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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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Characterization of MSL3 chromodomain mutants(a) Schematic of the structure of the MSL3TAP transgenic protein, with mutations indicated below. To force expression of only the short isoform, the start codon for MSL3 was changed to a serine (M1S). (b) Complementation analysis. Rescue frequencies were calculated by comparing the number of msl31 homozygous mutant males expressing one copy of the transgene to their brothers with the TM3, Sb, msl3+ balancer. Number of progeny scored for each genotype is noted on the right. Chromodomain mutant males also exhibited a 2-day developmental delay with respect to their msl3+ brothers. (c) Protein expression was assayed on Western blots, using anti-PAP antibody against the TAP epitope. All but the LYT30A construct were stably expressed. (d) Chromodomain mutants assemble into MSL complexes. S2 cells expressing msl3tap constructs were used to immunoprecipitate MSL3TAP. Immunoprecipitations (IP) were then analyzed by Western blot for MSL1 and MOF. Even lanes: Input sample; odd lanes: IP sample. The lower band in the MSL1 IP may be a degradation product. (e) Chromodomain mutants are indistinguishable from WT in their binding patterns on the male X chromosome at the resolution of polytene staining (red: MSL3TAP). Stainings were done in the absence of endogenous msl3. From top to bottom: WT; SYD62A; ΔCD; W59G.
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Figure 1: Characterization of MSL3 chromodomain mutants(a) Schematic of the structure of the MSL3TAP transgenic protein, with mutations indicated below. To force expression of only the short isoform, the start codon for MSL3 was changed to a serine (M1S). (b) Complementation analysis. Rescue frequencies were calculated by comparing the number of msl31 homozygous mutant males expressing one copy of the transgene to their brothers with the TM3, Sb, msl3+ balancer. Number of progeny scored for each genotype is noted on the right. Chromodomain mutant males also exhibited a 2-day developmental delay with respect to their msl3+ brothers. (c) Protein expression was assayed on Western blots, using anti-PAP antibody against the TAP epitope. All but the LYT30A construct were stably expressed. (d) Chromodomain mutants assemble into MSL complexes. S2 cells expressing msl3tap constructs were used to immunoprecipitate MSL3TAP. Immunoprecipitations (IP) were then analyzed by Western blot for MSL1 and MOF. Even lanes: Input sample; odd lanes: IP sample. The lower band in the MSL1 IP may be a degradation product. (e) Chromodomain mutants are indistinguishable from WT in their binding patterns on the male X chromosome at the resolution of polytene staining (red: MSL3TAP). Stainings were done in the absence of endogenous msl3. From top to bottom: WT; SYD62A; ΔCD; W59G.

Mentions: In order to study the contribution of the MSL3 chromodomain to targeting of the dosage compensation complex, we created transgenic lines expressing a deletion of the domain, as well as a set of targeted point mutants in potential methyl-lysine binding aromatic residues identified by alignment with other chromodomains22,23. The mutants were constructed in the context of a wild-type transgene fused to the TAP (Tandem Affinity Purification) epitope tag (Fig. 1a). The chromodomain deletion was created by mutating the start codon for full-length MSL3, ablating its expression and leading to overproduction of a naturally-occuring short form initiating at Met127, downstream of the chromodomain. The peptide composition of the short form was confirmed by TAP-tag affinity purification followed by mass spectrometry (Supplementary Fig. 1). The site-directed mutants designed to disrupt chromodomain function altered the following residues: 1) Y31 along with its neighboring residues L30 and T32 into alanines to obtain the LYT30A mutant; 2) Y63 along with S62 and D64 into alanines resulting in the SYD62A mutant; and 3) W59 into a glycine to create the W59G mutant (Fig. 1a).


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)

Characterization of MSL3 chromodomain mutants(a) Schematic of the structure of the MSL3TAP transgenic protein, with mutations indicated below. To force expression of only the short isoform, the start codon for MSL3 was changed to a serine (M1S). (b) Complementation analysis. Rescue frequencies were calculated by comparing the number of msl31 homozygous mutant males expressing one copy of the transgene to their brothers with the TM3, Sb, msl3+ balancer. Number of progeny scored for each genotype is noted on the right. Chromodomain mutant males also exhibited a 2-day developmental delay with respect to their msl3+ brothers. (c) Protein expression was assayed on Western blots, using anti-PAP antibody against the TAP epitope. All but the LYT30A construct were stably expressed. (d) Chromodomain mutants assemble into MSL complexes. S2 cells expressing msl3tap constructs were used to immunoprecipitate MSL3TAP. Immunoprecipitations (IP) were then analyzed by Western blot for MSL1 and MOF. Even lanes: Input sample; odd lanes: IP sample. The lower band in the MSL1 IP may be a degradation product. (e) Chromodomain mutants are indistinguishable from WT in their binding patterns on the male X chromosome at the resolution of polytene staining (red: MSL3TAP). Stainings were done in the absence of endogenous msl3. From top to bottom: WT; SYD62A; ΔCD; W59G.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636508&req=5

Figure 1: Characterization of MSL3 chromodomain mutants(a) Schematic of the structure of the MSL3TAP transgenic protein, with mutations indicated below. To force expression of only the short isoform, the start codon for MSL3 was changed to a serine (M1S). (b) Complementation analysis. Rescue frequencies were calculated by comparing the number of msl31 homozygous mutant males expressing one copy of the transgene to their brothers with the TM3, Sb, msl3+ balancer. Number of progeny scored for each genotype is noted on the right. Chromodomain mutant males also exhibited a 2-day developmental delay with respect to their msl3+ brothers. (c) Protein expression was assayed on Western blots, using anti-PAP antibody against the TAP epitope. All but the LYT30A construct were stably expressed. (d) Chromodomain mutants assemble into MSL complexes. S2 cells expressing msl3tap constructs were used to immunoprecipitate MSL3TAP. Immunoprecipitations (IP) were then analyzed by Western blot for MSL1 and MOF. Even lanes: Input sample; odd lanes: IP sample. The lower band in the MSL1 IP may be a degradation product. (e) Chromodomain mutants are indistinguishable from WT in their binding patterns on the male X chromosome at the resolution of polytene staining (red: MSL3TAP). Stainings were done in the absence of endogenous msl3. From top to bottom: WT; SYD62A; ΔCD; W59G.
Mentions: In order to study the contribution of the MSL3 chromodomain to targeting of the dosage compensation complex, we created transgenic lines expressing a deletion of the domain, as well as a set of targeted point mutants in potential methyl-lysine binding aromatic residues identified by alignment with other chromodomains22,23. The mutants were constructed in the context of a wild-type transgene fused to the TAP (Tandem Affinity Purification) epitope tag (Fig. 1a). The chromodomain deletion was created by mutating the start codon for full-length MSL3, ablating its expression and leading to overproduction of a naturally-occuring short form initiating at Met127, downstream of the chromodomain. The peptide composition of the short form was confirmed by TAP-tag affinity purification followed by mass spectrometry (Supplementary Fig. 1). The site-directed mutants designed to disrupt chromodomain function altered the following residues: 1) Y31 along with its neighboring residues L30 and T32 into alanines to obtain the LYT30A mutant; 2) Y63 along with S62 and D64 into alanines resulting in the SYD62A mutant; and 3) W59 into a glycine to create the W59G mutant (Fig. 1a).

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