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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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MSL3 chromodomain mutants disrupt spreading in vivoChromosomes from roX1 roX2 deficient 3rd instar larvae with the GMroX2-26D8 transgene on chromosome 2L were stained for either the TAP epitope (red) alone or for both anti-MSL2 (red) and anti-MSL3 (green) whose overlap is shown by yellow. Anti-MSL3 staining shows both TAP-tagged and untagged MSL3 proteins. The transgene insertion is indicated by a star and the chromosomes are aligned by the location of the transgene for comparison. (a) When a transgene harboring roX2 is put on an autosome in the absence of both roX genes on the X, the MSL complex spreads extensively around the transgene insertion. (b) In the presence of the WT transgene (red), 100% of nuclei show extensive spreading. Three representative chromosomes are shown. (c) ΔCD (red) shows mosaic spreading. Three representative nuclei are shown. One shows spreading, albeit limited, whereas the other two show binding to the transgene but no spreading. Endogenous complexes (yellow) are also affected. (d) W59G (red) also shows mosaic spreading, but a majority of the nuclei show limited spreading. (e) SYD62A generally fails to spread and interferes with endogenous spreading.
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Figure 4: MSL3 chromodomain mutants disrupt spreading in vivoChromosomes from roX1 roX2 deficient 3rd instar larvae with the GMroX2-26D8 transgene on chromosome 2L were stained for either the TAP epitope (red) alone or for both anti-MSL2 (red) and anti-MSL3 (green) whose overlap is shown by yellow. Anti-MSL3 staining shows both TAP-tagged and untagged MSL3 proteins. The transgene insertion is indicated by a star and the chromosomes are aligned by the location of the transgene for comparison. (a) When a transgene harboring roX2 is put on an autosome in the absence of both roX genes on the X, the MSL complex spreads extensively around the transgene insertion. (b) In the presence of the WT transgene (red), 100% of nuclei show extensive spreading. Three representative chromosomes are shown. (c) ΔCD (red) shows mosaic spreading. Three representative nuclei are shown. One shows spreading, albeit limited, whereas the other two show binding to the transgene but no spreading. Endogenous complexes (yellow) are also affected. (d) W59G (red) also shows mosaic spreading, but a majority of the nuclei show limited spreading. (e) SYD62A generally fails to spread and interferes with endogenous spreading.

Mentions: To further probe the ability of the SYD62A and ΔCD proteins to direct spreading, we asked whether expression of the mutant proteins might visibly interfere with the extensive wild-type spreading seen from an autosomal roX transgene insertion on polytene chromosomes. We used a transgenic line carrying a GMroX2 transgene at 26D8, which shows extensive spreading in the absence of roX1 and roX2 genes on the X (ref. 8, Fig. 4a). We analyzed the behavior of the mutant proteins and their effects on wild-type complexes, in a dominant assay with one functional copy of the endogenous wild-type msl3+ gene present. When stained for the TAP epitope, transgenic WT MSL3 showed consistent and extensive spreading around the roX2 insertion in close to 100% of nuclei, as expected (Fig. 4b). However, the chromodomain mutants failed to do so to varying extents. In msl31 heterozygotes carrying a ΔCD transgene, about half of all nuclei showed spreading of both MSL3TAP and MSL2, albeit not as extensive as the WT control (Fig. 4c). The remaining nuclei did not show any spreading for either MSL3TAP or MSL2 although both proteins were detected at the transgene insertion site. In the W59G point mutant, most nuclei showed spreading for MSL3TAP and MSL2 (Fig. 4d). The most pronounced defect was in the SYD62A mutant, where almost all nuclei lacked spreading for both MSL3TAP and MSL2 even though wild-type MSL3 protein was present (Fig. 4e). Therefore, chromodomain mutant proteins can interfere with ectopic spreading of endogenous MSL complexes, even in the presence of wild-type MSL3. This argues against a model in which simply lowering the level of complex results in the loss of binding seen in ΔCD mutants. Rather, the lack of an intact chromodomain is specifically implicated in diminished spreading from chromatin entry sites.


