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1.
Genes Dev ; 21(22): 2880-96, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17978103

ABSTRACT

Myb-MuvB (MMB)/dREAM is a nine-subunit complex first described in Drosophila as a repressor of transcription, dependent on 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 on 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 RNA interference and microarray expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupied 3538 chromosomal sites and was promoter-proximal to 32% of Drosophila genes. MMB contains multiple DNA-binding factors, and the data highlighted the combinatorial way by which the complex was targeted and utilized for regulation. Interestingly, only a subset of chromatin-bound complexes repressed genes normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression, whereas at other nonoverlapping sites, Myb was critical for repression. 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.


Subject(s)
Caspases/metabolism , Cell Cycle Proteins/metabolism , Cell Proliferation , Drosophila Proteins/metabolism , Drosophila/metabolism , Gene Expression Profiling/methods , Gene Expression , Proto-Oncogene Proteins c-myb/metabolism , Animals , Caspases/genetics , Cell Cycle Proteins/genetics , Cells, Cultured , Chromatin Immunoprecipitation , Drosophila/cytology , Drosophila Proteins/genetics , Genome, Insect , Models, Biological , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-myb/genetics , RNA Interference
2.
Genes Dev ; 21(8): 904-19, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17403774

ABSTRACT

The Drosophila Myb-Muv B (MMB)/dREAM complex regulates gene expression and DNA replication site-specifically, but its activities in vivo have not been thoroughly explored. In ovarian amplification-stage follicle cell nuclei, the largest subunit, Mip130, is a negative regulator of replication, whereas another subunit, Myb, is a positive regulator. Here, we identified a mutation in mip40 and generated a mutation in mip120, two additional MMB subunits. Both mutants were viable, but mip120 mutants had many complex phenotypes including shortened longevity and severe eye defects. mip40 mutant females had severely reduced fertility, whereas mip120 mutant females were sterile, substantiating ovarian regulatory role(s) for MMB. Myb accumulation and binding to polytene chromosomes was dependent on the core factors of the MMB complex. In contrast to the documented mip130 mutant phenotypes, both mip40 and mip120 mutant males were sterile. We purified Mip40-containing complexes from testis nuclear extracts and identified tMAC, a new testis-specific meiotic arrest complex that contained Mip40, Caf1/p55, the Mip130 family member, Always early (Aly), and a Mip120 family member, Tombola (Tomb). Together, these data demonstrate that MMB serves diverse roles in different developmental pathways, and members of MMB can be found in alternative, noninteracting complexes in different cell types.


Subject(s)
Chromatin/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Meiosis , Multiprotein Complexes/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Nucleus/chemistry , Cell-Free System , Chromosomal Proteins, Non-Histone/metabolism , Drosophila/genetics , Drosophila/physiology , Drosophila Proteins/genetics , Eye/anatomy & histology , Female , Male , Molecular Chaperones/metabolism , Molecular Sequence Data , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Reproduction , Retinoblastoma-Binding Protein 4 , Sequence Alignment , Spermatids/cytology , Testis/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Curr Biol ; 15(8): 755-9, 2005 Apr 26.
Article in English | MEDLINE | ID: mdl-15854909

ABSTRACT

The full complement of proteins required for the proper regulation of genome duplication are yet to be described. We employ a genetic DNA-replication model system based on developmental amplification of Drosophila eggshell (chorion) genes [1]. Hypomorphic mutations in essential DNA replication genes result in a distinct thin-eggshell phenotype owing to reduced amplification [2]. Here, we molecularly identify the gene, which we have named humpty dumpty (hd), corresponding to the thin-eggshell mutant fs(3)272-9 [3]. We confirm that hd is essential for DNA amplification in the ovary and show that it also is required for cell proliferation during development. Mosaic analysis of hd mutant cells during development and RNAi in Kc cells reveal that depletion of Hd protein results in severe defects in genomic replication and DNA damage. Most Hd protein is found in nuclear foci, and some may traverse the nuclear envelope. Consistent with a role in DNA replication, expression of Hd protein peaks during late G1 and S phase, and it responds to the E2F1/Dp transcription factor. Hd protein sequence is conserved from plants to humans, and published microarrays indicate that expression of its putative human ortholog also peaks at G1/S [4]. Our data suggest that hd defines a new gene family likely required for cell proliferation in all multicellular eukaryotes.


