MOLECULAR BASIS OF SUPPRESSION IN DROSOPHILA

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DESCRIPTION: Dr. Victor Corces' long-term goal is to gain some understanding of the role of higher order chromatin organization in the control of gene expression in eukaryotes. To this end he proposes to continue his studies on the nature of gypsy retrotransposon-induced mutations and their suppression. Some of these mutations are caused by a direct alteration of the open reading frame; the mutations of interest to Dr. Corces result from the insertion of gypsy in the regulatory region of the gene. In these cases, the presence of the element between the promoter and some of the transcriptional enhancers prevents the function of these enhancers. Using mutations, first in the yellow and later also in the forked genes of Drosophila, Dr. Corces has determined that gypsy contains 12 binding sites for the suppressor of Hairy-wing protein and that binding of this protein creates a structure that is an example of a class of chromatin elements called insulators. Insulators prevent the influence of active or repressed chromatin from extending across their location. As expected, loss-of-function mutations in the su(Hw) reverse the phenotypic effects of gypsy insertions. Dr. Corces has also determined that the su(Hw) protein has twelve zinc fingers that mediate its binding to DNA and two acidic and a leucine zipper regions that are necessary for its repression of gene expression. Others had discovered a gene, modifier of mdg4, whose mutation affects the phenotype of gypsy-induced mutations. Genetic analysis of double mutants performed in Corces' lab. indicates that the mod(mdg4) protein is necessary for the directionality of the gypsy effect on enhancer function: in absence of this protein, enhancers both upstream and downstream of the point of gypsy insertion are affected. Dr. Corces believes that he has cloned the mod(mdg4) gene although this has not been confirmed by mutant rescue. In order to determine what additional proteins are involved in the su(Hw) and mod(mdg4) interaction, Dr. Corces has screened for dominant suppressors of mod(mdg4) and has identified 3 genes that he has called Su(mod); the proteins appear to allow su(Hw) to form an insulator without directionality in the absence of the mod(mdg4) gene product. Others had discovered that, in a strain carrying the female-sterile mutation ovo[D], insertion of gypsy caused the frequent restoration of female fertility. In this strain, gypsy transposition in the germ line is caused by a maternal-effect mutation called flamenco. In collaboration with these workers, Dr. Corces has determined that the ovaries of flamenco females have a novel gypsy transcript that encodes an envelope protein of the type that is needed in other retroviruses to form infectious particles. He has carried out some fractionation, EM and feeding experiments whose results indicate the presence of infective gypsy particles in extracts of flam females. In the current application, Dr. Corces concentrates on the mechanism underlying the formation of chromatin boundaries by the su(Hw) protein. To begin, he proposes to continue the molecular genetic characterization of mod(mdg4) including the interaction of the three proteins that it encodes with the su(Hw) protein and its DNA binding site. Next he will characterize additional components that mediate the formation and function of the su(Hw) insulator. He will clone one gene from each of the two classes of mutations that modulate the mutagenic effect of the su(Hw) protein: one of the three e(y) genes whose mutations enhance the phenotype of the y[2] allele and one of the three Su(mod) genes whose mutations reverse the effect of mod(mdg4) mutations. Once these genes are cloned, he will determine the ability of the encoded proteins to interact with gypsy DNA and with the su(Hw) and mod(mdg4) proteins. Finally, he proposes to study the changes in chromatin structure that result from the formation of the su(Hw) insulator and that may be responsible for its directional interference with the effect of upstream enhancers on transcription. To this end, he will analyze changes in hypersensitive sites, in the positioning of nucleosomes and in the binding of specific transcription factors to the regulatory sequences of two genes mosaic genes especially assembled for this purpose.