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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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High resolution ChIP-chip mapping of MSL3 mutant binding sites(a) The number of genes within bound clusters on the X chromosome was compared among experiments in a Venn diagram. 98% (418/432) of ΔCD bound genes and 96% (327/340) of SYD62A bound genes are also WT sites; 92% (313/340) of SYD62A bound genes belong to the ΔCD set. (b) Distributions of WT log-ratios for genes from different categories. Among all X-linked genes (black), those identified as bound in WT (magenta) have high log-ratios. Importantly, most of the genes bound in ΔCD (green) or SYD62A (blue) mutants have the highest signals in WT. The color scheme corresponds to that in Fig. 2a; for this plot, we defined the signal for a gene as the maximum log-ratio over the gene. (c) Scatter-plot of ChIP-chip signals between WT and ΔCD at probe level. If the probe is equally bound in both WT and ΔCD experiments, the representative point should fall near the diagonal. The presence of two separate clusters of red probes on the X suggests that there are two types of bound sites in WT: chromodomain-independent (cluster 1) and chromodomain-dependent sites (cluster 2).
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Figure 2: High resolution ChIP-chip mapping of MSL3 mutant binding sites(a) The number of genes within bound clusters on the X chromosome was compared among experiments in a Venn diagram. 98% (418/432) of ΔCD bound genes and 96% (327/340) of SYD62A bound genes are also WT sites; 92% (313/340) of SYD62A bound genes belong to the ΔCD set. (b) Distributions of WT log-ratios for genes from different categories. Among all X-linked genes (black), those identified as bound in WT (magenta) have high log-ratios. Importantly, most of the genes bound in ΔCD (green) or SYD62A (blue) mutants have the highest signals in WT. The color scheme corresponds to that in Fig. 2a; for this plot, we defined the signal for a gene as the maximum log-ratio over the gene. (c) Scatter-plot of ChIP-chip signals between WT and ΔCD at probe level. If the probe is equally bound in both WT and ΔCD experiments, the representative point should fall near the diagonal. The presence of two separate clusters of red probes on the X suggests that there are two types of bound sites in WT: chromodomain-independent (cluster 1) and chromodomain-dependent sites (cluster 2).

Mentions: Each experiment was performed in duplicate per genotype and the signal intensities from both profiles were used to calculate a mean signal intensity value for each probe on the array. A computational algorithm was applied to locate statistically significant clusters of binding, assessing the minimum level of enrichment and the size of cluster (see Methods). The total number of bound clusters was calculated for each genotype and the number of bound genes within the clusters was determined. Using these criteria, WT MSL3-TAP protein bound to 1337 genes within 694 clusters on the X and 9 clusters were bound on chromosome arm 2L. Chromodomain mutants retained a strong bias for the X, but bound to only a subset of the WT targets. 340 genes were mapped within 338 clusters in the SYD62A mutant (with 13 clusters on 2L) and 432 genes were mapped within 444 clusters in the ΔCD mutant (with 15 clusters on 2L). Therefore, at least two-thirds of WT target genes were scored as unbound in the SYD62A and ΔCD mutants (Fig. 2a). The clusters in chromodomain mutants were also smaller and contained fewer genes on average than WT (~1.0 gene per cluster for SYD62A or ΔCD; ~1.9 genes per cluster for WT).


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)

High resolution ChIP-chip mapping of MSL3 mutant binding sites(a) The number of genes within bound clusters on the X chromosome was compared among experiments in a Venn diagram. 98% (418/432) of ΔCD bound genes and 96% (327/340) of SYD62A bound genes are also WT sites; 92% (313/340) of SYD62A bound genes belong to the ΔCD set. (b) Distributions of WT log-ratios for genes from different categories. Among all X-linked genes (black), those identified as bound in WT (magenta) have high log-ratios. Importantly, most of the genes bound in ΔCD (green) or SYD62A (blue) mutants have the highest signals in WT. The color scheme corresponds to that in Fig. 2a; for this plot, we defined the signal for a gene as the maximum log-ratio over the gene. (c) Scatter-plot of ChIP-chip signals between WT and ΔCD at probe level. If the probe is equally bound in both WT and ΔCD experiments, the representative point should fall near the diagonal. The presence of two separate clusters of red probes on the X suggests that there are two types of bound sites in WT: chromodomain-independent (cluster 1) and chromodomain-dependent sites (cluster 2).
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: High resolution ChIP-chip mapping of MSL3 mutant binding sites(a) The number of genes within bound clusters on the X chromosome was compared among experiments in a Venn diagram. 98% (418/432) of ΔCD bound genes and 96% (327/340) of SYD62A bound genes are also WT sites; 92% (313/340) of SYD62A bound genes belong to the ΔCD set. (b) Distributions of WT log-ratios for genes from different categories. Among all X-linked genes (black), those identified as bound in WT (magenta) have high log-ratios. Importantly, most of the genes bound in ΔCD (green) or SYD62A (blue) mutants have the highest signals in WT. The color scheme corresponds to that in Fig. 2a; for this plot, we defined the signal for a gene as the maximum log-ratio over the gene. (c) Scatter-plot of ChIP-chip signals between WT and ΔCD at probe level. If the probe is equally bound in both WT and ΔCD experiments, the representative point should fall near the diagonal. The presence of two separate clusters of red probes on the X suggests that there are two types of bound sites in WT: chromodomain-independent (cluster 1) and chromodomain-dependent sites (cluster 2).
Mentions: Each experiment was performed in duplicate per genotype and the signal intensities from both profiles were used to calculate a mean signal intensity value for each probe on the array. A computational algorithm was applied to locate statistically significant clusters of binding, assessing the minimum level of enrichment and the size of cluster (see Methods). The total number of bound clusters was calculated for each genotype and the number of bound genes within the clusters was determined. Using these criteria, WT MSL3-TAP protein bound to 1337 genes within 694 clusters on the X and 9 clusters were bound on chromosome arm 2L. Chromodomain mutants retained a strong bias for the X, but bound to only a subset of the WT targets. 340 genes were mapped within 338 clusters in the SYD62A mutant (with 13 clusters on 2L) and 432 genes were mapped within 444 clusters in the ΔCD mutant (with 15 clusters on 2L). Therefore, at least two-thirds of WT target genes were scored as unbound in the SYD62A and ΔCD mutants (Fig. 2a). The clusters in chromodomain mutants were also smaller and contained fewer genes on average than WT (~1.0 gene per cluster for SYD62A or ΔCD; ~1.9 genes per cluster for WT).

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
Related in: MedlinePlus