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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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The MSL3 chromodomain is important for spreading(a) 200kb of sample binding data from WT and mutants. The chromodomain mutant proteins bind in specific peaks, but fail to cover all WT sites. The H3K36 methylation profile from male tissue culture cells (S2) over the same region is shown for comparison. The H3K36me3 profile is very similar to the profile for WT MSL binding, extending beyond the chromatin entry sites. Boxes below the profiles represent annotated genes; red: transcribed, black: non-transcribed. (b) GA-rich motif identified by MEME from the SYD62A binding peaks. (c) The distribution of distances to the nearest chromatin entry site (CES) for the probes bound by WT (magenta), the ΔCD mutant (green) and the SYD62A mutant (navy), or the average of 10 random circular permutations of WT (light blue). The number of probes bound by the mutants (y axis) falls more sharply than WT as one moves farther from CES (X axis). (d) Alternative representation of the data in (c). The y-axis represents the % of bound probes (i.e. density) rather than absolute count. The binding of chromodomain mutants is clustered at or near the chromatin entry sites as shown by increased density of binding. (e) Representation of ChIP-chip data of MSL3TAP binding on polytene chromosomes shows that patterns are not distinguishable at 30kb resolution. Each black bar represents a cluster bound by MSL3TAP after merging neighboring clusters with gaps smaller than 30kb.
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Figure 3: The MSL3 chromodomain is important for spreading(a) 200kb of sample binding data from WT and mutants. The chromodomain mutant proteins bind in specific peaks, but fail to cover all WT sites. The H3K36 methylation profile from male tissue culture cells (S2) over the same region is shown for comparison. The H3K36me3 profile is very similar to the profile for WT MSL binding, extending beyond the chromatin entry sites. Boxes below the profiles represent annotated genes; red: transcribed, black: non-transcribed. (b) GA-rich motif identified by MEME from the SYD62A binding peaks. (c) The distribution of distances to the nearest chromatin entry site (CES) for the probes bound by WT (magenta), the ΔCD mutant (green) and the SYD62A mutant (navy), or the average of 10 random circular permutations of WT (light blue). The number of probes bound by the mutants (y axis) falls more sharply than WT as one moves farther from CES (X axis). (d) Alternative representation of the data in (c). The y-axis represents the % of bound probes (i.e. density) rather than absolute count. The binding of chromodomain mutants is clustered at or near the chromatin entry sites as shown by increased density of binding. (e) Representation of ChIP-chip data of MSL3TAP binding on polytene chromosomes shows that patterns are not distinguishable at 30kb resolution. Each black bar represents a cluster bound by MSL3TAP after merging neighboring clusters with gaps smaller than 30kb.

Mentions: Interestingly, when individual ChIP-chip profiles of chromodomain mutant binding sites are compared to WT, they show graphically what might be expected for initial recognition sites of the MSL complex: discrete peaks of binding in place of coverage of whole genes and neighboring genes (Fig. 3a). Noting the pattern, we wondered whether the discrete peaks might reflect binding to chromatin entry sites, with a loss of spreading to cover whole genes and flanking targets. Chromatin entry sites (CES) were originally defined as the 35–70 sites seen on polytene chromosomes of msl3 mutant larvae. However, we recently proposed that this was an underestimate based on ChIP-chip experiments of msl31 mutant embryos, in which we identified a set of at least 150 candidate entry sites containing a GA- or TC–rich MSL recognition element (MRE) required for MSL binding27. Therefore, we examined whether the ~400 binding sites detected in our chromodomain mutants included the 150 newly defined chromatin entry sites. We found that 98% of the CES were contained within the SYD62A and ΔCD sets of binding clusters. Furthermore, when we searched for ~8–25bp motifs using the MEME algorithm28, the top sequence identified using 500 bp peaks from either SYD62A or ΔCD clusters was a GA-rich motif that aligns extremely well with the MRE27 (Fig. 3b). However, not all ΔCD sites have MREs. When we surveyed 500 bp segments centered on the peaks within SYD62A or ΔCD clusters, ~190 sites in both cases contain an MRE at a p value of 10−5. Using a 1 Kb window, these numbers were ~240; further increasing the window size had minimal effect. This indicates that a substantial number of binding sites in chromodomain mutants (up to 200) do not encompass an MRE. These sites generally displayed a lower signal in our mutant ChIP-chip analysis (data not shown). Therefore, we wondered whether these additional binding sites might be the result of a limited amount of spreading retained in the chromodomain mutants.