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)

MSL3 chromodomain mutants disrupt spreading in vivoChromosomes from roX1 roX2 deficient 3rd instar larvae with the GMroX2-26D8 transgene on chromosome 2L were stained for either the TAP epitope (red) alone or for both anti-MSL2 (red) and anti-MSL3 (green) whose overlap is shown by yellow. Anti-MSL3 staining shows both TAP-tagged and untagged MSL3 proteins. The transgene insertion is indicated by a star and the chromosomes are aligned by the location of the transgene for comparison. (a) When a transgene harboring roX2 is put on an autosome in the absence of both roX genes on the X, the MSL complex spreads extensively around the transgene insertion. (b) In the presence of the WT transgene (red), 100% of nuclei show extensive spreading. Three representative chromosomes are shown. (c) ΔCD (red) shows mosaic spreading. Three representative nuclei are shown. One shows spreading, albeit limited, whereas the other two show binding to the transgene but no spreading. Endogenous complexes (yellow) are also affected. (d) W59G (red) also shows mosaic spreading, but a majority of the nuclei show limited spreading. (e) SYD62A generally fails to spread and interferes with endogenous spreading.
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Figure 4: MSL3 chromodomain mutants disrupt spreading in vivoChromosomes from roX1 roX2 deficient 3rd instar larvae with the GMroX2-26D8 transgene on chromosome 2L were stained for either the TAP epitope (red) alone or for both anti-MSL2 (red) and anti-MSL3 (green) whose overlap is shown by yellow. Anti-MSL3 staining shows both TAP-tagged and untagged MSL3 proteins. The transgene insertion is indicated by a star and the chromosomes are aligned by the location of the transgene for comparison. (a) When a transgene harboring roX2 is put on an autosome in the absence of both roX genes on the X, the MSL complex spreads extensively around the transgene insertion. (b) In the presence of the WT transgene (red), 100% of nuclei show extensive spreading. Three representative chromosomes are shown. (c) ΔCD (red) shows mosaic spreading. Three representative nuclei are shown. One shows spreading, albeit limited, whereas the other two show binding to the transgene but no spreading. Endogenous complexes (yellow) are also affected. (d) W59G (red) also shows mosaic spreading, but a majority of the nuclei show limited spreading. (e) SYD62A generally fails to spread and interferes with endogenous spreading.
Mentions: To further probe the ability of the SYD62A and ΔCD proteins to direct spreading, we asked whether expression of the mutant proteins might visibly interfere with the extensive wild-type spreading seen from an autosomal roX transgene insertion on polytene chromosomes. We used a transgenic line carrying a GMroX2 transgene at 26D8, which shows extensive spreading in the absence of roX1 and roX2 genes on the X (ref. 8, Fig. 4a). We analyzed the behavior of the mutant proteins and their effects on wild-type complexes, in a dominant assay with one functional copy of the endogenous wild-type msl3+ gene present. When stained for the TAP epitope, transgenic WT MSL3 showed consistent and extensive spreading around the roX2 insertion in close to 100% of nuclei, as expected (Fig. 4b). However, the chromodomain mutants failed to do so to varying extents. In msl31 heterozygotes carrying a ΔCD transgene, about half of all nuclei showed spreading of both MSL3TAP and MSL2, albeit not as extensive as the WT control (Fig. 4c). The remaining nuclei did not show any spreading for either MSL3TAP or MSL2 although both proteins were detected at the transgene insertion site. In the W59G point mutant, most nuclei showed spreading for MSL3TAP and MSL2 (Fig. 4d). The most pronounced defect was in the SYD62A mutant, where almost all nuclei lacked spreading for both MSL3TAP and MSL2 even though wild-type MSL3 protein was present (Fig. 4e). Therefore, chromodomain mutant proteins can interfere with ectopic spreading of endogenous MSL complexes, even in the presence of wild-type MSL3. This argues against a model in which simply lowering the level of complex results in the loss of binding seen in ΔCD mutants. Rather, the lack of an intact chromodomain is specifically implicated in diminished spreading from chromatin entry sites.

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