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Distinct contributions of MSL complex subunits to the transcriptional enhancement responsible for dosage compensation in Drosophila.

Dunlap D, Yokoyama R, Ling H, Sun HY, McGill K, Cugusi S, Lucchesi JC - Nucleic Acids Res. (2012)

Bottom Line: In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males.We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification.Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Department of Biology, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
The regulatory mechanism of dosage compensation is the paramount example of epigenetic regulation at the chromosomal level. In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males. We have investigated the function of various subunits of the complex in mediating dosage compensation. Our results confirm that the highly enriched specific acetylation of histone H4 at lysine 16 of compensated genes by the histone acetyl transferase subunit MOF induces a more disorganized state of their chromatin. We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification. Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

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The differences in the median linking number of topoisomers isolated from S2 cells treated with RNAi to knockdown the indicated proteins were determined with respect to the linking number of plasmids isolated from cells in which MSL2, and therefore the MSL complex, was absent. (A) MOF knockdown, which leaves a complex of MSL1 + MSL2 + MLE, did not significantly change the topology compared with plasmid from compensated cells (GFP). MLE knockdown, which leaves a complex of MSL1 + MSL2, resulted in a partial or intermediate reduction in negative supercoiling of the plasmid. (B) ISWI knockdown produced more relaxed noncompensated and compensated topoisomers that migrated one linking number ahead of the similar plasmids from untreated cells. However, ADA3 and GCN5 knockdown in S2 cells did not significantly change the topology nor the migration of the isolated topoisomers.
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gks890-F5: The differences in the median linking number of topoisomers isolated from S2 cells treated with RNAi to knockdown the indicated proteins were determined with respect to the linking number of plasmids isolated from cells in which MSL2, and therefore the MSL complex, was absent. (A) MOF knockdown, which leaves a complex of MSL1 + MSL2 + MLE, did not significantly change the topology compared with plasmid from compensated cells (GFP). MLE knockdown, which leaves a complex of MSL1 + MSL2, resulted in a partial or intermediate reduction in negative supercoiling of the plasmid. (B) ISWI knockdown produced more relaxed noncompensated and compensated topoisomers that migrated one linking number ahead of the similar plasmids from untreated cells. However, ADA3 and GCN5 knockdown in S2 cells did not significantly change the topology nor the migration of the isolated topoisomers.

Mentions: Using RNA interference, we selectively eliminated components of the MSL complex (Supplementary Figure S4). Assembly of the entire complex is prevented by the absence of MSL2 (40). In the absence of MOF, only MSL1 + MSL2 + MLE associate with the chromatin entry sites of the complex (41); in the absence of MLE, only MSL1 + MSL2 are at these sites (42). In the current set of experiments, the linking number difference between a compensated plasmid extracted from S2 cells that form a fully functional complex and a plasmid extracted from cells where the complex is absent was 1.08 ± 0.14 (P=0.0003; see Figure 5A). The mobility of the topoisomer distribution of a plasmid extracted from cells where a partial complex composed of MSL1 + MSL2 + MLE was similar to that of the fully compensated plasmid; the difference in the linking number between these two plasmids was 0.11 ± 0.29 (P=0.26). The linking number of a plasmid extracted from cells in which MSL1 + MSL2 are the only subunits that associate with the chromatin entry site differed from that of the noncompensated plasmid by 0.6 ± 0.07 (P=0.002). Therefore, under our experimental conditions, this plasmid exhibited a partial relaxation of negative supercoils. These data suggest that MOF, and therefore H4K16 acetylation, and MSL3 do not contribute to the topological changes exhibited by a plasmid exposed to a complete MSL complex. A partial complex made up of MSL1, MSL2 and MLE was sufficient to produce these changes. Finally, just the presence of MSL1 and MSL2, the two MSL subunits that bind DNA, was sufficient to partially alter the topology of the plasmid.Figure 5.


