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The MSL3 chromodomain directs a key targeting step for dosage compensation of the Drosophila melanogaster X chromosome.

Sural TH, Peng S, Li B, Workman JL, Park PJ, Kuroda MI - Nat. Struct. Mol. Biol. (2008)

Bottom Line: Using ChIP-chip analysis, we find that MSL3 chromodomain mutants retain binding to chromatin entry sites but show a clear disruption in the full pattern of MSL targeting in vivo, consistent with a loss of spreading.Furthermore, when compared to wild type, chromodomain mutants lack preferential affinity for nucleosomes containing H3K36me3 in vitro.Our results support a model in which activating complexes, similarly to their silencing counterparts, use the nucleosomal binding specificity of their respective chromodomains to spread from initiation sites to flanking chromatin.

View Article: PubMed Central - PubMed

Affiliation: Harvard-Partners Center for Genetics and Genomics, Division of Genetics, Department of Medicine, Brigham & Women's Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

ABSTRACT
The male-specific lethal (MSL) complex upregulates the single male X chromosome to achieve dosage compensation in Drosophila melanogaster. We have proposed that MSL recognition of specific entry sites on the X is followed by local targeting of active genes marked by histone H3 trimethylation (H3K36me3). Here we analyze the role of the MSL3 chromodomain in the second targeting step. Using ChIP-chip analysis, we find that MSL3 chromodomain mutants retain binding to chromatin entry sites but show a clear disruption in the full pattern of MSL targeting in vivo, consistent with a loss of spreading. Furthermore, when compared to wild type, chromodomain mutants lack preferential affinity for nucleosomes containing H3K36me3 in vitro. Our results support a model in which activating complexes, similarly to their silencing counterparts, use the nucleosomal binding specificity of their respective chromodomains to spread from initiation sites to flanking chromatin.

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Chromodomain mutants show loss of preference for H3K36me3 containing nucleosomesA range of concentrations of recombinant MSL3 proteins was tested for binding to nucleosomes in gel shift assays. (a) Binding to recombinant nucleosomes methylated at H3K36. SYD62A and W59G point mutants can still bind unmodified nucleosomes albeit to a lesser extent than the WT (compare lanes 4, 7 and 10). WT has higher affinity for modified nucleosomes (compare lanes 4 and 14). The point mutants show no difference in binding to modified vs. unmodified nucleosomes (compare lanes 7 and 17 for SD62A; 10 and 20 for W59G). (b) WT MSL3 can bind native nucleosomes efficiently in gel shift assays. W59G has decreased binding and ΔCD has no detectable binding. (c) Chromatin entry sites are preferentially found in H3K36me3-enriched regions. Sequences matching the consensus for MSL recognition elements (MREs) were ordered by their enrichment for H3K36me3 and grouped into 10 bins (x-axis). The likelihood of an MRE being in a functional chromatin entry site (CES) (y-axis) increases as the level of H3K36me3 enrichment increases. H3K36me3 enrichment was computed by averaging the signal of the 3 probes nearest to each MRE.
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Figure 5: Chromodomain mutants show loss of preference for H3K36me3 containing nucleosomesA range of concentrations of recombinant MSL3 proteins was tested for binding to nucleosomes in gel shift assays. (a) Binding to recombinant nucleosomes methylated at H3K36. SYD62A and W59G point mutants can still bind unmodified nucleosomes albeit to a lesser extent than the WT (compare lanes 4, 7 and 10). WT has higher affinity for modified nucleosomes (compare lanes 4 and 14). The point mutants show no difference in binding to modified vs. unmodified nucleosomes (compare lanes 7 and 17 for SD62A; 10 and 20 for W59G). (b) WT MSL3 can bind native nucleosomes efficiently in gel shift assays. W59G has decreased binding and ΔCD has no detectable binding. (c) Chromatin entry sites are preferentially found in H3K36me3-enriched regions. Sequences matching the consensus for MSL recognition elements (MREs) were ordered by their enrichment for H3K36me3 and grouped into 10 bins (x-axis). The likelihood of an MRE being in a functional chromatin entry site (CES) (y-axis) increases as the level of H3K36me3 enrichment increases. H3K36me3 enrichment was computed by averaging the signal of the 3 probes nearest to each MRE.

Mentions: When tested for binding to methylated H3K36, WT protein shows increased binding to modified nucleosomes vs. unmodified ones (Fig. 5a, comparing lane 14 to lane 4), consistent with previous data that MSL3 has a higher affinity for nucleosomes with H3K36 methylation8. The SYD62A and the W59G mutants showed no difference in binding to methylated vs. unmodified nucleosomes, suggesting that general affinity for nucleosomes was retained, but specificity for H3K36 methylation in these mutants is lost (Fig. 5a). The ΔCD recombinant protein lacked even general binding to nucleosomes (Fig. 5b), as reported previously21. These data demonstrate that recombinant MSL3 recognizes nucleosomes via its chromodomain, with increased affinity for octamers containing the methylated H3K36 mark.


