Project description:Myb-MuvB (MMB)/dREAM is a nine subunit complex first described in Drosophila as a repressor of transcription, dependent upon E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent upon DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNAi and micro-array expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupies thousands of chromosomal sites where a substantial number are proximal to repressed genes that are normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression whereas at other non-overlapping sites, Myb was critical for repression. These data highlight that the MMB factors are utilized in a combinatorial way for targeting gene regulation. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants. Keywords: Drosophila Myb-MuvB/dREAM, RNAi, ChIP-chip
Project description:Myb-MuvB (MMB)/dREAM is a nine subunit complex first described in Drosophila as a repressor of transcription, dependent upon E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent upon DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNAi and micro-array expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupies thousands of chromosomal sites where a substantial number are proximal to repressed genes that are normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression whereas at other non-overlapping sites, Myb was critical for repression. These data highlight that the MMB factors are utilized in a combinatorial way for targeting gene regulation. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants. Experiment Overall Design: RNAi to deplete Lin-52, Mip40, Myb, Mip120, Mip130, E2F2, both RBFs (RBF1 and RBF2) and L(3)MBT were performed in triplicate. RNAi with a nonspecific RNA derived from a pBSK+ plasmid (named SK+) was used as control. Total RNA was extracted from RNAi-transfected cells after 4 days using RNeasy Mini Kit (QIAGEN).
Project description:Myb-MuvB (MMB)/dREAM is a nine subunit complex first described in Drosophila as a repressor of transcription, dependent upon E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent upon DNA sites and developmental context. Keywords: Drosophila Myb-MuvB/dREAM, ChIP-chip
Project description:The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner.