Studies on the mechanisms of action of the male specific lethal complex in Drosophila
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Dosage compensation is achieved by a twofold up-regulation of the single X chromosome in male Drosophila. The inverse dosage effect produced by the single dose of the X chromosome increases the expression of the X chromosome and autosomes. In order to equalize the autosomal expression in males and females, the MSL complex, formed by the active MSL2 protein in males, is sequestered from the autosomes to the X chromosome, including the histone acetylase MOF, to nullify the overexpression of autosomal genes but allows this effect to act on the X chromosome. The X linked genes in males are up-regulated twofold to reach the level of two X chromosomes in females. To investigate the repressive property of the MSL complex, selected protein components of the complex were combined with the yeast GAL4 binding domain. When targeted to a UAS-minimal promoter mini-white reporter, which was recovered at several X and autosomal positions, GAL4-MOF causes up-regulation in females but down-regulation in males. By using immuno-staining and FISH, it was found that the whole MSL complex is recruited to the sites of the reporter genes in males and conditions an increased level of histone acetylation, but in females only the high acetylation co-localized with the reporter, which was further confirmed by ChIP (Chromatin Immunoprecipitation). Using a GAL4-MSL2 construct does not cause dosage compensation on X and autosomal reporters in females, although the ectopically expressed MSL causes the organization of the MSL complex on the reporter genes with an increased histone acetylation. When the ectopically expressing MSL2 without GAL4 DNA binding domain in GAL4-MOF targeted systems, we found that the increased expression in females and the reduced expression in males, caused by the MOF fusion protein, have been reversed. Global gene expression on both the X chromosome and autosomes are increased in mle/mle mutant males, in which the MSL complex is destroyed. This result is consistent with the expression analyses of the X-linked and autosomal reporter genes in the mle/mle mutant background. The high level of histone acetylation on the autosomes, produced by the release of histone modifier, MOF, induces the elevated expression and the inverse dosage effect is allowed to operate on the X chromosome to maintain dosage compensation. All these data indicate that the MSL complex does not condition dosage compensation directly, but rather its repressive activity overrides the high level of histone acetylation and counteracts the over-expression of X-linked genes to achieve the proper two-fold up-regulation in males.
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