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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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In roX mutants Hoppel is enriched in MSL-complex.(A) Sequence motif enriched in MSL-bound regions on heterochromatin of roX mutants. (B) MOF and MSL1 ChIP-seq enrichment profiles, with a 2000 bp smoothing, for the representative chromosome arm 3L from wild type and roX mutant males. Numbers along the x-axis denote chromosomal positions along the chromosome in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. The mapping of the motif along 3L is indicated. Below the motif track the ratio roX-MOF/WT-MOF is plotted. Note that in roX mutants MSL is enriched in the pericentromeric region (indicated by a yellow box). (C) DNA-FISH with a 33 nucleotide long probe against the heterochromatic motif shown in A combined with MSL1 immunostaining, on polytene chromosomes of roX mutant males. Note the overall colocalization between the MSL1 bound regions and the motif hybridisation.
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pgen-1004865-g004: In roX mutants Hoppel is enriched in MSL-complex.(A) Sequence motif enriched in MSL-bound regions on heterochromatin of roX mutants. (B) MOF and MSL1 ChIP-seq enrichment profiles, with a 2000 bp smoothing, for the representative chromosome arm 3L from wild type and roX mutant males. Numbers along the x-axis denote chromosomal positions along the chromosome in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. The mapping of the motif along 3L is indicated. Below the motif track the ratio roX-MOF/WT-MOF is plotted. Note that in roX mutants MSL is enriched in the pericentromeric region (indicated by a yellow box). (C) DNA-FISH with a 33 nucleotide long probe against the heterochromatic motif shown in A combined with MSL1 immunostaining, on polytene chromosomes of roX mutant males. Note the overall colocalization between the MSL1 bound regions and the motif hybridisation.

Mentions: The pericentromeric regions and the 4th chromosome are both heterochromatic regions of the D. melanogaster genome that are targeted by MSL in roX mutants and are enriched in satellite repeat sequences, transposable elements, and the heterochromatic proteins HP1a and HP2, among others. Our ChIP-seq results show that in the absence of roX RNAs, the MSL-complex targets the pericentromeric regions of all chromosomes and that its abundance increases gradually on moving towards the centromere. This tendency is illustrated for chromosome 3L in Fig. 4B. One possible mechanism underlying this binding is that MSL recognizes a specific recruitment element but that its binding to this element is blocked by the presence of roX RNAs. Another possibility is that the MSL-complex has an intrinsic affinity for heterochromatic sequences or repeats in general. Using the MEME software, we analyzed the identified MSL1-bound heterochromatic sequences of each chromosome (2LHet, 2RHet, 3LHet, 3RHet, 4Het, XHet). A specific motif corresponding to repeats in the Hoppel (1360) transposable element was found to be significantly enriched (Fig. 4A and B). In situ DNA hybridization experiments using the identified motif as a probe in conjunction with MSL staining revealed a high degree of colocalization between the MSL-complex and the motif in both the pericentromeric heterochromatin and the three specific bands on the 4th chromosome (Fig. 4C). To determine whether this motif can act as an MSL-recruitment element, we generated a construct containing three tandemly repeated copies of a 108-nucleotide Hoppel element featuring the motif in question. This repeat segment was placed upstream of a cDNA copy of ankyrin (a gene on the 4th targeted by MSL in roX mutants) under an endogenous promoter. The construct was inserted into the 3L:65B2 PhiC landing platform and tested for MSL binding in a roX mutant background. The transgene was visualized using mini-white DNA-FISH and MSL-complex was not detected on the target (S3 Figure). These results suggest that the repeat motif from the Hoppel transposable element, which is enriched at MSL-targeted regions (4th and pericentromeric) in roX mutants, is not by itself sufficient to recruit the MSL-complex. However, we cannot exclude the possibility that recruitment might be achieved with a greater number of motif copies.


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)

In roX mutants Hoppel is enriched in MSL-complex.(A) Sequence motif enriched in MSL-bound regions on heterochromatin of roX mutants. (B) MOF and MSL1 ChIP-seq enrichment profiles, with a 2000 bp smoothing, for the representative chromosome arm 3L from wild type and roX mutant males. Numbers along the x-axis denote chromosomal positions along the chromosome in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. The mapping of the motif along 3L is indicated. Below the motif track the ratio roX-MOF/WT-MOF is plotted. Note that in roX mutants MSL is enriched in the pericentromeric region (indicated by a yellow box). (C) DNA-FISH with a 33 nucleotide long probe against the heterochromatic motif shown in A combined with MSL1 immunostaining, on polytene chromosomes of roX mutant males. Note the overall colocalization between the MSL1 bound regions and the motif hybridisation.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004865-g004: In roX mutants Hoppel is enriched in MSL-complex.(A) Sequence motif enriched in MSL-bound regions on heterochromatin of roX mutants. (B) MOF and MSL1 ChIP-seq enrichment profiles, with a 2000 bp smoothing, for the representative chromosome arm 3L from wild type and roX mutant males. Numbers along the x-axis denote chromosomal positions along the chromosome in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. The mapping of the motif along 3L is indicated. Below the motif track the ratio roX-MOF/WT-MOF is plotted. Note that in roX mutants MSL is enriched in the pericentromeric region (indicated by a yellow box). (C) DNA-FISH with a 33 nucleotide long probe against the heterochromatic motif shown in A combined with MSL1 immunostaining, on polytene chromosomes of roX mutant males. Note the overall colocalization between the MSL1 bound regions and the motif hybridisation.
Mentions: The pericentromeric regions and the 4th chromosome are both heterochromatic regions of the D. melanogaster genome that are targeted by MSL in roX mutants and are enriched in satellite repeat sequences, transposable elements, and the heterochromatic proteins HP1a and HP2, among others. Our ChIP-seq results show that in the absence of roX RNAs, the MSL-complex targets the pericentromeric regions of all chromosomes and that its abundance increases gradually on moving towards the centromere. This tendency is illustrated for chromosome 3L in Fig. 4B. One possible mechanism underlying this binding is that MSL recognizes a specific recruitment element but that its binding to this element is blocked by the presence of roX RNAs. Another possibility is that the MSL-complex has an intrinsic affinity for heterochromatic sequences or repeats in general. Using the MEME software, we analyzed the identified MSL1-bound heterochromatic sequences of each chromosome (2LHet, 2RHet, 3LHet, 3RHet, 4Het, XHet). A specific motif corresponding to repeats in the Hoppel (1360) transposable element was found to be significantly enriched (Fig. 4A and B). In situ DNA hybridization experiments using the identified motif as a probe in conjunction with MSL staining revealed a high degree of colocalization between the MSL-complex and the motif in both the pericentromeric heterochromatin and the three specific bands on the 4th chromosome (Fig. 4C). To determine whether this motif can act as an MSL-recruitment element, we generated a construct containing three tandemly repeated copies of a 108-nucleotide Hoppel element featuring the motif in question. This repeat segment was placed upstream of a cDNA copy of ankyrin (a gene on the 4th targeted by MSL in roX mutants) under an endogenous promoter. The construct was inserted into the 3L:65B2 PhiC landing platform and tested for MSL binding in a roX mutant background. The transgene was visualized using mini-white DNA-FISH and MSL-complex was not detected on the target (S3 Figure). These results suggest that the repeat motif from the Hoppel transposable element, which is enriched at MSL-targeted regions (4th and pericentromeric) in roX mutants, is not by itself sufficient to recruit the MSL-complex. However, we cannot exclude the possibility that recruitment might be achieved with a greater number of motif copies.

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