Subject(s)
Cell Proliferation , Chorion/cytology , DNA Replication/genetics , Drosophila Proteins/genetics , Drosophila/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Brain/metabolism , Cells, Cultured , Cloning, Molecular , DNA Primers , Drosophila/physiology , Drosophila Proteins/metabolism , Female , Flow Cytometry , Microscopy, Fluorescence , Molecular Sequence Data , Multigene Family/genetics , Ovary/metabolism , RNA Interference , Sequence Alignment , Sequence Analysis, DNA , Transgenes/genetics
4.
Genes Dev ; 18(23): 2929-40, 2004 Dec 01.
Article in English | MEDLINE | ID: mdl-15545624

ABSTRACT

The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. To further understand biochemically the functions of the Myb complex, we fractionated Drosophila embryo extracts relying upon affinity chromatography. We found that E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, we found that the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, associated with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription.


Subject(s)
Drosophila Proteins/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Animals , Chromatography, Gel , Drosophila , E2F2 Transcription Factor , Immunoprecipitation , Retinoblastoma Protein
5.
Genes Dev ; 18(14): 1667-80, 2004 Jul 15.
Article in English | MEDLINE | ID: mdl-15256498

ABSTRACT

Gene amplification at the chorion loci in Drosophila ovarian follicle cells is a model for the developmental regulation of DNA replication. Previously, we showed that the Drosophila homolog of the Myb oncoprotein family (DmMyb) is tightly associated with four additional proteins and that DmMyb is required for this replication-mediated amplification. Here we used targeted mutagenesis to generate a mutant in the largest subunit of the DmMyb complex, the Aly and Lin-9 family member, Myb-interacting protein 130 (Mip130). We found that mip130 mutant females are sterile and display inappropriate bromodeoxyuridine (BrdU) incorporation throughout the follicle cell nuclei at stages undergoing gene amplification. Whereas mutations in Dm-myb are lethal, mutations in mip130 are viable. Surprisingly, Dm-myb mip130 double mutants are also viable and display the same phenotypes as mip130 mutants alone. This suggests that Mip130 activity without DmMyb counteraction may be responsible for the Dm-myb mutant lethality. RNA interference (RNAi) to selectively remove each DmMyb complex member revealed that DmMyb protein levels are dependent upon the presence of several of the complex members. Together, these data support a model in which DmMyb activates a repressive complex containing Mip130 into a complex competent to support replication at specific loci in a temporally and developmentally proscribed manner.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , DNA Replication/physiology , Drosophila Proteins/metabolism , Gene Amplification/physiology , Ovarian Follicle/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Animals , Bromodeoxyuridine , Cell Cycle Proteins/physiology , DNA Primers , Drosophila , Drosophila Proteins/physiology , Female , Immunoblotting , Mutagenesis, Site-Directed , Mutation/physiology , Ovarian Follicle/physiology , Plasmids/genetics , Proto-Oncogene Proteins c-myb/physiology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction
6.
EMBO J ; 23(4): 897-907, 2004 Feb 25.
Article in English | MEDLINE | ID: mdl-14765124

ABSTRACT

Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator-initiator interactions and that in vivo other proteins must determine ORC's localization.


Subject(s)
DNA, Superhelical/chemistry , DNA-Binding Proteins/chemistry , Drosophila Proteins/chemistry , Animals , Base Sequence , Binding Sites , Drosophila melanogaster , Electrophoretic Mobility Shift Assay , Nucleic Acid Conformation , Origin Recognition Complex , Plasmids , Replication Origin
7.
Nature ; 420(6917): 833-7, 2002.
Article in English | MEDLINE | ID: mdl-12490953

ABSTRACT

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.


Subject(s)
DNA Replication , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Animals , Binding Sites , Chromatin/genetics , Chromatin/metabolism , DNA Footprinting , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic/genetics , Female , Gene Amplification , Genes, Insect/genetics , Macromolecular Substances , Origin Recognition Complex , Precipitin Tests , Protein Binding , Regulatory Sequences, Nucleic Acid/genetics , Replication Origin , Substrate Specificity , Transgenes/genetics
8.
Genetics ; 162(1): 217-27, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12242235

ABSTRACT

Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI(3)-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.


Subject(s)
Drosophila/genetics , Transposases/genetics , Amino Acid Sequence , Animals , Drosophila/embryology , Germ Cells , Microinjections , Molecular Sequence Data , Mutagenesis , Phosphorylation , Transposases/chemistry
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