Limits...
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.

Show MeSH

Related in: MedlinePlus

MSL-complex targets PROTOP and NTS in roX mutants.(A) Reads from the ChIP samples mapped to repeat classes from Repbase Update are shown on the x-axis as RPKM values (Reads Per Kilobase per Million mapped reads) and related to ChIP/input enrichment ratios, which are shown on the y-axis. The left part shows MSL1 ChIP in roX mutants compared to MSL2 in wild type and the right part shows MOF ChIP in roX mutants and wild type. Note, that NTS is enriched also in MOF wild type. (B) Enrichment ratio (IP/input) of the overrepresented repeats from A (NTS, PROTOP_A, PROTOP_B) in roX mutants compared to the wild type. Rectangles represent the mean values of the IP/input enrichment ratios of all mapped and unmapped reads matching to each repeat type, and error bars indicate 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263465&req=5

pgen-1004865-g005: MSL-complex targets PROTOP and NTS in roX mutants.(A) Reads from the ChIP samples mapped to repeat classes from Repbase Update are shown on the x-axis as RPKM values (Reads Per Kilobase per Million mapped reads) and related to ChIP/input enrichment ratios, which are shown on the y-axis. The left part shows MSL1 ChIP in roX mutants compared to MSL2 in wild type and the right part shows MOF ChIP in roX mutants and wild type. Note, that NTS is enriched also in MOF wild type. (B) Enrichment ratio (IP/input) of the overrepresented repeats from A (NTS, PROTOP_A, PROTOP_B) in roX mutants compared to the wild type. Rectangles represent the mean values of the IP/input enrichment ratios of all mapped and unmapped reads matching to each repeat type, and error bars indicate 95% confidence intervals.

Mentions: DNA sequences from centromeres, telomeres, the Y-chromosome and other heterochromatic regions are not assembled to any region of the D. melanogaster genome due to their highly repeated nature, and the mapping of sequences recovered in the ChIP-seq normally discards the large number of repeated sequences in the genome. We suspected that the non-mapped reads recovered by ChIP-seq might hold information about other transposable elements targeted by MSL in roX mutants in the above-mentioned heterochromatic regions. To test this hypothesis, we aligned all of the ChIP-seq reads to the repeat class sequences from the Repbase Update database and calculated RPKM values for each repeat class. Using this approach we found that in roX mutants there were strong enrichments of three repeat classes: PROTOP_B, PROTOP_A and NTS (Non-transcribed Spacer) (Fig. 5). Interestingly, the PROTOP is a family of autonomous DNA transposons that have been suggested to be ancient ancestors of the P-element and Hoppel element transposon families [59] and PROTOP_A and PROTOP_B are listed as synonyms of Hoppel[60]. Our results confirm that MSL has an affinity for regions enriched in repeats from Hoppel and PROTOP transposable elements and for NTS. All of these are highly repeated elements that are present in heterochromatic regions of the genome.


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 targets PROTOP and NTS in roX mutants.(A) Reads from the ChIP samples mapped to repeat classes from Repbase Update are shown on the x-axis as RPKM values (Reads Per Kilobase per Million mapped reads) and related to ChIP/input enrichment ratios, which are shown on the y-axis. The left part shows MSL1 ChIP in roX mutants compared to MSL2 in wild type and the right part shows MOF ChIP in roX mutants and wild type. Note, that NTS is enriched also in MOF wild type. (B) Enrichment ratio (IP/input) of the overrepresented repeats from A (NTS, PROTOP_A, PROTOP_B) in roX mutants compared to the wild type. Rectangles represent the mean values of the IP/input enrichment ratios of all mapped and unmapped reads matching to each repeat type, and error bars indicate 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004865-g005: MSL-complex targets PROTOP and NTS in roX mutants.(A) Reads from the ChIP samples mapped to repeat classes from Repbase Update are shown on the x-axis as RPKM values (Reads Per Kilobase per Million mapped reads) and related to ChIP/input enrichment ratios, which are shown on the y-axis. The left part shows MSL1 ChIP in roX mutants compared to MSL2 in wild type and the right part shows MOF ChIP in roX mutants and wild type. Note, that NTS is enriched also in MOF wild type. (B) Enrichment ratio (IP/input) of the overrepresented repeats from A (NTS, PROTOP_A, PROTOP_B) in roX mutants compared to the wild type. Rectangles represent the mean values of the IP/input enrichment ratios of all mapped and unmapped reads matching to each repeat type, and error bars indicate 95% confidence intervals.
Mentions: DNA sequences from centromeres, telomeres, the Y-chromosome and other heterochromatic regions are not assembled to any region of the D. melanogaster genome due to their highly repeated nature, and the mapping of sequences recovered in the ChIP-seq normally discards the large number of repeated sequences in the genome. We suspected that the non-mapped reads recovered by ChIP-seq might hold information about other transposable elements targeted by MSL in roX mutants in the above-mentioned heterochromatic regions. To test this hypothesis, we aligned all of the ChIP-seq reads to the repeat class sequences from the Repbase Update database and calculated RPKM values for each repeat class. Using this approach we found that in roX mutants there were strong enrichments of three repeat classes: PROTOP_B, PROTOP_A and NTS (Non-transcribed Spacer) (Fig. 5). Interestingly, the PROTOP is a family of autonomous DNA transposons that have been suggested to be ancient ancestors of the P-element and Hoppel element transposon families [59] and PROTOP_A and PROTOP_B are listed as synonyms of Hoppel[60]. Our results confirm that MSL has an affinity for regions enriched in repeats from Hoppel and PROTOP transposable elements and for NTS. All of these are highly repeated elements that are present in heterochromatic regions of the genome.

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