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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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The MSL-complex is redistributed in roX mutants.(A) MSL3 immunostaining on polytene chromosomes from wild type, mle, mof and roX mutant males. Note that MSL3 only targets a subset of sites on the X-chromosome, the 4th chromosome and the chromocenter (indicated by the arrow) in roX mutants. (B) DNA-FISH with a probe against the 1.686 g/cm3 satellite repeat from pericentromeric regions of chromosomes 2 and 3 (1686 probe) combined with MSL3 immunostaining, on interphase nuclei of brain cells from third instar male larvae wild type, mof and roX mutants. (C) Percentage colocalization of 1686 probe signal and MSL3 staining from 8 replicates each of the genotypes: wild type, mof and roX (30–50 nuclei scored per replicate). The bars indicate the mean colocalization and the whiskers indicate standard deviations. Significant differences are indicated by *** (Independent two-sample t-test, p<0.001). (D) DNA-FISH with the 1686 probe combined with MSL3 immunostaining on metaphase nuclei of brain cells from third instar larvae wild type and roX mutant males. Note that on metaphase chromosomes MSL3 colocalization with centromeres is not detected in roX mutants.
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pgen-1004865-g001: The MSL-complex is redistributed in roX mutants.(A) MSL3 immunostaining on polytene chromosomes from wild type, mle, mof and roX mutant males. Note that MSL3 only targets a subset of sites on the X-chromosome, the 4th chromosome and the chromocenter (indicated by the arrow) in roX mutants. (B) DNA-FISH with a probe against the 1.686 g/cm3 satellite repeat from pericentromeric regions of chromosomes 2 and 3 (1686 probe) combined with MSL3 immunostaining, on interphase nuclei of brain cells from third instar male larvae wild type, mof and roX mutants. (C) Percentage colocalization of 1686 probe signal and MSL3 staining from 8 replicates each of the genotypes: wild type, mof and roX (30–50 nuclei scored per replicate). The bars indicate the mean colocalization and the whiskers indicate standard deviations. Significant differences are indicated by *** (Independent two-sample t-test, p<0.001). (D) DNA-FISH with the 1686 probe combined with MSL3 immunostaining on metaphase nuclei of brain cells from third instar larvae wild type and roX mutant males. Note that on metaphase chromosomes MSL3 colocalization with centromeres is not detected in roX mutants.

Mentions: To test the role of roX RNAs in MSL targeting, we performed immunostaining experiments on polytene chromosomes of roX1 roX2 double mutants (hereafter called roX mutants). In the absence of roX RNAs, the extent of MSL-complex targeting to the X-chromosome was dramatically reduced and the complex was relocalized to the chromocenter and to three distinct regions on the 4th chromosome (Fig. 1A). The disruption of MSL targeting seen in roX mutants is clearly different from the disturbance that occurs when the protein components of the complex are removed: in msl1 or msl2 mutants, no MSL-complexes are formed on the X-chromosome at all [25]. Conversely, as shown in Fig. 1A, in mle or mof mutants, the MSL-complex is exclusively targeted to a limited number of bands on the X-chromosome. This shows that the roX RNAs and the protein components of the complex have different functional roles in MSL chromatin targeting.


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)

The MSL-complex is redistributed in roX mutants.(A) MSL3 immunostaining on polytene chromosomes from wild type, mle, mof and roX mutant males. Note that MSL3 only targets a subset of sites on the X-chromosome, the 4th chromosome and the chromocenter (indicated by the arrow) in roX mutants. (B) DNA-FISH with a probe against the 1.686 g/cm3 satellite repeat from pericentromeric regions of chromosomes 2 and 3 (1686 probe) combined with MSL3 immunostaining, on interphase nuclei of brain cells from third instar male larvae wild type, mof and roX mutants. (C) Percentage colocalization of 1686 probe signal and MSL3 staining from 8 replicates each of the genotypes: wild type, mof and roX (30–50 nuclei scored per replicate). The bars indicate the mean colocalization and the whiskers indicate standard deviations. Significant differences are indicated by *** (Independent two-sample t-test, p<0.001). (D) DNA-FISH with the 1686 probe combined with MSL3 immunostaining on metaphase nuclei of brain cells from third instar larvae wild type and roX mutant males. Note that on metaphase chromosomes MSL3 colocalization with centromeres is not detected in roX mutants.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004865-g001: The MSL-complex is redistributed in roX mutants.(A) MSL3 immunostaining on polytene chromosomes from wild type, mle, mof and roX mutant males. Note that MSL3 only targets a subset of sites on the X-chromosome, the 4th chromosome and the chromocenter (indicated by the arrow) in roX mutants. (B) DNA-FISH with a probe against the 1.686 g/cm3 satellite repeat from pericentromeric regions of chromosomes 2 and 3 (1686 probe) combined with MSL3 immunostaining, on interphase nuclei of brain cells from third instar male larvae wild type, mof and roX mutants. (C) Percentage colocalization of 1686 probe signal and MSL3 staining from 8 replicates each of the genotypes: wild type, mof and roX (30–50 nuclei scored per replicate). The bars indicate the mean colocalization and the whiskers indicate standard deviations. Significant differences are indicated by *** (Independent two-sample t-test, p<0.001). (D) DNA-FISH with the 1686 probe combined with MSL3 immunostaining on metaphase nuclei of brain cells from third instar larvae wild type and roX mutant males. Note that on metaphase chromosomes MSL3 colocalization with centromeres is not detected in roX mutants.
Mentions: To test the role of roX RNAs in MSL targeting, we performed immunostaining experiments on polytene chromosomes of roX1 roX2 double mutants (hereafter called roX mutants). In the absence of roX RNAs, the extent of MSL-complex targeting to the X-chromosome was dramatically reduced and the complex was relocalized to the chromocenter and to three distinct regions on the 4th chromosome (Fig. 1A). The disruption of MSL targeting seen in roX mutants is clearly different from the disturbance that occurs when the protein components of the complex are removed: in msl1 or msl2 mutants, no MSL-complexes are formed on the X-chromosome at all [25]. Conversely, as shown in Fig. 1A, in mle or mof mutants, the MSL-complex is exclusively targeted to a limited number of bands on the X-chromosome. This shows that the roX RNAs and the protein components of the complex have different functional roles in MSL chromatin targeting.

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