The MSL3 chromodomain directs a key targeting step for dosage compensation of the Drosophila melanogaster X chromosome.

Sural TH, Peng S, Li B, Workman JL, Park PJ, Kuroda MI - Nat. Struct. Mol. Biol. (2008)

Chromodomain mutants show loss of preference for H3K36me3 containing nucleosomesA range of concentrations of recombinant MSL3 proteins was tested for binding to nucleosomes in gel shift assays. (a) Binding to recombinant nucleosomes methylated at H3K36. SYD62A and W59G point mutants can still bind unmodified nucleosomes albeit to a lesser extent than the WT (compare lanes 4, 7 and 10). WT has higher affinity for modified nucleosomes (compare lanes 4 and 14). The point mutants show no difference in binding to modified vs. unmodified nucleosomes (compare lanes 7 and 17 for SD62A; 10 and 20 for W59G). (b) WT MSL3 can bind native nucleosomes efficiently in gel shift assays. W59G has decreased binding and ΔCD has no detectable binding. (c) Chromatin entry sites are preferentially found in H3K36me3-enriched regions. Sequences matching the consensus for MSL recognition elements (MREs) were ordered by their enrichment for H3K36me3 and grouped into 10 bins (x-axis). The likelihood of an MRE being in a functional chromatin entry site (CES) (y-axis) increases as the level of H3K36me3 enrichment increases. H3K36me3 enrichment was computed by averaging the signal of the 3 probes nearest to each MRE.
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Figure 5: Chromodomain mutants show loss of preference for H3K36me3 containing nucleosomesA range of concentrations of recombinant MSL3 proteins was tested for binding to nucleosomes in gel shift assays. (a) Binding to recombinant nucleosomes methylated at H3K36. SYD62A and W59G point mutants can still bind unmodified nucleosomes albeit to a lesser extent than the WT (compare lanes 4, 7 and 10). WT has higher affinity for modified nucleosomes (compare lanes 4 and 14). The point mutants show no difference in binding to modified vs. unmodified nucleosomes (compare lanes 7 and 17 for SD62A; 10 and 20 for W59G). (b) WT MSL3 can bind native nucleosomes efficiently in gel shift assays. W59G has decreased binding and ΔCD has no detectable binding. (c) Chromatin entry sites are preferentially found in H3K36me3-enriched regions. Sequences matching the consensus for MSL recognition elements (MREs) were ordered by their enrichment for H3K36me3 and grouped into 10 bins (x-axis). The likelihood of an MRE being in a functional chromatin entry site (CES) (y-axis) increases as the level of H3K36me3 enrichment increases. H3K36me3 enrichment was computed by averaging the signal of the 3 probes nearest to each MRE.
Mentions: When tested for binding to methylated H3K36, WT protein shows increased binding to modified nucleosomes vs. unmodified ones (Fig. 5a, comparing lane 14 to lane 4), consistent with previous data that MSL3 has a higher affinity for nucleosomes with H3K36 methylation8. The SYD62A and the W59G mutants showed no difference in binding to methylated vs. unmodified nucleosomes, suggesting that general affinity for nucleosomes was retained, but specificity for H3K36 methylation in these mutants is lost (Fig. 5a). The ΔCD recombinant protein lacked even general binding to nucleosomes (Fig. 5b), as reported previously21. These data demonstrate that recombinant MSL3 recognizes nucleosomes via its chromodomain, with increased affinity for octamers containing the methylated H3K36 mark.

Bottom Line: Using ChIP-chip analysis, we find that MSL3 chromodomain mutants retain binding to chromatin entry sites but show a clear disruption in the full pattern of MSL targeting in vivo, consistent with a loss of spreading.Furthermore, when compared to wild type, chromodomain mutants lack preferential affinity for nucleosomes containing H3K36me3 in vitro.Our results support a model in which activating complexes, similarly to their silencing counterparts, use the nucleosomal binding specificity of their respective chromodomains to spread from initiation sites to flanking chromatin.

View Article: PubMed Central - PubMed

Affiliation: Harvard-Partners Center for Genetics and Genomics, Division of Genetics, Department of Medicine, Brigham & Women's Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

ABSTRACT
The male-specific lethal (MSL) complex upregulates the single male X chromosome to achieve dosage compensation in Drosophila melanogaster. We have proposed that MSL recognition of specific entry sites on the X is followed by local targeting of active genes marked by histone H3 trimethylation (H3K36me3). Here we analyze the role of the MSL3 chromodomain in the second targeting step. Using ChIP-chip analysis, we find that MSL3 chromodomain mutants retain binding to chromatin entry sites but show a clear disruption in the full pattern of MSL targeting in vivo, consistent with a loss of spreading. Furthermore, when compared to wild type, chromodomain mutants lack preferential affinity for nucleosomes containing H3K36me3 in vitro. Our results support a model in which activating complexes, similarly to their silencing counterparts, use the nucleosomal binding specificity of their respective chromodomains to spread from initiation sites to flanking chromatin.

Show MeSH