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Nucleic Acids Res
2008 Jul 01;3612:3905-15. doi: 10.1093/nar/gkn291.
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Transcriptional regulation of the Drosophila moira and osa genes by the DREF pathway.
Nakamura K
,
Ida H
,
Yamaguchi M
.
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The DNA replication-related element binding factor (DREF) plays an important role in regulation of cell proliferation in Drosophila, binding to DRE and activating transcription of genes carrying this element in their promoter regions. Overexpression of DREF in eye imaginal discs induces a rough eye phenotype in adults, which can be suppressed by half dose reduction of the osa or moira (mor) genes encoding subunits of the BRM complex. This ATP-dependent chromatin remodeling complex is known to control gene expression and the cell cycle. In the 5'' flanking regions of the osa and mor genes, DRE and DRE-like sequences exist which contribute to their promoter activities. Expression levels and promoter activities of osa and mor are decreased in DREF knockdown cells and our results in vitro and in cultured cells indicate that transcription of osa and mor is regulated by the DRE/DREF regulatory pathway. In addition, mRNA levels of other BRM complex subunits and a target gene, string/cdc25, were found to be decreased by knockdown of DREF. These results indicate that DREF is involved in regulation of the BRM complex and thereby the cell cycle.
Figure 1. osa and mor genes genetically interact with DREF. Scanning electron micrographs of adult eyes. DREF overexpressing flies (GMR-GAL4/GMR-GAL4; UAS-DREF/UAS-DREF) were crossed with osa or mor mutant flies and developed at 28°C. (A, B) GMR-GAL4/+; UAS-DREF/+; +/+. (C, D) GMR-GAL4/+; UAS-DREF/+; osa2/+. (E, F) GMR-GAL4/+; UAS-DREF/+; P{PZ}osa00090/+. (G, H) GMR-GAL4/+; UAS-DREF/+; mor1/+. (A, C, E, G) Scale bars are for 50 μm. (B, D, F, H) Scale bars are for 12.5 μm.
Figure 2. DRE and DRE-like sequences in the 5′ flanking regions of the osa and mor genes. Transcription initiation site is numbered as +1. (A) A schematic representation of DREs in the osa gene. (B) A schematic representation of DREs in the mor gene.
Figure 3. Effects of mutations in DRE sites in the 5′ flanking region of the osa and mor genes on their promoter activities. osa or mor promoter–luciferase fusion plasmids were transfected into S2 cells. Luciferase activities are expressed relative to wild-type osa (A) and mor (B) promoters. Mean values with standard deviations from three independent transfections are shown. The white box, wild-type DRE; the black box, mutant DRE.
Figure 4. DREFdsRNA treatment affects mRNA levels and promoter activities of osa and mor. (A) cDNAs were prepared from total RNA isolated from dsRNA-treated S2 cells and levels of DREF, osa and mor mRNAs were measured by quantitative RT-PCR. Fold differences against the amplification with no treatment are shown with standard deviations from three independent dsRNA treatments. β-tubulin was used as a negative control. (B) Cell extracts were analyzed by western blotting with antiDREF antibodies. (C) Promoter activities of osa and mor in dsRNA-treated cells. Luciferase activities relative to that of wild-type promoter in non-dsRNA-treated cells are shown with standard deviations from five independent transfections.
Figure 5. Complex formation between osa DREs and Kc cell nuclear extracts. 32P-labeled double-stranded oligonucleotides osaDRE1 (A) and osaDRE2 (B) were incubated with Kc cell nuclear extracts in the presence of the indicated competitor oligonucleotides or antiDREF monoclonal antibodies. The amounts of competitors were 25-, 50- or 100-fold molar ratios for the osaDRE1 probe (A) and 100- or 400-fold molar ratios for the osaDRE2 probe (B). Mock, normal mouse IgG; Mab1, antiDREF monoclonal antibody 1; Mab4, antiDREF monoclonal antibody 4.
Figure 6. Complex formation between mor DREs and Kc cell nuclear extracts. 32P-labeled double-stranded oligonucleotides morDRE1 (A), morDRE2 (B) and morDRE3 (C) were incubated with Kc cell nuclear extracts in the presence of the indicated competitor oligonucleotides or antiDREF monoclonal antibodies. The amounts of competitors were 25-, 50- or 100-fold molar ratios for the morDRE1 probe (A) and 100- or 400-fold molar ratios for the morDRE2 (B) and morDRE3 probes (C). Mock, normal mouse IgG; Mab1, antiDREF monoclonal antibody 1; Mab4, antiDREF monoclonal antibody 4.
Figure 7. Binding of DREF to the DRE-containing genomic regions of the osa and mor genes. Crosslinked chromatin of S2 cells was immunoprecipitated with either antiDREF IgG or control rabbit IgG. The genomic regions containing osaDRE1, osaDRE2, morDRE1, 2 or morDRE3 were amplified by real-time PCR and compared with the amplification from the immunoprecipitates with the control IgG.
Figure 8. Effects of DREF knockdown on mRNA levels of other BRM complex components. (A) Total RNA was isolated from S2 cells treated with dsRNA and cDNA was prepared. mRNA levels were determined by quantitative RT-PCR. Fold differences versus no dsRNA treatment are shown as mean values with standard deviations from three independent dsRNA transfections. (B) DRE-containing regions were amplified from immunoprecipitates with antiDREF IgG. (C) mRNA level of stg in DREFdsRNA-treated cells.
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