The interaction between the coactivator dCBP and Modulo, a chromatin-associated factor, affects segmentation and melanotic tumor formation in Drosophila

Abstract

The development of Drosophila requires the function of the CREB-binding protein, dCBP. In flies, dCBP serves as a coactivator for the transcription factors Cubitus interruptus, Dorsal, and Mad, and as a cosuppressor of Drosophila T cell factor. Current models propose that CBP, through its intrinsic and associated histone acetyltransferase activities, affects transient chromatin changes that allow the preinitiation complex to access the promoter. In this report, we provide evidence that dCBP may regulate the formation of chromatin states through interactions with the modulo (mod) gene product, a protein that is thought to be involved in chromatin packaging. We demonstrate that dCBP and Modulo bind in vitro and in vivo, that mutations in mod enhance the embryonic phenotype of a dCBP mutation, and that dCBP mutations enhance the melanotic tumor phenotype characteristic of mod homozygous mutants. These results imply that, in addition to its histone acetyltransferase activity, dCBP may affect higher-order chromatin structure.

Figure 1

dCBP interacts with Modulo. Two-hybrid interactions between different pLexA-dCBP baits and the pACT-Modulo clone isolated by the two-hybrid screen (A) or between different pACT-Modulo preys and the pLexA-dCBP-2278–2678 bait (B). The dCBP baits and the Modulo preys used are represented, the numbers show the amino acid positions, and the black box in dCBP represents the C/H3 domain. The results of the two-hybrid interactions obtained by using a β-gal filter assay are represented on the right. ++, Strong interaction; +, weak interaction; −, no interaction. (C) In vitro binding between dCBP and Modulo. Equimolar amounts of immobilized GST and GST-dCBP-2278-2678 were incubated with in vitro translated ³⁵S-labeled Modulo proteins. Some (10%) of the total reaction was loaded in the input lanes, and all samples were run on the same gel. (D) Coimmunoprecipitation of the dCBP-Modulo complex from Kc cell extracts by using a dCBP antisera. Some (2%) of the unprecipitated extract was loaded in the input lane and run on the same gel as the immunoprecipitated complexes.

Figure 2

Denticle belt fusion phenotype of nej¹; L8 double mutant embryos. Cuticle preparation of late-stage embryos photographed with dark-field optics; anterior is to the left. (a) nej¹/Y embryo. (b) L8 embryo. (c and d) nej¹/Y; L8 double mutant embryos showing head defects and fusions of segment denticle belts: fusion A2–A3 (c), fusion A3–A4 (d).

Figure 3

Laser confocal microscopy of Modulo and dCBP in the epidermis of stage 14 embryos. (a–f) Colocalization in wild-type embryos. Mod immunostaining with FITC (a and d), dCBP immunostaining with rhodamine (b and e), merged image from a and b (c), and merged image from d and e (f). Arrows in f indicate potential colocalization of Mod and dCBP at the periphery of the nucleolus. A 20× objective was used for a–c and a 60× objective with lasersharp to zoom 3× for d–f. (g and h) In green fluorescence, Mod immunostaining of L8 heterozygous embryos (g) or L8 homozygous embryos (h). The third chromosome balancer TM3, Sb Ser carrying a ftz-lacZ gene was used to distinguish the two populations of embryos, and the red stripes represent the β-gal staining of the balanced heterozygous animals (g). Anterior is to the right.

Figure 4

Microscopy of wild-type polytene chromosomes. (A) Portion of chromosome arm showing localization of Mod. (B) Chromosome arm in A showing localization of dCBP. (C) Merged image of A and B. Large arrowhead indicates locus that binds Mod and not dCBP. Small arrowhead indicates band that binds dCBP and not Mod. Small arrows indicated loci that colocalize both dCBP and Mod, and are shown in yellow. Images were taken with a 40× objective.

Figure 5

Phenotypic and statistical analysis of y¹w¹/FM7c; L8 and nej¹/y¹w¹; L8 third-instar larvae.