The multisubunit MSL dosage compensation complex binds to hundreds of sites along the Drosophila single male X chromosome, mediating its hypertranscription. The male X chromosome is also coated with noncoding roX RNAs. When either msl3, mle, or mof is mutant, a partial MSL complex is bound at only approximately 35 unusual sites distributed along the X. We show that two of these sites are the roX1 and roX2 genes and postulate that one of their functions is to provide entry sites for the MSL complex to recognize the X chromosome. The roX1 gene provides a nucleation site for extensive spreading of the MSL complex into flanking chromatin even when moved to an autosome. The spreading can occur in cis or in trans between paired homologs. We present a model for how the dosage compensation complex recognizes X chromatin.

The roX1 and roX2 genes of Drosophila produce malespeci®c non-coding RNAs that co-localize with the Male-Speci®c Lethal (MSL) protein complex. This complex mediates up-regulation of the male X chromosome by increasing histone H4 acetylation, thus contributing to the equalization of X-linked gene expression between the sexes. Both roX genes overlap two of~35 chromatin entry sites, DNA sequences proposed to act in cis to direct the MSL complex to the X chromosome. Although dosage compensation is essential in males, an intact roX1 gene is not required by either sex. We have generated¯ies lacking roX2 and ®nd that this gene is also non-essential. However, simultaneous removal of both roX RNAs causes a striking male-speci®c reduction in viability accompanied by relocation of the MSL proteins and acetylated histone H4 from the X chromosome to autosomal sites and heterochromatin. Males can be rescued by roX cDNAs from autosomal transgenes, demonstrating the genetic separation of the chromatin entry and RNA-encoding functions. Therefore, the roX1 and roX2 genes produce redundant, male-speci®c lethal transcripts required for targeting the MSL complex.

The Drosophila roX1 gene is X-linked and produces RNAs that are male-specific, somatic, and preferentially expressed in the central nervous system. These RNAs are retained in the nucleus and lack any significant open reading frame. Although all sexually dimorphic characteristics in Drosophila were thought to be controlled by the sex determination pathway through the gene transformer (tra), the expression of roX1 is independent of tra activity. Instead, the dosage compensation system is necessary and sufficient for the expression of roX1. Consistent with a potential function in dosage compensation, roX1 RNAs localize specifically to the male X chromosome. This localization occurs even when roX1 RNAs are expressed from autosomal locations in X-to-autosome translocations. The novel regulation and subnuclear localization of roX1 RNAs makes them candidates for an RNA component of the dosage compensation machinery.

Our results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome.

The untranslated roX1 and roX2 RNAs are components of the Drosophila male-specific lethal (MSL) complex, which modifies histones to up-regulate transcription of the male X chromosome. roX genes are normally located on the X chromosome, and roX transgenes can misdirect the dosage compensation machinery to spread locally on other chromosomes. Here we define MSL protein abundance as a determinant of whether the MSL complex will spread in cis from an autosomal roX transgene. The number of expressed roX genes in a nucleus was inversely correlated with spreading from roX transgenes. We suggest a model in which MSL proteins assemble into active complexes by binding nascent roX transcripts. When MSL protein/roX RNA ratios are high, assembly will be efficient, and complexes may be completed while still tethered to the DNA template. We propose that this local production of MSL complexes determines the extent of spreading into flanking chromatin.