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. 2004 Dec 1;18(23):2929-40.
doi: 10.1101/gad.1255204. Epub 2004 Nov 15.

Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex

Peter W Lewis  1 , Eileen L Beall, Tracey C Fleischer, Daphne Georlette, Andrew J Link, Michael R Botchan
Affiliations

Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex

Peter W Lewis et al. Genes Dev. .

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.

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Figures

Figure 1.
Figure 1.
The Drosophila Myb complex copurifies with E2F2/RBF. (A) The fractionation scheme used to purify Mip120-associated proteins from Drosophila embryo nuclear extract is shown. An anti-Mip120 affinity column was used as the final step in the purification. (B) SDS-PAGE and silver stain analysis of the anti-Mip120 (p120) or nonspecific immunoglobulin (cIgG) affinity column eluates. Proteins from the Mip120 affinity column eluate were identified by direct analysis of large protein complexes (DALPC) mass spectrometry (Link et al. 1999) and are indicated at the right. (C) Verification of the proteins identified in the anti-Mip120 or the nonspecific immunoglobulin (cIgG) eluate. Shown are immunoblots performed with the antibodies indicated on the right of each panel confirming that all members of the Myb and E2F2 complex were present in the supercomplex. (D) Data from duplicate control (cIgG and Flag) and experimental (Mip120 affinity and Flag-Mip130) samples were analyzed using mass spectroscopy. Briefly, a protein abundance factor (PAF) for each protein was expressed as the total number of nonredundant peptide hits normalized to the molecular weight of the cognate protein. An average PAF for each protein found in duplicate samples was then calculated. Average PAF values from experimental samples were normalized to that from the control samples to determine a relative abundance factor (RAF), shown as the ratio of the experimental/control samples.
Figure 2.
Figure 2.
The Mip130-associated proteins form a large stable complex. (A) Shown are immunoblot analyses of sephacryl S-400 gel filtration column fractions using antibodies against the proteins listed on the left. The eluate from the affinity-purified Flag-Mip130 column was used as the starting material for the column profile. (B) Shown is a silver-stained gradient SDS-PAGE gel of fractions derived from glycerol gradient centrifugation of the anti-Flag-Mip130 eluate. Approximately 6 pmol of Flag-Mip130 protein was loaded onto a 15%-40% gradient and the sample centrifuged at 150,000g for 12 h. The protein components of the peak fractions are indicated on the right. (C) Partially clarified embryo nuclear extracts were immunoprecipitated using the antibodies indicated on the top. The precipitates were washed with buffer containing 0.5M KCl, and the coimmunoprecipitated proteins were eluted with 0.4% sarcosyl. All immunoprecipitations were performed in the presence of 50 μg/mL ethidium bromide. Shown are immunoblot analyses of the immunoprecipitated material using antibodies directed against each of the super-complex members as indicated on the right.
Figure 5.
Figure 5.
The Drosophila Myb complex interacts with the histone deacetylase, Rpd3. (A) Immunoprecipitates using antibodies against either Mip120 or nonspecific IgG (cIgG) and Drosophila 0-12-h embryo nuclear extract were incubated with 3H-acetylated core Drosophila histones. Released 3H-acetic acid was measured by scintillation counting, and the average and standard deviations from three independent experiments are indicated by the bar graph. (B) Immunoprecipitations as in A were analyzed for the presence of Rpd3 following immunoblot analysis. Specific enrichment for Rpd3 was detected in the anti-Mip120 IP versus the control IgG. (C) The histone deacetylase inhibitor, TSA, resulted in abnormal BrdU incorporation in follicle cell nuclei. Both wild-type (TM3) and Rpd3303 heterozygous mutant flies were grown in the presence of various concentrations of TSA as indicated. Shown are confocal images taken of stage-10 egg chambers from heterozygous Rpd3303 females treated either with ethanol alone (left) or with 20 μM TSA in ethanol (right) and stained for anti DmOrc2 (red), anti-BrdU (green), and DAPI (blue). The percentage of egg chambers displaying one or more follicle cells exhibiting overall genomic BrdU incorporation for each sample is indicated at the bottom. The total number of egg chambers scored are as follows: for wild type: EtOH alone, 149; 10 μM TSA, 267; and 20 μM TSA, 279; for Rpd3303: EtOH alone, 160; 10 μM TSA, 289; and 20 μM TSA, 310.
Figure 5.
Figure 5.
