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Non-coding roX RNAs Prevent the Binding of the MSL-complex to Heterochromatic Regions

ABSTRACT: Long non-coding RNAs are involved in dosage compensation both in mammals and in Drosophila by inducing changes in the X-chromosome chromatin structure. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increased transcriptional output. It has been shown that in polytene chromosomes, in absence of both roX1 and roX2, the MSL-complex is decreased on the male X-chromosome and found relocated to the chromocenter, and the 4th chromosome. Here we address the role of roX RNAs in MSL-complex targeting and in the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments and show that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent on roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4th chromosome. Interestingly, our sequence analysis shows that in the absence of roX RNAs, the MSL-complex has affinity to regions enriched in Hoppel transposable elements and to repeats in general. We hypothesize that roX mutants reveal an ancient targeting of the MSL-complex and propose that the role of roX RNAs is to restrict MSL-complex from binding to heterochromatin.

ORGANISM(S): Drosophila simulans Drosophila melanogaster

PROVIDER: GSE58768 | GEO | 2014/11/07

SECONDARY ACCESSION(S): PRJNA253407

REPOSITORIES: GEO

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Project description:roX RNAs are involved in the chromosome-wide gene regulation that occurs during dosage compensation in Drosophila. Dosage compensation equalizes expression of X-linked and autosomal genes. Drosophila males increase transcription two-fold from their single X chromosome. This is mediated by the MSL complex, which is composed of the male-specific lethal (MSL) proteins and two noncoding roX RNAs, roX1 and roX2. Upon elimination of both roX transcripts, a global decrease of X-linked gene expression is observed in males. Expression of the genes on the entire 4th chromosome also decreased in the absence of both roX transcripts. roX1 RNA also presents in females in the early stages. To investigate the effect of loss of roX transcripts on gene expression in females, gene expression was analyzed by microarrays in roX1-roX2- female flies. To eliminate inconsistency caused by differences in genetic background, expression of roX1-roX2- females with females of virtually identical genetic background but carrying the [hsp83-roX1+] transgene were compared. Expression of any chromosome did not change in roX1-roX2- females. It was concluded that roX RNAs only effect in males . Experiment Overall Design: Total RNA was prepared from groups of 50 third instar larvae by TRIzol (Invitrogen) extraction and purified using the RNeasy kit (Qiagen). Three independent RNA preparations for each genotype served as templates for probe synthesis. These probes were hybridized to Affymetrix Drosophila Genome 2.0 chips (Santa Clara, CA). Genes were filtered for present/absent calls by a PM-MM (Perfect match-Mismatch) comparison. Affymetrix Gene expression data was background corrected, normalized and summarized into a one expression value per replicate sample and probeset using the RMA (robust multi-array average) algorithm. Changes in gene expression were determined by comparing the mean signal intensities of genes on arrays hybridized with roX1- roX2- (mutant) probes to those hybridized with roX1- roX2-; [hsp83-roX1+] (control) probes.

Project description:Drosophila males double transcription of their single X chromosome to equalize X-linked gene expression with females, which carry two X chromosomes. Increased transcription requires the Male-Specific Lethal (MSL) complex. One of the primary functions of the MSL complex is thought to be enrichment of H4Ac16 on the male X chromosome, a modification linked to elevated transcription. The roX1 and roX2 RNAs are essential but redundant components of the MSL complex. Simultaneous removal of both roX RNAs reduces MSL X-localization and leads to ectopic binding of these proteins at autosomal sites and to the chromocenter. Some H4Ac16 accumulates at these ectopic sites in roX1- roX2- males, suggesting the possibility of increased expression. The global effect of roX mutations on gene expression was measured by microarray analysis. We found that expression of the X chromosome was decreased by 26% in roX1- roX2- male larvae, supporting the involvement of roX RNAs in the up-regulation of X-linked genes. This finding is broadly comparable to reports of reduced X chromosome expression following msl2 RNAi knockdown in S2 cells. In spite of strong MSL binding and H4Ac16 accumulation at autosomal sites in roX1- roX2- males, enhanced gene expression could not be detected at these sites by microarray analysis or reverse northern blotting. Thus, failure to compensate X-linked genes, rather than inappropriate up-regulation of autosomal genes at ectopic sites of MSL binding, appears to cause male lethality upon loss of roX RNAs. Experiment Overall Design: Total RNA was prepared from groups of 50 third instar larvae by TRIzol (Invitrogen) extraction and purified using the RNeasy kit (Qiagen). Three independent RNA preparations for each genotype served as templates for probe synthesis. Affymetrix Drosophila Genome 2.0 chips were hybridized to these probes (Santa Clara, CA). The affymertrix Drosophila annotation of December 2004 was used to map genes to their cytological locations. Genes were filtered for present/absent calls by a PM-MM (Perfect match- Mismatch) comparison. Autosomal transcripts were normalized on a chip-by-chip basis to bring their median values to 100. The identical degree of adjustment was used to normalize X-linked transcripts. Changes in gene expression were determined by comparing the mean signal intensities of genes on arrays hybridized with roX1SMC17A roX2- probes to those hybridized with roX1+ roX2- probes.

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Non-coding roX RNAs Prevent the Binding of the MSL-complex to Heterochromatic Regions

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