Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut

Abstract

The gene promoter of Drosophila proliferating cell nuclear antigen ( dPCNA ) contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replicationrelated element (DRE, 5′ ′ ′ ′ -TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in dPCNA promoter as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut bound to both DRE and the sequence 5′ ′ ′ ′ -AATCAAAC in URE, with much higher affinity to the former. Measurement of dPCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the dPCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the dPCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of dPCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of dPCNA during cell proliferation.

Evidence for Autoregulation and Cell Signaling Pathway Regulation From Genome-Wide Binding of theDrosophilaRetinoblastoma Protein

“…We searched the motifs in the JASPAR and TRANSFAC databases, and one of the motifs was similar to the E2F site, as expected. Another bears clear similarity to the DREF site, which is bound by the DNA replication-related element-binding factor, involved in the expression of a wide variety of proliferation-related genes and whose function in the context of cell-cycle regulation has been investigated on the PCNA and DNApol alpha promoters (Yamaguchi et al 1995; Takahashi et al 1996; Seto et al 2006). We also found a motif similar to the binding sequence of the mammalian Forkhead transcription factor FOXJ2, and one novel motif that did not closely match any other sequence in the database, which we designated RAM (Rbf associated motif).…”

Identification of common and cell type specific LXXLL motif EcR cofactors using a bioinformatics refined candidate RNAi screen in Drosophila melanogastercell lines

“…Mor encodes for a component of the ATP-dependent chromatin remodeling BRM complex, and is thought to be essential for complex integrity [55,56]. Mor transcription is regulated by the DRE/DREF regulatory pathway, which is required for expression of genes involved in cell proliferation [56] and implicated in ecdysone regulation [57]. MED23 has RNA Pol II transcription mediator activity and is involved in transcription initiation from the RNA Pol II promoter.…”

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway

“…Transfection of various DNA mixtures was performed using Cell-Fectin reagent (Invitrogen) and cells were harvested 48 h thereafter. Luciferase activity was measured as described earlier 21, 52, 55 and normalized to Renilla luciferase activity using pAct5C-seapansy as an internal control. 18 All plasmids for transfection were prepared using a QIAGEN plasmid Kit (Qiagen, Venlo, Netherlands).…”