Academic journal article Genetics

roX RNAs Are Required for Increased Expression of X-Linked Genes in Drosophila Melanogaster Males

Academic journal article Genetics

roX RNAs Are Required for Increased Expression of X-Linked Genes in Drosophila Melanogaster Males

Article excerpt

ABSTRACT

The male-specific lethal (MSL) ribonucleoprotein complex is necessary for equalization of X:A expression levels in Drosophila males, which have a single X chromosome. It binds selectively to the male X chromosome and directs acetylation of histone H4 at lysine 16 (H4Ac16), a modification linked to elevated transcription. roX1 and roX2 noncoding RNAs are essential but redundant components of this complex. Simultaneous removal of both roX RNAs reduces X localization of the MSL proteins and permits their ectopic binding to autosomal sites and the chromocenter. However, the MSL proteins still colocalize, and low levels of H4Ac16 are detected at ectopic sites of MSL binding and residual sites on the X chromosome of roX1^sup -^ roX2^sup -^ males. Microarray analysis was performed to reveal the effect of roX1 and roX2 elimination on X-linked and autosomal gene expression. Expression of the X chromosome is decreased by 26% in roX1^sup -^ roX2^sup -^ male larvae. Enhanced expression could not be detected at autosomal sites of MSL binding in roX1^sup -^ roX2^sup -^ males. These results implicate failure to compensate X-linked genes, rather than inappropriate upregulation of autosomal genes at ectopic sites of MSL binding, as the primary cause of male lethality upon loss of roX RNAs.

DROSOPHILA males have one X chromosome and two sets of autosomes (A). The imbalance in X:A gene dosage is addressed by the male-specific lethal (MSL) complex that binds to hundreds of sites along the male X chromosome. While there is general agreement that this complex is responsible for dosage compensation, disagreement about how compensation is accomplished remains (BIRCHLER et al. 2003; STRUB et al. 2005a). Two recent studies provide strong evidence that binding of the MSL complex to the X chromosome of Drosophila males increases transcription from almost all X-linked genes (HAMADA et al. 2005; STRUB et al. 2005b). The MSL complex is composed of five proteins, encoded by the genes maleless (mle), malespecific lethal 1, 2, and 3 (msl1, -2, and -3), and males absent on first (mof) (FUKUNAGA et al. 1975; BELOTE and LUCCHESI 1980; UCHIDA et al. 1981; HILFIKER et al. 1997). A sixth protein, JIL-1, is required in both sexes but is enriched on the male X chromosome (JIN et al. 2000). One of the primary functions of the MSL complex is thought to be acetylation of histone H4 at lysine 16 (H4Ac16), a modification associated with increased expression and attributable to MOF, a histone acetyltransferase (AKHTAR and BECKER 2000; SMITH et al. 2000). Two noncoding RNAs, roX1 and roX2, are integral components of the MSL complex and necessary for exclusive binding to the X chromosome (MELLER and RATTNER 2002). Simultaneous mutation of both roX genes causes male lethality, but mutation of a single roX gene is without phenotype.

Removal of MSL1 or MSL2 prevents any chromatin binding by the remaining subunits. Mutants of mle, msl3, or mof retain a partial MSL complex at ~35 sites on the X chromosome (GORMAN et al. 1995; LYMAN et al. 1997; GU et al. 1998). By contrast, simultaneous mutation of both roX genes prevents the exclusive binding of MSL proteins to the X chromosome. Reduced levels of MSL proteins are retained at some sites on the X chromosome, but they are now detected at a number of ectopic autosomal sites (MELLER and RATTNER 2002; DENG et al. 2005). Some of these sites are puffed, a chromatin state usually associated with active transcription. Failure to increase expression of X-linked genes could cause male lethality. However, it is possible that elevated transcription at a small number of autosomal sites contributes to male lethality.

To address global changes in gene expression in roX1^sup -^ roX2^sup -^ males, we turned to microarray analysis. Previous studies examining the expression of a limited number of genes in msl mutant larvae produced complex, and sometimes conflicting, data (BHADRA et al. 1999, 2000, 2005; CHIANG and KURNIT 2003). …

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