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.     

Modulation of Heterochromatin by Male Specific Lethal Proteins and roX RNA in Drosophila melanogaster Males

The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila melanogaster males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. To explore the molecular basis of this observation we examined additional proteins that participate in compensation and found that MLE, but not Jil-1 kinase, contributes to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, we tested a panel of roX1 transgenes and deletions and find that the X chromosome and heterochromatin functions are separable by some mutations. Chromatin immunoprecipitation of staged embryos revealed widespread autosomal binding of MSL3 before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. The broad localization of these proteins early in embryogenesis supports the idea of direct action at autosomal sites. We postulate that this may contribute to the sex-specific differences in heterochromatin that we, and others, have noted.     

Incorporation of the noncoding roX RNAs alters the chromatin-binding specificity of the Drosophila MSL1/MSL2 complex

The male-specific lethal (MSL) protein-RNA complex is required for X chromosome dosage compensation in Drosophila melanogaster. The MSL2 and MSL1 proteins form a complex and are essential for X chromosome binding. In addition, the MSL complex must integrate at least one of the noncoding roX RNAs for normal X chromosome binding. Here we find the amino-terminal RING finger domain of MSL2 binds as a complex with MSL1 to the heterochromatic chromocenter and a few sites on the chromosome arms. This binding required the same amino-terminal basic motif of MSL1 previously shown to be essential for binding to high-affinity sites on the X chromosome. While the RING finger domain of MSL2 is sufficient to increase the expression of roX1 in females, activation of roX2 requires motifs in the carboxyl-terminal domain. Binding to hundreds of sites on the X chromosome and efficient incorporation of the roX RNAs into the MSL complex require proline-rich and basic motifs in the carboxyl-terminal domain of MSL2. We suggest that incorporation of the roX RNAs into the MSL complex alters the binding specificity of the chromatin-binding module formed by the amino-terminal domains of MSL1 and MSL2.     

A sequence motif within chromatin entry sites directs MSL establishment on the Drosophila X chromosome

The Drosophila MSL complex associates with active genes specifically on the male X chromosome to acetylate histone H4 at lysine 16 and increase expression approximately 2-fold. To date, no DNA sequence has been discovered to explain the specificity of MSL binding. We hypothesized that sequence-specific targeting occurs at "chromatin entry sites," but the majority of sites are sequence independent. Here we characterize 150 potential entry sites by ChIP-chip and ChIP-seq and discover a GA-rich MSL recognition element (MRE). The motif is only slightly enriched on the X chromosome ( approximately 2-fold), but this is doubled when considering its preferential location within or 3' to active genes (>4-fold enrichment). When inserted on an autosome, a newly identified site can direct local MSL spreading to flanking active genes. These results provide strong evidence for both sequence-dependent and -independent steps in MSL targeting of dosage compensation to the male X chromosome.