DNA-binding activity of the transcription factor upstream stimulatory factor 1 (USF-1) is regulated by cyclin-dependent phosphorylation

Abstract

The ubiquitous transcription factor upstream stimulatory factor (USF) 1 is a member of the bzHLH (leucine zipper-basic-helix-loop-helix) family, which is structurally related to the Myc family of proteins. It plays a role in the regulation of many genes, including the cyclin B1 gene, which is active during the G2/M and M phases of the cell cycle and may also play a role in the regulation of cellular proliferation. We show that the affinity of recombinant USF-1 for DNA is greatly increased by treatment with active cyclin A2-p34(cdc2) or cyclin B1-p34(cdc2) complexes and that its interaction with DNA is dependent on p34(cdc2)-mediated phosphorylation. We have localized the phosphorylation site(s) to a region that lies outside the minimal DNA-binding domain but overlaps with the previously identified USF-specific region. Deletion studies of USF-1 suggest that amino acids 143-197 regulate DNA-binding activity in a phosphorylation-dependent manner.

Involvement of an Upstream Stimulatory Factor as Well as cAMP-responsive Element-binding Protein in the Activation of Brain-derived Neurotrophic Factor Gene Promoter I

“…As described in the present study, it is highly possible that USF1/USF2 also functions as a Ca 2ϩ signal-stimulated transcription factor in neurons. It has been reported that the phosphorylation of USF1 enhanced its DNA binding activity (36); quite recently, Galibert et al (37) reported that phosphorylation of USF1 at Thr-153 increased its transcriptional activity. In addition, the activation of gene transcription by USF1/USF2 could be mediated by CBP/p300 (38).…”

Novel CAR-mediated Mechanism for Synergistic Activation of Two Distinct Elements within the Human Cytochrome P450 2B6 Gene in HepG2 Cells

“…E boxes consist of the core hexanucleotide sequence CANNTG and can bind proteins from the basic helix-loop-helix (bHLH) family that includes the aryl hydrocarbon receptor, upstream stimulatory factor, and sterol regulatory elementbinding protein (32). Protein phosphorylation has been shown to either enhance (30,33) or disrupt the DNA binding (34) of some bHLH factors and/or to modify their transcriptional activity (35,36). In fact the bHLH factors heart and neural crest derivatives expressed 1 and 2 interact with the protein phosphatase 2A targeting subunit B56␦ (37).…”

Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene

“…and P.J.H., unpublished). Phosphorylation of USF-1 is necessary for DNA binding (Cheung et al, 1999) and, consistent with this requirement, the predominant form of USF-1 eluted from PAI-1 deoxyoligonucleotide affinity columns co-migrated with the 45 kDa (phospho-USF-1) species (Fig. 5).…”

“…The upper-lower doublet retardation pattern was evident as early as 2 hours after scrape injury (2 hr edge) and, like growing RK cultures (G), addition of USF-1 antibodies specifically supershifted this upper complex. phosphorylation requirement of certain HLH factors for E-box motif recognition (Nozaki et al, 1997;Cheung et al, 1999).…”

“…Nuclei were collected by centrifugation for 30 seconds at 14,000 g, resuspended in 50 µl of cold lysis buffer (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) containing leupeptin, aprotinin, chymostatin, pepstatin A, antipain (each at a final concentration of 10 µg/ml), rocked at 4°C for 15 minutes and extracts clarified at 10,000 g for 5 minutes. For phosphatase treatments, isolated nuclei were lysed (Cheung et al, 1999) and 5 µg nuclear extract protein incubated with potato acid phosphatase in PIPES/KOH digestion buffer, pH 6.5, for 2 hours at 37°C prior to electrophoresis on SDS/12% acrylamide gels and western blotting for USF-1 (as indicated below).…”