The ubiquitously expressed upstream stimulatory factor (USF) involved in the transcription of a wide variety of cellular
genes consists of 2 related polypeptides; USF1, of 43 kD and USF2, of 44 kD. USF1, also known as major
late transcription factor (MLTF), was originally described by its ability to bind to and stimulate transcription from the
adenovirus major late promoter.
General function
Nucleic acid binding, DNA binding, Transcription factor
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Cellular localization
Nuclear
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Ovarian function
Ovulation
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Liu J, et al reported that the delayed activation of the prostaglandin G/H synthase-2
promoter in bovine granulosa cells is associated with
down-regulation of truncated upstream stimulatory factor-2.
The high
levels of amino-terminal truncated USF-2 and C/EBPbeta in bovine granulosa
cells prior to hCG treatment could repress gene expression, and be involved in the
delayed induction of PGHS-2 in species with a long ovulatory process.
Expression regulated by
LH
Comment
Role of upstream stimulatory factor phosphorylation in the regulation of the prostaglandin G/H synthase-2 promoter in granulosa cells Sayasith K, et al .
To investigate the role of upstream stimulatory factor 1 (USF1) and USF2 phosphorylation in the regulation of the prostaglandin G/H synthase-2 promoter in granulosa cells, promoter activity assays were performed in cultures of bovine granulosa cells transfected with the PGHS-2 promoter/luciferase (LUC) construct -149/-2PGHS-2.LUC. Transfections were done in the absence or presence of forskolin, the cAMP-dependent proteine kinase (PKA) inhibitor H89 or expression vectors encoding USF1, USF2, the catalytic subunit of PKA (cPKA) or a PKA inhibitor protein (PKI). Electrophoretic mobility shift assays (EMSAs) were performed to study USF/DNA interactions using granulosa cell nuclear extracts and a proximal PGHS-2 promoter fragment containing the E-box element. Results showed that forskolin stimulation and cPKA overexpression caused a significant increase of USF-dependent DNA binding and PGHS-2 promoter activities (p < 0.05). In contrast, both activities were decreased by H89 treatment or PKI overexpression. RT-PCR analyses revealed that these treatments had similar effects (induction by forskolin and cPKA, and inhibition by H89 and PKI) on endogenous PGHS-2 mRNA levels in granulosa cells. Cotransfection studies with a USF2 mutant lacking N-terminal activation domains (U2D1-220) repressed forskolin-, cPKA- and USF-dependent PGHS-2 promoter activities. EMSAs showed that U2D1-220 competed with full-length USF proteins and saturated the E-box element. Immunoprecipitation/Western blot analyses revealed an increase in levels of phosphorylated USF1 and USF2 after forskolin treatment, whereas chromatin immunoprecipitation assays showed that binding of USF proteins to the endogenous PGHS-2 promoter was stimulated by forskolin. Site-directed mutagenesis of a consensus PKA phosphorylation site within USF proteins abolished their trans-activating capacity. Collectively, these results characterize for the first time the role of USF phosphorylation in PGHS-2 expression, and identify the phosphorylation-dependent increase in USF binding to the E-box as a putative molecular basis for the rise in PGHS-2 promoter trans-activation in granulosa cells after the preovulatory gonadotropin surge.
Ovarian localization
Granulosa
Comment
Kazuya Yamada et al 2001 reported the cloning and Functional Expression of an E
Box-Binding Protein from Rat Granulosa Cells.
They cloned the cDNA(s) encoding E box (5'-CACGTG-3')-binding protein from a rat granulosa cell cDNA library using a yeast one-hybrid system. When multiple E box sequences were
used as target, they obtained a positive clone that encodes the rat homologue of upstream stimulatory factor 2 (USF2). An
analysis of the nucleotide sequence and its deduced amino acid sequence reveals that rat USF2 protein consists of 346 amino acid residues and belongs to the basic helix-loop-helix/leucine zipper protein family. Northern blot analysis
shows that rat USF2 mRNA exists as multiple forms between 1.6 and 2.2 kilobases. Electrophoretic mobility shift assays showed that in vitro-translated
rat USF2 specifically binds to the E box. In addition, cotransfection experiments with luciferase-reporter constructs in HepG2 cells reveal that the overexpression of rat USF2 leads to an increase of luciferase activity in the E box
sequence-dependent manner.
Follicle stages
Preovulatory
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In vivo regulation of FSH-receptor(Fshr) by the transcription factors USF1 and USF2 is cell specific. Hermann BP et al. Pituitary FSH promotes pubertal timing and normal gametogenesis by binding its receptor (FSHR) located on Sertoli and granulosa cells of the testis and ovary, respectively. Studies on Fshr transcription provide substantial evidence that Upstream stimulatory factor 1 (USF1) and USF2, basic helix-loop-helix leucine zipper proteins, regulate Fshr through an E-box within its promoter. However, despite the strong in vitro support for USF1 and USF2 in Fshr regulation, there is currently no in vivo corroborating evidence. In the present study, chromatin immunoprecipitation (ChIP) demonstrated specific binding of USF1 and USF2 to the Fshr promoter in both Sertoli and granulosa cells, in vivo. Control cells lacking Fshr expression showed no USF-Fshr promoter binding, thus correlating USF-promoter binding to gene activity. Evaluation of Fshr expression in Usf1 and Usf2 null mice further explored USF's role in Fshr transcription. Loss of either gene significantly reduced ovarian Fshr levels, while testis levels were unaltered. ChIP analysis of USF-Fshr promoter binding in Usf-null mice indicated differences in the composition of promoter-bound USF dimers in granulosa and Sertoli cells. Promoter-bound USF dimer levels declined in granulosa cells from both null mice, despite increased USF2 levels in Usf1-null ovaries. However, compensatory increases in promoter-bound USF homodimers were evident in Usf-null Sertoli cells. In summary, this study provides the first in vivo evidence that USF1 and USF2 bind the Fshr promoter and revealed differences between Sertoli and granulosa cells in compensatory responses to USF loss and the USF dimeric composition required for Fshr transcription.