The Drosophila Myb complex interacts with the histone deacetylase, Rpd3. (A) Immunoprecipitates using antibodies against either Mip120 or nonspecific IgG (cIgG) and Drosophila 0-12-h embryo nuclear extract were incubated with 3H-acetylated core Drosophila histones. Released 3H-acetic acid was measured by scintillation counting, and the average and standard deviations from three independent experiments are indicated by the bar graph. (B) Immunoprecipitations as in A were analyzed for the presence of Rpd3 following immunoblot analysis. Specific enrichment for Rpd3 was detected in the anti-Mip120 IP versus the control IgG. (C) The histone deacetylase inhibitor, TSA, resulted in abnormal BrdU incorporation in follicle cell nuclei. Both wild-type (TM3) and Rpd3303 heterozygous mutant flies were grown in the presence of various concentrations of TSA as indicated. Shown are confocal images taken of stage-10 egg chambers from heterozygous Rpd3303 females treated either with ethanol alone (left) or with 20 μM TSA in ethanol (right) and stained for anti DmOrc2 (red), anti-BrdU (green), and DAPI (blue). The percentage of egg chambers displaying one or more follicle cells exhibiting overall genomic BrdU incorporation for each sample is indicated at the bottom. The total number of egg chambers scored are as follows: for wild type: EtOH alone, 149; 10 μM TSA, 267; and 20 μM TSA, 279; for Rpd3303: EtOH alone, 160; 10 μM TSA, 289; and 20 μM TSA, 310.
Figure 3.
Figure 3.
The Myb-MuvB complex is involved in transcriptional corepression. (A) Chromatin immunoprecipitation (ChIP) was performed using the antibodies indicated at the top, and the isolated DNA was analyzed using PCR for the presence of promoter fragments derived from the E2F2 regulated genes indicated on the right. Shown is an ethidium bromide-stained gel of PCR reactions performed with both gene-specific and actin primers simultaneously. The ratio of intensities of the particular gene promoter to the actin PCR signal in the ChIP and input DNA lanes were used to calculate the relative fold enrichment, as indicated below each lane. (B) RNAi was used to specifically eliminate members of the Myb-MuvB complexes as indicated on the top. Total RNA isolated from the RNAi-treated samples was analyzed for the abundance of E2F2 regulated transcripts using probes specific for the genes indicated on the right. nsRNA indicates RNAi performed using a double-stranded RNA against a region of the plasmid, pBSKS(+). GADPH was used as a loading control. (C) Signals derived from three independent Northern blots for the genes indicated were quantitated using PhosphorImager analysis and compared to the signal obtained for GADPH. Shown are histograms and standard deviations for the average normalized signal.
Figure 4.
Figure 4.
Simultaneous double RNAi directed at Myb-MuvB complex members show some additive repressive effects. (A) RNAi directed at E2F2 and Mip130 or E2F2 and Mip120 displayed a small additive effect of derepression on the four genes we tested as mentioned in Figure 3. (B) Signals derived from five independent Northern blots for the genes indicated were quantitated using PhosphorImager analysis and compared to the signal obtained for GADPH. Shown are histograms and standard deviations for the average normalized signal.
Figure 6.
Figure 6.
Cdk phosphorylated RBF1 does not dissociate from the Myb-MuvB complex. (A) Flag-Mip130-purified proteins were incubated with purified recombinant Cyclin E-Cdk2. Immunoblot analysis using anti-RBF1 antibodies as indicated showed that the mobility of RBF1 was retarded in SDS-PAGE upon phosphorylation. Incubation with λ-phosphatase restored the normal mobility of the RBF1 protein. Incubation with 32P-autoradiographs indicated that RBF1 was phosphorylated in the presence of cyclin-Cdk. (B) Immunoprecipitations of Flag-Mip130 proteins were performed after mock (odd lanes) or Cyclin E-Cdk2 phosphorylation (even lanes). Shown is an anti-RBF1 immunoblot analysis performed after immunoprecipitation with the various antibodies indicated on the top. The presence of phosphorylated RBF1 is indicated by the autoradiograph. (C) A titration of the Cyclin E-Cdk2 levels was performed using a constant amount of purified Myb-MuvB complex. PhosphorImager analysis indicated that the RBF1 phosphorylation levels plateaued at the kinase concentration used in the experiments (3 pmol Cyclin E-Cdk2). (D) E2F1/RBF1 was immunoprecipitated from 0- to 12-h Drosophila embryo nuclear extract. The immunoprecipitated complex was washed and equilibrated with the kinase reaction buffer. Following immunoprecipitation using either anti-E2F1 or control nonspecific IgG, the pellet was washed and incubated for 30 min at 30°C with Cyclin E-Cdk2. Shown is an immunoblot analysis using anti-RBF1 antibodies in which Cylin E-Cdk2 treatment dirupts the interaction of E2F1 with RBF1.
Figure 7.
Figure 7.
A model for Drosophila Myb complex activity. The Myb complex may serve dual functions in both the activation and repression of transcription and DNA replication that may depend upon the presence or absence of other factors at a given chromosomal location and/or developmental context. The conversion between activator and repressor functions may alter the subunit composition of the Myb complex, favoring either coactivator or corepressor interactions. Drosophila E2F1 may preferentially interact with the Myb complex to promote activation of both transcription and replication. Ultimately, the complex acts through recruitment of chromatin modifying enzymes, such as the histone deacetylase, Rpd3, to alter local chromatin architecture in order to influence both origin usage and promoter activity.
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