Multiple classes of MSL binding sites target dosage compensation to the X chromosome of Drosophila

MSL complexes bind hundreds of sites along the single male X chromosome to achieve dosage compensation in Drosophila. Previously, we proposed that approximately 35 "high-affinity" or "chromatin entry" sites (CES) might nucleate spreading of MSL complexes in cis to paint the X chromosome. This was based on analysis of the first characterized sites roX1 and roX2. roX transgenes attract MSL complex to autosomal locations where it can spread long distances into flanking chromatin. roX1 and roX2 also produce noncoding RNA components of the complex. Here we identify a third site from the 18D10 region of the X chromosome. Like roX genes, 18D binds full and partial MSL complexes in vivo and encompasses a male-specific DNase I hypersensitive site (DHS). Unlike roX genes, the 510 bp 18D site is apparently not transcribed and shows high affinity for MSL complex and spreading only as a multimer. While mapping 18D, we discovered MSL binding to X cosmids that do not carry one of the approximately 35 high-affinity sites. Based on additional analyses of chromosomal transpositions, we conclude that spreading in cis from the roX genes or the approximately 35 originally proposed "entry sites" cannot be the sole mechanism for MSL targeting to the X chromosome.     

Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome to increase its expression approximately 2-fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at approximately 35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are sufficient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows male-specific DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions.     

Functional redundancy within roX1, a noncoding RNA involved in dosage compensation in Drosophila melanogaster

Drosophila melanogaster males dosage compensate by twofold upregulation of the expression of genes on their single X chromosome. This process requires at least five proteins and two noncoding RNAs, roX1 and roX2, which paint the male X chromosome. We used a deletion analysis to search for functional RNA domains within roX1, assaying RNA stability, targeting of the MSL proteins to the X, and rescue of male viability in a roX1(-) roX2(-) mutant background. We found that deletion of 10% segments of the RNA did not dramatically reduce function in most cases, suggesting extensive internal redundancy. The 3' 600 nt of roX1 were most sensitive to mutations, affecting proper localization and 3' processing of the RNA. Disruption of an inverted repeat predicted to form a stem-loop structure was found partially responsible for the defects observed.