designated by an ‘X’ over them. (A, left) The sal 328 u5 single binding site mutant element drives reporter activity in the wing in a pattern similar to that of the wild-type element (Fig. 2E). (A, right) The sal 328 u5 element drives reporter activity in the haltere, demonstrating that Ubx binding site 5 is necessary for complete repression of the sal 328 element. (B, left) The sal 328 u6 element drives reporter gene activity in the wing in a pattern similar to the wild-type element. (B, right) The sal 328 u6 element drives some reporter activity in the haltere, and it is weaker than the sal 328 u5 element (compare to A, right). Therefore, Ubx binding site 6 is necessary to completely repress the sal 328 element, but binding site 5 mediates stronger repression. (C, left) The sal 328 u7 element drives ß-galactosidase activity in the wing. Because this pattern is similar to that of the sal 328 u5-7 element (see Fig. 3G), it appears that the mutant Ubx binding site 7 is probably responsible for the difference in the reporter expression patterns driven in the wing by the sal 328 wild-type and sal 328 u5-7 elements, as well as other elements in which binding site 7 is mutant. (C, right) The sal 328 u7 element drives barely detectable levels of reporter activity. The small region of reporter activity has also been observed by overstaining of discs carrying the wild-type sal 328 element. Binding site 7 appears not to be necessary for complete repression of the sal 328 element. (D, left) The sal 328 u6&7 mutant element bearing only Ubx site 5 drives reporter activity in the wing in a pattern that is similar to that of the wild-type sal 328 element (Fig. 2E), but it is expanded towards the AP boundary. (D, right) The sal 328 u6&7 element drives reporter activity in the haltere, but not to the level observed for the sal 328 u5-7 element (compare to Fig. 3H). Therefore, Ubx binding site 5 alone can mediate partial repression of the sal 328 element by Ubx. (E, left) The sal 328 u5&7 element bearing only Ubx site 6 drives reporter activity in a pattern similar to that of the sal 328 u6&7 element in the wing. (E, right) The sal 328 u6&7 element drives reporter activity in the haltere at nearly the level observed for the triple mutant sal 328 u5-7 element. This indicates that Ubx binding site 6 alone can mediate only a small degree of repression of the sal 328 element. (F, left) The sal 328 u5&6 element bearing only Ubx site 7 drives ß-galactosidase activity in the wing in a pattern very similar to that of the wild-type sal 328 element. (F, right) The sal 328 u5&6 element drives reporter activity in the haltere at a lower level than the sal 328 u5-7 element, but at a higher level than the sal u6&7 element. Therefore, Ubx binding site 7 can mediate partial repression of the sal 328 element in the haltere.

View larger version (49K): [in a new window]   Fig. 5. The activity of a Ubx protein lacking the YPWM Exd-interaction motif. (A) Electromobility shift analysis of an oligonucleotide probe bearing a consensus composite Ubx/Exd binding site using wild-type and YPWM-YAAA mutant Ubx proteins with and without Exd. Exd alone does not bind the oligo (lane 2). Together with Exd, wild-type Ubx protein binds with higher affinity than Ubx alone (compare lanes 3 and 4). Surprisingly, the Ubx YPWM-YAAA mutant protein also exhibits an increased binding affinity for a composite Ubx/Exd binding site when complexed with Exd (compare lanes 5 and 6), although not as great an increase as exhibited by wild-type Ubx and Exd (20% less) (compare lanes 4 and 6). The Ubx YAAA mutant protein alone exhibits a slight increase in binding to the probe than the wild-type protein (1.5-fold) (compare lanes 3 and 5). The closed arrowhead indicates the position of shifts due to Ubx/Exd complexes, the open arrowhead indicates Ubx shifts, and the arrow indicates the position of free probe. (B-D) Dark-field photomicrographs of cuticle preparations showing ventral denticle belts in the third thoracic segment (T3) and the first and second abdominal segments (A1 and A2, respectively), from left to right. Segmental identities are indicated next to each of the three denticle belts in each panel. (E-G) Confocal photomicrographs of the three thoracic segments in embryos carrying a ß-galactosidase reporter trans-gene driven by the Dll embryonic limb enhancer and stained with anti-ß-galactosidase antibody (green). (H-J) Confocal photomicrographs of embryos stained for Ubx protein (green). White arrowheads indicate the boundary between A1 and T3. Ectopic expression of the Ubx proteins is driven by arm11-Gal4. In all images, anterior is to the left and ventral is down. (B) In wild-type larvae, the T3, A1 and A2 denticle belts each have distinct morphologies. The T3 denticle belt comprises two rows of small hairs, the A1 denticle belt comprises four rows of larger hairs, and the A2 denticle belt comprises six rows arranged in a trapezoidal shape. (C) The ectopic expression of wild-type Ubx protein transforms T3 segmental identity to that of A1. Thus, wild-type Ubx specifies A1 segmental identity. (D) Ectopically expressing the Ubx YAAA mutant protein induces segmental identity transformations to A2 in the T3 and A1 segments. Therefore, the Ubx YAAA mutant protein specifies A2 segmental identity, a phenotype consistent with the inability of this protein to physically interact with Exd. (E) The Dll304 embryonic limb enhancer drives reporter gene expression in the three limb primordia in wild-type embryos. (F) Ecoptic expression of Ubx strongly represses the Dll304 enhancer. (G) Ectopic expression of the Ubx YAAA mutant protein similarly represses the Dll304 enhancer, indicating that an interaction between Ubx and Exd is not required to repress an embryonic target gene. (H) In a wild-type embryo, the anterior boundary of Ubx expression is posterior T2. (I) Ectopic expression of wild-type UbxIa protein occurs anterior to its normal anterior boundary in thoracic and head segments. (J) The Ubx YAAA protein is ectopically expressed at levels similar to ectopic UbxIa (I).

View larger version (18K): [in a new window]   Fig. 6. The evolution of Hox target gene regulation by the stepwise accumulation of monomer binding sites. Represented are two serially homologous structures, one of which is under Hox control (shaded red). The schematic of a cis-regulatory element is shown to the right of each pair of serial homologs. Its expression is indicated by the blue pattern in each structure and is mediated through a binding site (blue circle) for a transcriptional activator. Our model posits that the repression of a target gene by a Hox protein begins with the evolution of a single Hox monomer binding site (red circle) that can mediate partial repression of the activity of a cis-regulatory element. If the binding site becomes fixed, then directional selection for a further decrease in gene activity can fix additional Hox monomer sites that increase the repression of the cis-regulatory element and eventually lead to its qualitative repression. Thus, the qualitative regulation of Hox target genes evolves in a gradual, stepwise fashion and not all at once (the pathway indicated by the crossed out, dotted arrow).