Male-specific lethal-2 (msl-2) is a RING finger protein that is required for X chromosome dosage compensation in Drosophila males. Consistent with the formation of a dosage compensation protein complex, msl-2 colocalizes with the other MSL proteins on the male X chromosome and coimmunoprecipitates with msl-1 from male larval extracts. Ectopic expression of msl-2 in females results in the appearance of the other MSL dosage compensation regulators on the female X chromosomes and decreased female viability. We suggest that msl-2 RNA is the primary target of SxI regulation in the dosage compensation pathway and present a speculative model for the regulation of two distinct modes of dosage compensation by SxI.

Drosophila MSL proteins are thought to act within a complex to elevate transcription from the male X chromosome. We found that the MSL1, MSL2 and MSL3 proteins are associated in immunoprecipitations, chromatographic steps and in the yeast two-hybrid system, but that the MLE protein is not tightly complexed in these assays. We focused our analysis on the MSL2-MSL1 interaction, which is postulated to play a critical role in MSL complex association with the X chromosome. Using a modified two-hybrid assay, we isolated missense mutations in MSL2 that disrupt its interaction with MSL1. Eleven out of 12 mutated residues clustered around the first zinc-binding site of the RING finger domain were conserved in a Drosophila virilis MSL2 homolog. Two pre-existing msl2 alleles, which fail to support male viability in vivo, have lesions in the same region of the RING finger. We tested these in the two-hybrid system and found that they are also defective in interaction with MSL1. Mutation of the second zinc-binding site had little effect on MSL1 binding, suggesting that this portion of the RING finger may have a distinct function. Our data support a model in which MSL2-MSL1 interaction nucleates assembly of an MSL complex, with which MLE is weakly or transiently associated.

MSL-2 is required for the male-specific assembly of a dosage compensation regulatory complex on the X chromosome of Drosophila melanogaster. We found that MSL-2 binds in a reproducible, partial pattern to the male Xchromosome in the absence of MLE or MSL-3, or when ectopically expressed at a low level in females. Moreover, the pattern of MSL-2 binding corresponds precisely in each case to that of MSL-1, suggesting that the two proteins function together to associate with the X. Consistent with this hypothesis, we isolated EMSinduced loss of function msl-1 and msl-2 alleles in a screen for suppressors of the toxic effects of MSL-2 expression in females. We also used sitedirected mutagenesis to determine the importance of the MSL-2 RING finger domain and second cysteine-rich motif. The mutations, including those in conserved zinc coordinating cysteines, confirm that the RING finger is essential for MSL-2 function, while suggesting a less stringent requirement for an intact second motif.