Insulation of enhancer-promoter communication by a gypsy transposon insert in the Drosophila cut gene: cooperation between suppressor of hairy-wing and modifier of mdg4 proteins - PubMed (original) (raw)

Insulation of enhancer-promoter communication by a gypsy transposon insert in the Drosophila cut gene: cooperation between suppressor of hairy-wing and modifier of mdg4 proteins

M Gause et al. Mol Cell Biol. 2001 Jul.

Abstract

The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Here we provide evidence that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. We find that the C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. We infer that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. We theorize that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein.

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Figures

FIG. 1

FIG. 1

Dominant effects of mod(mdg4) mutations on the ct K cut wing phenotype. (A) Representative wings from the indicated genotypes. The mod(mdg4) u1 and mod(mdg4) T6 mutations strongly suppress the cut wing phenotype, and the mod(mdg4) bpdEX57 mutation and the Df(3R)e-BS2 deficiency that deletes the mod(mdg4) gene weakly suppress it. Rare mod(mdg4) neo129 homozygotes generated at 29°C show strong but incomplete suppression. The wings shown represent the median phenotypes for the indicated genotypes. (B) Box plot of the suppression of the ct K mutant phenotype by selected mod(mdg4) alleles. The percentage of the wing margin lacking bristles was measured from 10 randomly chosen wings for each genotype, and a box plot was generated. The five horizontal lines from top to bottom for each genotype are the 90th, 75th, 50th, 25th, and 10th percentiles. The circles are the minimum and maximum values observed for each genotype.

FIG. 2

FIG. 2

Effects of mod(mdg4) mutations on the In(1)w m4 variegation phenotype. The panels show representative eyes from In(1)w m4 males with the indicated combinations of mod(mdg4) alleles. mod(mdg4) neo129 and Df(3R)e-BS2 strongly enhance the variegation when heterozygous with mod(mdg4) u1 . mod(mdg4) neo129 and deficiencies also strongly enhance the variegation when they are heterozygous with wild-type mod(mdg4) (10). The eyes shown represent the median phenotype displayed by the indicated genotype.

FIG. 3

FIG. 3

The Mod(mdg4)-67.2 protein interacts with a region of Su(Hw) that is required for insulation. The Su(Hw) protein contains 12 zinc fingers (gray boxes) and an enhancer-blocking region within residues 737 to 880 (black box). The Su(Hw)-CTD and the indicated mutant forms fused to GST were expressed in E. coli and bound to glutathione beads. The autoradiograms of SDS-PAGE gels show the amount of 35S-Mod(mdg4)-67.2 and 35S-luciferase control protein (luc) prepared by translation in vitro that bound to the various GST-Su(Hw)-CTD beads and to GST-only control beads. The left-hand lane (load, lane 1) shows the amount of input labeled proteins. Mod(mdg4)-67.2 binds to the GST-Su(Hw)-CTD beads (lane 3), but deletions that affect the enhancer-blocking region of Su(Hw) prevent binding of Mod(mdg4)-67.2 (lanes 4, 5 and 6). All the results were reproduced in multiple independent experiments.

FIG. 4

FIG. 4

The truncated Mod(mdg4)-67.2u1 and Mod(mdg4)T6 proteins do not interact with Su(Hw). The Mod(mdg4)-67.2 protein contains a BTB/POZ motif (gray box) at the N terminus common to most if not all Mod(mdg4) proteins, a glutamine-rich (Q-rich) region, and a unique C terminus (cross-hatched box) (3). 35S-labeled fragments of Mod(mdg4)-67.2, including fragments that mimic the predicted mutant proteins produced by the mod(mdg4) u1 and mod(mdg4) T6 alleles, were prepared by translation in vitro and tested for their ability to bind GST-Su(Hw)-CTD beads and GST-only control beads. The autoradiograms of SDS-PAGE gels show the amount of input labeled protein (load, lane 1) and the amount that bound to the beads indicated at the tops of lanes 2 and 3. The full-length Mod(mdg4)-67.2 and the C-terminal fragment of Mod(mdg4)-7.2 both bound to the GST-Su(Hw)-CTD beads (lane 3), while the truncated Mod(mdg4)-67.2u1, Mod(mdg4)-67.2T6, and N-terminal fragment of Mod(mdg4)-67.2 did not (lane 3). As expected, the luciferase (luc) control protein also did not bind to any of the beads. In the lowest panel, nearly equal amounts of labeled full-length Mod(mdg4)-67.2 and Mod(mdg4)-67.2u1 were prepared by cotranslation in vitro. In this case, a small but significant amount of Mod(mdg4)-67.2u1 was retained (lane 6, arrow). All the results shown were reproducible in independent experiments.

FIG. 5

FIG. 5

Mod(mdg4)-67.2 interacts with the Chip facilitator protein. The Chip protein contains multiple protein interaction regions (gray boxes) including the SID, the LID, and the OID that interacts with several homeodomain proteins and the Su(Hw) zinc finger region (55). 35S-labeled N-terminal and C-terminal fragments of Mod(mdg4)-67.2 (see Fig. 4) and a luciferase control protein (luc) were prepared by translation in vitro and tested for their ability to bind to GST control beads and GST-Chip fusion protein beads (55). The autoradiograms of SDS-PAGE gels on the left (lanes 1, 2, and 3) show the amount of input protein (load, lane 1) and the amount that was retained by the indicated beads. The N-terminal fragment bound to the GST-Chip beads, but the C-terminal fragment and the luciferase control did not (lane 3). The autoradiogram of an SDS-PAGE gel on the right (lanes 4 through 10) shows the binding of full-length 35S-labeled Mod(mdg4)-67.2 prepared by in vitro translation to beads containing fusions of GST to wild-type Chip and various deletion mutants of Chip (55). The load lane (lane 4) contains a tenth of the input protein. Deletions affecting only the SID (lane 10) or only the LID (lane 7) did not affect binding of Mod(mdg4)-67.2, while deletions affecting the OID (lanes 8 and 9) displayed substantially reduced binding. All the results shown were reproduced in independent experiments.

FIG. 6

FIG. 6

Proposed model for the role of Mod(mdg4)-67.2 protein in insulation. (A) To explain why truncated Mod(mdg4)-67.2 proteins have dominant-negative (antimorphic) effects on insulator activity, we propose, as shown on the left, that a multimer of Mod(mdg4)-67.2 interacts with more than one DNA-bound molecule of Su(Hw) to form a stable insulator complex. As shown on the right, truncated Mod(mdg4)-67.2u1 or Mod(mdg4)-67.2T6 protein would destabilize the complex, leading to reduced interaction between Mod(mdg4)-67.2 and Su(Hw) and/or reduced binding of Su(Hw) to DNA. (B) To explain how the Mod(mdg4)-67.2 protein contributes to the insulator activity of Su(Hw), we propose, as depicted on the left, that it traps facilitators such as Chip and blocks the previously postulated Chip-assisted spread of homeodomain protein (HD) binding between the enhancer and promoter (12, 55). As depicted on the right, this spread could create a series of loops that brings the enhancer and promoter closer together or could aid the binding of a surrogate activator near the promoter. Like the Mod(mdg4) proteins, GAGA factor is a group of BTB/POZ-containing trxG proteins that can also both aid activation and help insulate (44, 45). GAGA factor binds proximal to several promoters and potentiates activation of the engrailed promoter by a distal enhancer (46). We speculate, as shown with the promoter on the right, that when BTB/POZ proteins such as GAGA bind just upstream of a promoter, they anchor activators close to the promoter and thereby aid activation.

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