Integrity of the Mod(mdg4)-67.2 BTB Domain Is Critical to Insulator Function in Drosophila melanogaster

Abstract

The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function.

SUPPLEMENTAL MATERIAL

We thank Paul Fisher for the anti-lamin antibody, Dale Dorsett for providing the mod(mdg4)-67.2, full-length su(Hw) cDNA clone, and plasmid with the Su(Hw) promoter, and Mark Brennan for providing the mod(mdg4)^T6 line.

This work was supported by the Molecular and Cellular Biology program of Russian Academy of Science and by a stipend from the Center for Medical Studies, University of Oslo to A.G., by a Joint Research Project SCOPES grant from the Swiss National Science Foundation to V.P and P.G., and by an International Research Scholar award from the Howard Hughes Medical Institute to P.G. The work was also supported by the National Institute of Health grant GM42539 to P.G.