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)

The MSL3 chromodomain is important for spreading(a) 200kb of sample binding data from WT and mutants. The chromodomain mutant proteins bind in specific peaks, but fail to cover all WT sites. The H3K36 methylation profile from male tissue culture cells (S2) over the same region is shown for comparison. The H3K36me3 profile is very similar to the profile for WT MSL binding, extending beyond the chromatin entry sites. Boxes below the profiles represent annotated genes; red: transcribed, black: non-transcribed. (b) GA-rich motif identified by MEME from the SYD62A binding peaks. (c) The distribution of distances to the nearest chromatin entry site (CES) for the probes bound by WT (magenta), the ΔCD mutant (green) and the SYD62A mutant (navy), or the average of 10 random circular permutations of WT (light blue). The number of probes bound by the mutants (y axis) falls more sharply than WT as one moves farther from CES (X axis). (d) Alternative representation of the data in (c). The y-axis represents the % of bound probes (i.e. density) rather than absolute count. The binding of chromodomain mutants is clustered at or near the chromatin entry sites as shown by increased density of binding. (e) Representation of ChIP-chip data of MSL3TAP binding on polytene chromosomes shows that patterns are not distinguishable at 30kb resolution. Each black bar represents a cluster bound by MSL3TAP after merging neighboring clusters with gaps smaller than 30kb.
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Figure 3: The MSL3 chromodomain is important for spreading(a) 200kb of sample binding data from WT and mutants. The chromodomain mutant proteins bind in specific peaks, but fail to cover all WT sites. The H3K36 methylation profile from male tissue culture cells (S2) over the same region is shown for comparison. The H3K36me3 profile is very similar to the profile for WT MSL binding, extending beyond the chromatin entry sites. Boxes below the profiles represent annotated genes; red: transcribed, black: non-transcribed. (b) GA-rich motif identified by MEME from the SYD62A binding peaks. (c) The distribution of distances to the nearest chromatin entry site (CES) for the probes bound by WT (magenta), the ΔCD mutant (green) and the SYD62A mutant (navy), or the average of 10 random circular permutations of WT (light blue). The number of probes bound by the mutants (y axis) falls more sharply than WT as one moves farther from CES (X axis). (d) Alternative representation of the data in (c). The y-axis represents the % of bound probes (i.e. density) rather than absolute count. The binding of chromodomain mutants is clustered at or near the chromatin entry sites as shown by increased density of binding. (e) Representation of ChIP-chip data of MSL3TAP binding on polytene chromosomes shows that patterns are not distinguishable at 30kb resolution. Each black bar represents a cluster bound by MSL3TAP after merging neighboring clusters with gaps smaller than 30kb.
Mentions: Interestingly, when individual ChIP-chip profiles of chromodomain mutant binding sites are compared to WT, they show graphically what might be expected for initial recognition sites of the MSL complex: discrete peaks of binding in place of coverage of whole genes and neighboring genes (Fig. 3a). Noting the pattern, we wondered whether the discrete peaks might reflect binding to chromatin entry sites, with a loss of spreading to cover whole genes and flanking targets. Chromatin entry sites (CES) were originally defined as the 35–70 sites seen on polytene chromosomes of msl3 mutant larvae. However, we recently proposed that this was an underestimate based on ChIP-chip experiments of msl31 mutant embryos, in which we identified a set of at least 150 candidate entry sites containing a GA- or TC–rich MSL recognition element (MRE) required for MSL binding27. Therefore, we examined whether the ~400 binding sites detected in our chromodomain mutants included the 150 newly defined chromatin entry sites. We found that 98% of the CES were contained within the SYD62A and ΔCD sets of binding clusters. Furthermore, when we searched for ~8–25bp motifs using the MEME algorithm28, the top sequence identified using 500 bp peaks from either SYD62A or ΔCD clusters was a GA-rich motif that aligns extremely well with the MRE27 (Fig. 3b). However, not all ΔCD sites have MREs. When we surveyed 500 bp segments centered on the peaks within SYD62A or ΔCD clusters, ~190 sites in both cases contain an MRE at a p value of 10−5. Using a 1 Kb window, these numbers were ~240; further increasing the window size had minimal effect. This indicates that a substantial number of binding sites in chromodomain mutants (up to 200) do not encompass an MRE. These sites generally displayed a lower signal in our mutant ChIP-chip analysis (data not shown). Therefore, we wondered whether these additional binding sites might be the result of a limited amount of spreading retained in the chromodomain mutants.

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