Dosage compensation of X-chromosome gene expression has been an intriguing regulatory problem for several decades but not until the past few years has much progress been made to understand the underlying genetic system controlling it. It is now clear that the mechanisms of dosage compensation are quite distinct in the three most studied eukaryotic system, Drosophila C. elegans, and man. Drosophila dosage compensates by hyperactivation of the single male X chromosome, C. elegans reduces the expression of each of the two females X chromosomes, and human dosage compensation is achieved via inactivation of one of the female X-chromosomes. The genetic analysis of dosage compensation in Drosophila has lead to the discovery of four male specific lethal genes and which appear to be the primary mediators of dosage compensation while the sex-lethal gene, also a determinant of sex-determination, lies upstream in the regulatory hierarchy. The four male-lethal genes have been cloned and characterized in recent years with Dr. Baker's lab playing a prominent role in this work. In one of the more dramatic visual demonstrations , it was shown that these proteins bind at numerous site over the X chromosome but only a few site on the autosomes. The male-lethal proteins appear to bind to the same site and their binding is inter-dependent. Attempts by the Baker lab and other labs to identify the specific binding site sequences have been less successful, but recently Dr. Baker has developed FISH based method to finally provide the technical means of deletion mapping the binding sites. The first specific aim is to determine how the MSL-2 gene is regulated. Preliminary studies indicate that MSL-2 is negatively regulated by Sxl to prevent dosage compensation in females. Sxl's regulation of MSL-2 appears to be mediated via the 5' and 3' untranslated regions of MSL-2. A male-specific intron is retained in females and this retention requires Sxl. Potential Sxl binding sites are present within this intron as well as the 3'UTR. The MSL-2 gene will be dissected to map sequence element that mediated post-transcriptional regulation. The second specific aim is to identify the X-chromosome sequences that mediate dosage compensation. Two approaches will be used: search for sequences that bind to the putative MSL-2 DNA binding domain and use chromosomal rearrangements to delimit a cosmid size segment of DNA that contains a strong MSL band at its normal position on the X chromosome and then introduce a cLone of this DNA into autosomal sites. In addition a mixture of MSL-1 and MSL-2 will be used in selection experiments on pools of random 25-mer oligonucleotides to identify specific sequence elements. The third specific aim is to identify other components of the dosage compensation pathway by using genetic screens. Dr. Baker argues that the other components may have be involved in general aspects of transcription and chromatin structure. Consequently, mutations in such genes would be expected to be lethal in both sexes, unlike the four previously identified msls. The genetic screen will be for mutations that dominantly suppress or enhance ectopic expression of MSL-2 in females. Replacement of either the 5' or 3' UTR of MSL-2 results in ectopic expression in females and, if expressed at high enough levels, reduces female viability. Lower ectopic expression leads to some MSL complex formation but does not affect female viability. Loss of function mutations in genes that function positively with MSL-2 should be detected as dominant suppressors of impaired female viability whereas loss of function mutations in the genes that function negatively should act as dominant enhancers and lead to impaired female viability. The fourth specific aim is related to several issues related to dosage compensation during embryogenesis and in non-polytene cells. Apparently dosage compensation at the earliest stages of embryogenesis is controlled by Sxl but is independent of the msl genes and the developmental stage at which msl mediate dosage compensation begins has not been determined. Experiments are proposed to determine whether or not the MSL proteins are localized to the male X-chromosomes in non-polytene chromosomes during embryogenesis. The fifth specific aim is to investigate the evolution of dosage compensation in the genus Drosophila and in other closely related genera. Immuno detection of MSL-2 proteins on salivary gland proteins of other species will be performed and msl genes from other species will be cloned. Of particularly interest is the notion that dosage compensation may have evolved independently several times since the X-chromosomes of Drosophila have undergone dramatic episodes of evolution necessitating subsequent addition of dosage compensation to chromosome arms that were previously autosomal.
