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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 recruitment to the conserved HAS is independent of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for a representative region of the X-chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote positions along the chromosome in kb. The y-axis shows the ChIP enrichment over input as log2 ratios. Genes expressed from left to right and vice versa are shown above and below the horizontal lines, respectively. The HAS locations, previously defined by [34], [37], [38], are indicated by grey boxes. (B) Fraction of sites on the X-chromosome from roX mutants that are bound by MSL1, compared to random sites along the X-chromosome (red), sorted by distance to HAS. (C) MSL1 ChIP-seq enrichment profile, with a 500 bp smoothing, for the entirety of chromosomes X and 2L in salivary glands from D. simulans wild type males. Numbers along the x-axis denote chromosomal positions in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. (D) Sequence motifs enriched in MSL-bound regions of the X-chromosome in D. melanogaster wild type [37], roX mutants and D. simulans wild type.
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pgen-1004865-g002: MSL recruitment to the conserved HAS is independent of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for a representative region of the X-chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote positions along the chromosome in kb. The y-axis shows the ChIP enrichment over input as log2 ratios. Genes expressed from left to right and vice versa are shown above and below the horizontal lines, respectively. The HAS locations, previously defined by [34], [37], [38], are indicated by grey boxes. (B) Fraction of sites on the X-chromosome from roX mutants that are bound by MSL1, compared to random sites along the X-chromosome (red), sorted by distance to HAS. (C) MSL1 ChIP-seq enrichment profile, with a 500 bp smoothing, for the entirety of chromosomes X and 2L in salivary glands from D. simulans wild type males. Numbers along the x-axis denote chromosomal positions in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. (D) Sequence motifs enriched in MSL-bound regions of the X-chromosome in D. melanogaster wild type [37], roX mutants and D. simulans wild type.

Mentions: To better understand how the genome-wide targeting of MSL depends on roX RNAs, we performed MSL1, MSL2 and MOF ChIP-seq experiments on salivary glands from wild type individuals and roX mutants. These experiments confirmed the results of the immunostaining studies, showing that there is a pronounced decrease in MSL binding along the X-chromosome in roX mutants although binding persists at specific locations. Visual inspection demonstrates that the MSL enrichment peaks along the X-chromosome in roX mutants coincide with the previously defined HAS (Fig. 2A) [34], [37], [38]. Notably, although all previously defined HAS are not recognized by MSL enrichment in roX mutants, all enrichment peaks coincide with HAS. To verify that the roX RNA-independent MSL enrichment peaks on the X-chromosome correspond to the previously mapped HAS, we calculated the shortest distance between the coordinates of the HAS and those of the MSL1 binding sites on the X-chromosome in roX mutants identified in our ChIP-seq experiments. The fractions of sites bound by MSL1 in roX mutants were plotted against distance to nearest HAS, and the distances between the coordinates of HAS and random positions on the X-chromosome were used as controls. As seen in Fig. 2B, the largest fraction of the MSL binding sites in roX mutants overlap with HAS. This is in clear contrast to the control, in which the largest fraction of random X-chromosome sites are>35 kb away from HAS. Taken together this means that although all of the 263 previously defined HAS are not bound by MSL in roX mutants, in principle all of our 208 defined MSL binding sites in roX mutants target HAS. These results demonstrate that MSL binding to HAS on the X-chromosome occurs independently of roX RNAs.


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 recruitment to the conserved HAS is independent of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for a representative region of the X-chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote positions along the chromosome in kb. The y-axis shows the ChIP enrichment over input as log2 ratios. Genes expressed from left to right and vice versa are shown above and below the horizontal lines, respectively. The HAS locations, previously defined by [34], [37], [38], are indicated by grey boxes. (B) Fraction of sites on the X-chromosome from roX mutants that are bound by MSL1, compared to random sites along the X-chromosome (red), sorted by distance to HAS. (C) MSL1 ChIP-seq enrichment profile, with a 500 bp smoothing, for the entirety of chromosomes X and 2L in salivary glands from D. simulans wild type males. Numbers along the x-axis denote chromosomal positions in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. (D) Sequence motifs enriched in MSL-bound regions of the X-chromosome in D. melanogaster wild type [37], roX mutants and D. simulans wild type.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004865-g002: MSL recruitment to the conserved HAS is independent of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for a representative region of the X-chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote positions along the chromosome in kb. The y-axis shows the ChIP enrichment over input as log2 ratios. Genes expressed from left to right and vice versa are shown above and below the horizontal lines, respectively. The HAS locations, previously defined by [34], [37], [38], are indicated by grey boxes. (B) Fraction of sites on the X-chromosome from roX mutants that are bound by MSL1, compared to random sites along the X-chromosome (red), sorted by distance to HAS. (C) MSL1 ChIP-seq enrichment profile, with a 500 bp smoothing, for the entirety of chromosomes X and 2L in salivary glands from D. simulans wild type males. Numbers along the x-axis denote chromosomal positions in Mb. The y-axis shows the ChIP enrichment over input as log2 ratios. (D) Sequence motifs enriched in MSL-bound regions of the X-chromosome in D. melanogaster wild type [37], roX mutants and D. simulans wild type.
Mentions: To better understand how the genome-wide targeting of MSL depends on roX RNAs, we performed MSL1, MSL2 and MOF ChIP-seq experiments on salivary glands from wild type individuals and roX mutants. These experiments confirmed the results of the immunostaining studies, showing that there is a pronounced decrease in MSL binding along the X-chromosome in roX mutants although binding persists at specific locations. Visual inspection demonstrates that the MSL enrichment peaks along the X-chromosome in roX mutants coincide with the previously defined HAS (Fig. 2A) [34], [37], [38]. Notably, although all previously defined HAS are not recognized by MSL enrichment in roX mutants, all enrichment peaks coincide with HAS. To verify that the roX RNA-independent MSL enrichment peaks on the X-chromosome correspond to the previously mapped HAS, we calculated the shortest distance between the coordinates of the HAS and those of the MSL1 binding sites on the X-chromosome in roX mutants identified in our ChIP-seq experiments. The fractions of sites bound by MSL1 in roX mutants were plotted against distance to nearest HAS, and the distances between the coordinates of HAS and random positions on the X-chromosome were used as controls. As seen in Fig. 2B, the largest fraction of the MSL binding sites in roX mutants overlap with HAS. This is in clear contrast to the control, in which the largest fraction of random X-chromosome sites are>35 kb away from HAS. Taken together this means that although all of the 263 previously defined HAS are not bound by MSL in roX mutants, in principle all of our 208 defined MSL binding sites in roX mutants target HAS. These results demonstrate that MSL binding to HAS on the X-chromosome occurs independently of roX RNAs.

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