Physical and Functional Interactions between USF and Sp1 Proteins Regulate Human Deoxycytidine Kinase Promoter Activity

Abstract

Deoxycytidine kinase (EC 2.7.1.74, dCK) is central to drug activity of anticancer and antiviral agents such as cytosine arabinoside (araC) and gemcitabine. HepG2 hepatocellular carcinoma cells were used to study the transcriptional regulation of dCK. 5-Deletion and sitedirected mutagenesis of the dCK upstream region (positions ؊464 to ؊27) confirmed the importance of two GC-boxes (positions ؊317 to ؊309 and ؊213 to ؊206) and two E-boxes (positions ؊302 to ؊297 and ؊278 to ؊273). In vitro electromobility shift assays with HepG2 nuclear extracts and in vivo chromatin immunoprecipitation assays with HepG2 chromatin extracts confirmed the presence of bound Sp1/Sp3 and USF1/2. Co-transfections in HepG2 cells showed that USF1 and USF2a stimulated and Sp1 repressed promoter activity from a dCK-luciferase reporter gene construct. In Sp-and USF-null Drosophila Mel-2 cells, both Sp1 and USF1 stimulated dCK promoter activity in a dose-dependent manner, however, both Sp3 and USF2a were effectively inert. Combined Sp1 and USF1 showed additive transactivation at lower concentrations of Sp1. Sp1 was inhibitory at higher levels. Stimulation by combined USF1/USF2a with Sp1 was similar to that for USF1 alone with Sp1, whereas transactivation by Sp1 and USF2a without USF1 was synergistic. Physical interactions between USF and Sp proteins were confirmed by immunoprecipitations with Sp-and USF-specific antibodies. Domain mapping of USF1 and USF2a localized the functional interactions between USF and Sp proteins to the DNA binding domain of USF. Identifying the physical and functional interactions between Sp and USF proteins may lead to a better understanding of the basis for differential expression of the dCK gene in tumor cells and may foster strategies for up-regulating dCK gene expression and improving chemotherapy with araC and gemcitabine.

v-Src-mediated Down-regulation of SSeCKS Metastasis Suppressor Gene Promoter by the Recruitment of HDAC1 into a USF1-Sp1-Sp3 Complex

“…Although USF1 was first identified as a transcriptional activator for the adenovirus late promoter (59), recent studies demonstrate that USF1 is also involved in transcriptional repression of certain genes (60 -62). Moreover, Ge et al (63) showed physical interaction between USF1 and Sp1, and at low Sp1 concentrations, Sp1 and USF1 could cooperatively transactivate the deoxycytidine kinase promoter, whereas at higher levels of Sp1, USF1 helps form a repressor complex. Therefore, although v-Src does not alter USF1 binding to the E-box on the ␣ promoter, it is likely that the enhanced Sp1 binding to the adjacent GC-box in v-Src/3T3 cells converts the USF1 from a transcriptional activator to a repressor.…”

The Role of Cytidine Deaminase and GATA1 Mutations in the Increased Cytosine Arabinoside Sensitivity of Down Syndrome Myeloblasts and Leukemia Cell Lines

“…THP-1, HL60, and U937 leukemia cell lines and the human HT1080 fibrosarcoma cell line were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 containing 10% heat-inactivated calf serum (Hyclone Labs, Logan, UT), 100 units/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere at 37°C in the presence of 5% CO 2 /95% air. D. Mel-2 cells were purchased from Invitrogen (Carlsbad, CA) and maintained as described previously (34).…”

“…Mel-2 cells were cotransfacted with 1 g of the pBS-1451/-442ϩL-40/ ϩ400 reporter gene construct and 200 -800 ng of GATA1-L (pPacGATA1) or GATA1-S (pPacGATA1-S), using Fugene 6 reagent (Roche Diagnostics Corporation, Indianapolis, IN), as described previously (34). Luciferase activities were assayed using the Single Luciferase Assay System (Promega) and normalized to total cell protein, measured by the Bio-Rad protein assay system.…”

Role of Helix-Loop-Helix Proteins during Differentiation of Erythroid Cells

“…Both methylated H3K4 and H4 asymmetrically dimethylated at R3 are associated with permissive or actively transcribed gene loci (44,67). USF proteins also interact with DNA-binding transcription factors, including the ubiquitously expressed proteins TFII-I (112), nuclear factor Y (NF-Y), Sp1, and the AP1-like transcription factors MafB and NF-E2, an erythroid/myeloid cell-specific heterodimer composed of p45 and a small Maf protein (41,107,154,155).…”