Recruitment of the MSL proteins to X chromatin requires DNA sequences within the roX genes. H. Amrein , L. Dunipace. Dept Genetics, Duke Univ Medical Ctr, Durham, NC.

   Dosage compensation requires the function of at least five proteins encoded by the male-specific lethal genes (msls). These proteins all colocalize to hundreds of sites on the male X-chromosome where they modify chromatin such that most X-linked genes are hypertranscribed by about twofold. We are interested in how these proteins specifically recognize the X-chromosome and avoid association with the autosomes. The identification and characterization of two X-linked genes, roX1 and roX2, has provided a means to explain this X-chromosome specificity (Meller et al., Amrein and Axel, 1997). roX1 and roX2 are regulatory RNAs that are only expressed in the male where they colocalize with the MSL proteins to the single male X chromosome. It has been shown recently that autosomal roX1 transgenes can recruit MSL proteins to their autosomal locations and spread up to several 100 kb into autosomal DNA (Kelley et al. 1999). Using inducible roX transgenes, we present evidence that roX transcription is not necessary for recruitment of the MSL proteins to autosomal roX transgenes. Thus, DNA within the roX genes rather then roX RNA is responsible for initial recruitment of the MSL protein complex. A deletion analysis of the roX genes and the ability of such deletion constructs to recruit the MSL complex in vivo indicate that at least two distinct DNA sequence motifs are sufficient to recruit the MSL complex to autosomal sites. Furthermore, we investigated the consequences of multiple autosomal roX transgenes on both autosomal MSL complex formation and male viability. Taken together, our data lead us to propose a two step mechanism of male X chromatin-MSL complex association. First, the MSL complex is recruited to high affinity sites - among them the roX genes - via DNA-protein interactions and second, the MSL-roX riboprotein complex spreads from these sites in cis along the entire X-chromosome, which requires RNA/DNA or riboprotein/DNA interactions, or both.