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Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions.

Figueiredo ML, Kim M, Philip P, Allgardsson A, Stenberg P, Larsson J - PLoS Genet. (2014)

Bottom Line: We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of 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.We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.

ABSTRACT
Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. 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 increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4th chromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of 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 showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

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MSL-complex has affinity to repeats.(A) Percentage of repeat masked sequences (from UCSC), in 20 bins of 1 kb distance from the transcription start site (TSS) of X-chromosome expressed genes, autosomal expressed genes, MSL1-bound/unbound genes in roX mutants, on the X-chromosome and on autosomes. Rectangles represent the mean values of repeats for all expressed genes in each distance bin, and error bars indicate 95% confidence interval. (B) DNA-FISH with a probe against the mini-white gene combined with MSL2 immunostaining, on polytene chromosomes of wild type and roX mutant males. C2 is a cluster of seven tandemly repeated copies of P[lacW] transgene, the 1A-6 cluster consists of two copies. Note that the transgene cluster consisting of seven copies recruits MSL2 in a roX mutant background. The site of the construct insertion is indicated by arrows.
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pgen-1004865-g006: MSL-complex has affinity to repeats.(A) Percentage of repeat masked sequences (from UCSC), in 20 bins of 1 kb distance from the transcription start site (TSS) of X-chromosome expressed genes, autosomal expressed genes, MSL1-bound/unbound genes in roX mutants, on the X-chromosome and on autosomes. Rectangles represent the mean values of repeats for all expressed genes in each distance bin, and error bars indicate 95% confidence interval. (B) DNA-FISH with a probe against the mini-white gene combined with MSL2 immunostaining, on polytene chromosomes of wild type and roX mutant males. C2 is a cluster of seven tandemly repeated copies of P[lacW] transgene, the 1A-6 cluster consists of two copies. Note that the transgene cluster consisting of seven copies recruits MSL2 in a roX mutant background. The site of the construct insertion is indicated by arrows.

Mentions: In addition to the Hoppel transposable element repeats, the analysis of the mapped and unmapped sequences bound by MSL in roX mutants recovered NTS sequences, which occur between ribosomal DNA genes which are organized in tandem repeats. Because MSL targets some autosomal sites across the genome in the absence of roX, we wondered whether these sites were also enriched in repeats and whether repeats in general were enough to recruit MSL. To test this hypothesis, we analyzed the enrichment of repeat masked sequences around MSL targeted regions. Since the MSL-complex mainly targets expressed genes we calculated the enrichment of repeats surrounding the TSS (transcription start site) of genes that are expressed in salivary glands (the tissue of our binding data) and are located in either MSL-bound or MSL-unbound regions of the genome. We examined both the X-chromosome and the autosomes (excluding chromosome 4 and the mapped pericentromeric regions) in this analysis, taking into account the MSL-bound regions in wild type and roX mutants that were identified based on our ChIP-seq data. The density of satellite repeats on the X-chromosome is reportedly greater than on the 2nd and 3rd chromosomes [61]–[63]. Our results are consistent with this finding and show that the regions surrounding expressed genes on the X-chromosome have a somewhat higher repeat content than those surrounding autosomal expressed genes (Fig. 6A). Strikingly, autosomal expressed genes bound by MSL in roX mutants are enriched in surrounding repeats whereas the regions surrounding unbound autosomal expressed genes have a low repeat content. The repeat content of regions surrounding X-chromosomal genes bound by MSL was also higher than that of unbound regions, but the difference was less pronounced than for autosomal genes. Our results show that MSL targeting of autosomal sites in roX mutants correlates with high repeat content.


Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions.

Figueiredo ML, Kim M, Philip P, Allgardsson A, Stenberg P, Larsson J - PLoS Genet. (2014)

MSL-complex has affinity to repeats.(A) Percentage of repeat masked sequences (from UCSC), in 20 bins of 1 kb distance from the transcription start site (TSS) of X-chromosome expressed genes, autosomal expressed genes, MSL1-bound/unbound genes in roX mutants, on the X-chromosome and on autosomes. Rectangles represent the mean values of repeats for all expressed genes in each distance bin, and error bars indicate 95% confidence interval. (B) DNA-FISH with a probe against the mini-white gene combined with MSL2 immunostaining, on polytene chromosomes of wild type and roX mutant males. C2 is a cluster of seven tandemly repeated copies of P[lacW] transgene, the 1A-6 cluster consists of two copies. Note that the transgene cluster consisting of seven copies recruits MSL2 in a roX mutant background. The site of the construct insertion is indicated by arrows.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263465&req=5

pgen-1004865-g006: MSL-complex has affinity to repeats.(A) Percentage of repeat masked sequences (from UCSC), in 20 bins of 1 kb distance from the transcription start site (TSS) of X-chromosome expressed genes, autosomal expressed genes, MSL1-bound/unbound genes in roX mutants, on the X-chromosome and on autosomes. Rectangles represent the mean values of repeats for all expressed genes in each distance bin, and error bars indicate 95% confidence interval. (B) DNA-FISH with a probe against the mini-white gene combined with MSL2 immunostaining, on polytene chromosomes of wild type and roX mutant males. C2 is a cluster of seven tandemly repeated copies of P[lacW] transgene, the 1A-6 cluster consists of two copies. Note that the transgene cluster consisting of seven copies recruits MSL2 in a roX mutant background. The site of the construct insertion is indicated by arrows.
Mentions: In addition to the Hoppel transposable element repeats, the analysis of the mapped and unmapped sequences bound by MSL in roX mutants recovered NTS sequences, which occur between ribosomal DNA genes which are organized in tandem repeats. Because MSL targets some autosomal sites across the genome in the absence of roX, we wondered whether these sites were also enriched in repeats and whether repeats in general were enough to recruit MSL. To test this hypothesis, we analyzed the enrichment of repeat masked sequences around MSL targeted regions. Since the MSL-complex mainly targets expressed genes we calculated the enrichment of repeats surrounding the TSS (transcription start site) of genes that are expressed in salivary glands (the tissue of our binding data) and are located in either MSL-bound or MSL-unbound regions of the genome. We examined both the X-chromosome and the autosomes (excluding chromosome 4 and the mapped pericentromeric regions) in this analysis, taking into account the MSL-bound regions in wild type and roX mutants that were identified based on our ChIP-seq data. The density of satellite repeats on the X-chromosome is reportedly greater than on the 2nd and 3rd chromosomes [61]–[63]. Our results are consistent with this finding and show that the regions surrounding expressed genes on the X-chromosome have a somewhat higher repeat content than those surrounding autosomal expressed genes (Fig. 6A). Strikingly, autosomal expressed genes bound by MSL in roX mutants are enriched in surrounding repeats whereas the regions surrounding unbound autosomal expressed genes have a low repeat content. The repeat content of regions surrounding X-chromosomal genes bound by MSL was also higher than that of unbound regions, but the difference was less pronounced than for autosomal genes. Our results show that MSL targeting of autosomal sites in roX mutants correlates with high repeat content.

Bottom Line: We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of 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.We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.

ABSTRACT
Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. 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 increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4th chromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of 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 showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

Show MeSH
Related in: MedlinePlus