Distinct contributions of MSL complex subunits to the transcriptional enhancement responsible for dosage compensation in Drosophila.

Dunlap D, Yokoyama R, Ling H, Sun HY, McGill K, Cugusi S, Lucchesi JC - Nucleic Acids Res. (2012)

The differences in the median linking number of topoisomers isolated from S2 cells treated with RNAi to knockdown the indicated proteins were determined with respect to the linking number of plasmids isolated from cells in which MSL2, and therefore the MSL complex, was absent. (A) MOF knockdown, which leaves a complex of MSL1 + MSL2 + MLE, did not significantly change the topology compared with plasmid from compensated cells (GFP). MLE knockdown, which leaves a complex of MSL1 + MSL2, resulted in a partial or intermediate reduction in negative supercoiling of the plasmid. (B) ISWI knockdown produced more relaxed noncompensated and compensated topoisomers that migrated one linking number ahead of the similar plasmids from untreated cells. However, ADA3 and GCN5 knockdown in S2 cells did not significantly change the topology nor the migration of the isolated topoisomers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526317&req=5

gks890-F5: The differences in the median linking number of topoisomers isolated from S2 cells treated with RNAi to knockdown the indicated proteins were determined with respect to the linking number of plasmids isolated from cells in which MSL2, and therefore the MSL complex, was absent. (A) MOF knockdown, which leaves a complex of MSL1 + MSL2 + MLE, did not significantly change the topology compared with plasmid from compensated cells (GFP). MLE knockdown, which leaves a complex of MSL1 + MSL2, resulted in a partial or intermediate reduction in negative supercoiling of the plasmid. (B) ISWI knockdown produced more relaxed noncompensated and compensated topoisomers that migrated one linking number ahead of the similar plasmids from untreated cells. However, ADA3 and GCN5 knockdown in S2 cells did not significantly change the topology nor the migration of the isolated topoisomers.
Mentions: Using RNA interference, we selectively eliminated components of the MSL complex (Supplementary Figure S4). Assembly of the entire complex is prevented by the absence of MSL2 (40). In the absence of MOF, only MSL1 + MSL2 + MLE associate with the chromatin entry sites of the complex (41); in the absence of MLE, only MSL1 + MSL2 are at these sites (42). In the current set of experiments, the linking number difference between a compensated plasmid extracted from S2 cells that form a fully functional complex and a plasmid extracted from cells where the complex is absent was 1.08 ± 0.14 (P=0.0003; see Figure 5A). The mobility of the topoisomer distribution of a plasmid extracted from cells where a partial complex composed of MSL1 + MSL2 + MLE was similar to that of the fully compensated plasmid; the difference in the linking number between these two plasmids was 0.11 ± 0.29 (P=0.26). The linking number of a plasmid extracted from cells in which MSL1 + MSL2 are the only subunits that associate with the chromatin entry site differed from that of the noncompensated plasmid by 0.6 ± 0.07 (P=0.002). Therefore, under our experimental conditions, this plasmid exhibited a partial relaxation of negative supercoils. These data suggest that MOF, and therefore H4K16 acetylation, and MSL3 do not contribute to the topological changes exhibited by a plasmid exposed to a complete MSL complex. A partial complex made up of MSL1, MSL2 and MLE was sufficient to produce these changes. Finally, just the presence of MSL1 and MSL2, the two MSL subunits that bind DNA, was sufficient to partially alter the topology of the plasmid.Figure 5.

Bottom Line: In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males.We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification.Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Department of Biology, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
The regulatory mechanism of dosage compensation is the paramount example of epigenetic regulation at the chromosomal level. In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males. We have investigated the function of various subunits of the complex in mediating dosage compensation. Our results confirm that the highly enriched specific acetylation of histone H4 at lysine 16 of compensated genes by the histone acetyl transferase subunit MOF induces a more disorganized state of their chromatin. We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification. Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

Show MeSH