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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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A complete and enzymatically active MSL-complex is assembled in the absence of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for the entire 4th chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote chromosomal 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. Note that the genes targeted in roX mutants (indicated by red boxes) correspond to the three bands seen in polytene chromosome staining (below). (B) MSL1, MSL2, MSL3, MLE, MOF and H4K16ac immunostaining on polytene chromosomes from wild type males, with X-chromosome targeting, and from roX mutant males, showing the 4th chromosome and chromocenter targeting. (C) JIL1 immunostaining on polytene chromosomes from wild type and roX mutants males, shows targeting to the chromocenter and to the same chromosome 4 bands as the MSL-complex. (D) Mean levels of mRNA from the six genes targeted by MSL in roX mutants and from a control gene on the 4th chromosome that is not bound by MSL (MED26), determined by rt-qPCR (black). The corresponding mean expression of the same genes in wild type is shown in grey. The mRNA levels measured by qPCR were normalized against RpL32 mRNA in each replicate. Error bars represent the standard deviation of three biological replicates.
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pgen-1004865-g003: A complete and enzymatically active MSL-complex is assembled in the absence of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for the entire 4th chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote chromosomal 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. Note that the genes targeted in roX mutants (indicated by red boxes) correspond to the three bands seen in polytene chromosome staining (below). (B) MSL1, MSL2, MSL3, MLE, MOF and H4K16ac immunostaining on polytene chromosomes from wild type males, with X-chromosome targeting, and from roX mutant males, showing the 4th chromosome and chromocenter targeting. (C) JIL1 immunostaining on polytene chromosomes from wild type and roX mutants males, shows targeting to the chromocenter and to the same chromosome 4 bands as the MSL-complex. (D) Mean levels of mRNA from the six genes targeted by MSL in roX mutants and from a control gene on the 4th chromosome that is not bound by MSL (MED26), determined by rt-qPCR (black). The corresponding mean expression of the same genes in wild type is shown in grey. The mRNA levels measured by qPCR were normalized against RpL32 mRNA in each replicate. Error bars represent the standard deviation of three biological replicates.

Mentions: The binding of MSL to the 4th chromosome in the absence of roX RNAs is intriguing because there are several lines of evidence suggesting an evolutionary relationship between the 4th chromosome and the X-chromosome [1], [50]–[52]. Our ChIP-seq profiles show that the MSL-complex binds specifically to six genes on the 4th chromosome in roX mutants: Ankyrin, Rad23, CG2177, PMCA, Mitf and Dyrk3. The locations of these genes correspond to those of the MSL-stained bands seen on polytene chromosomes (Fig. 3A). One important question when considering the binding of MSL outside the X-chromosome is whether a complete and functional MSL-complex is formed at these locations. Our immunostaining experiments in roX mutants showed that all of the complex's protein components (MSL1, MSL2, MSL3, MLE and MOF) colocalize perfectly at the chromocenter and at the three bands on the 4th chromosome (Fig. 3B). In addition H4K16ac is also enriched at these three bands in roX mutants, which indicates that the MSL-complex is complete and active (Fig. 3B and S1 Figure). Note that H4K16ac on the 4th chromosome shows a broader enrichment pattern compared to the MSL proteins in similarity to what previously have been observed for H4K16ac in relation to MSL on the male X-chromosome in wild type [10]. Next we tested the H3S10 kinase JIL1, previously shown to be enriched on the male X-chromosome and dependent on a functional MSL-complex for its targeting [53]–[55]. JIL1 has previously been shown to co-immunoprecipitate with the MSL-complex under low stringency conditions or after formaldehyde cross-linking [54], [56]. Interestingly, like the MSL-complex, JIL1 is also relocalized to the chromocenter and the three regions on the 4th in the absence of roX RNAs (Fig. 3C).


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)

A complete and enzymatically active MSL-complex is assembled in the absence of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for the entire 4th chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote chromosomal 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. Note that the genes targeted in roX mutants (indicated by red boxes) correspond to the three bands seen in polytene chromosome staining (below). (B) MSL1, MSL2, MSL3, MLE, MOF and H4K16ac immunostaining on polytene chromosomes from wild type males, with X-chromosome targeting, and from roX mutant males, showing the 4th chromosome and chromocenter targeting. (C) JIL1 immunostaining on polytene chromosomes from wild type and roX mutants males, shows targeting to the chromocenter and to the same chromosome 4 bands as the MSL-complex. (D) Mean levels of mRNA from the six genes targeted by MSL in roX mutants and from a control gene on the 4th chromosome that is not bound by MSL (MED26), determined by rt-qPCR (black). The corresponding mean expression of the same genes in wild type is shown in grey. The mRNA levels measured by qPCR were normalized against RpL32 mRNA in each replicate. Error bars represent the standard deviation of three biological replicates.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1004865-g003: A complete and enzymatically active MSL-complex is assembled in the absence of roX.(A) MOF and MSL1 ChIP-seq enrichment profiles, with a 500 bp smoothing, for the entire 4th chromosome in salivary gland tissue from wild type and roX mutant males. Numbers along the x-axis denote chromosomal 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. Note that the genes targeted in roX mutants (indicated by red boxes) correspond to the three bands seen in polytene chromosome staining (below). (B) MSL1, MSL2, MSL3, MLE, MOF and H4K16ac immunostaining on polytene chromosomes from wild type males, with X-chromosome targeting, and from roX mutant males, showing the 4th chromosome and chromocenter targeting. (C) JIL1 immunostaining on polytene chromosomes from wild type and roX mutants males, shows targeting to the chromocenter and to the same chromosome 4 bands as the MSL-complex. (D) Mean levels of mRNA from the six genes targeted by MSL in roX mutants and from a control gene on the 4th chromosome that is not bound by MSL (MED26), determined by rt-qPCR (black). The corresponding mean expression of the same genes in wild type is shown in grey. The mRNA levels measured by qPCR were normalized against RpL32 mRNA in each replicate. Error bars represent the standard deviation of three biological replicates.
Mentions: The binding of MSL to the 4th chromosome in the absence of roX RNAs is intriguing because there are several lines of evidence suggesting an evolutionary relationship between the 4th chromosome and the X-chromosome [1], [50]–[52]. Our ChIP-seq profiles show that the MSL-complex binds specifically to six genes on the 4th chromosome in roX mutants: Ankyrin, Rad23, CG2177, PMCA, Mitf and Dyrk3. The locations of these genes correspond to those of the MSL-stained bands seen on polytene chromosomes (Fig. 3A). One important question when considering the binding of MSL outside the X-chromosome is whether a complete and functional MSL-complex is formed at these locations. Our immunostaining experiments in roX mutants showed that all of the complex's protein components (MSL1, MSL2, MSL3, MLE and MOF) colocalize perfectly at the chromocenter and at the three bands on the 4th chromosome (Fig. 3B). In addition H4K16ac is also enriched at these three bands in roX mutants, which indicates that the MSL-complex is complete and active (Fig. 3B and S1 Figure). Note that H4K16ac on the 4th chromosome shows a broader enrichment pattern compared to the MSL proteins in similarity to what previously have been observed for H4K16ac in relation to MSL on the male X-chromosome in wild type [10]. Next we tested the H3S10 kinase JIL1, previously shown to be enriched on the male X-chromosome and dependent on a functional MSL-complex for its targeting [53]–[55]. JIL1 has previously been shown to co-immunoprecipitate with the MSL-complex under low stringency conditions or after formaldehyde cross-linking [54], [56]. Interestingly, like the MSL-complex, JIL1 is also relocalized to the chromocenter and the three regions on the 4th in the absence of roX RNAs (Fig. 3C).

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