The upstream stimulatory factor is a ubiquitously expressed cellular transcription factor that binds to a symmetrical
DNA sequence that is found in a variety of viral and cellular promoters.
General function
DNA binding, Transcription factor
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Cellular localization
Nuclear
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Ovarian function
Luteinization, Early embryo development
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Requirement of the transcription factor USF1 in bovine oocyte and early embryonic development. Datta TK et al. (2015) Upstream stimulating factor 1 (USF1) is a basic helix-loop-helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8-16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation of JY1, GDF9, and FST genes associated with oocyte competence.//////////////////
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.
Expression regulated by
Eicosanoids
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Ovarian localization
Luteal cells
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Wu YL, et al reported the transcriptional regulation of cyclooxygenase-2 gene in ovine
large luteal cells.
There is positive feedback pathway in the ovine large luteal cell, such that
prostaglandin (PG) F,, stimulation induces intraluteal PGF(2 alpha) production
as the result of induction of one of the rate-limiting enzymes in PG
production, cyclooxygenase-2 (Cox-2). In transient transfection assays, Cox-2 promoter was
rapidly induced (4 h) by phorbol didecanoate (a protein kinase [PK] C
activator), ionomycin, and cloprostenol (PGF(2 alpha) analogue), with a peak
induction at 12 h.
The Cox-2 promoter could be reduced to 282 base pairs (bp) of the 5 ' flanking
sequence with retention of full inducibility by cloprostenol. Mutation of
three critical cis-responsive elements within this 282-bp region (C/EBP, cAMP
responsive element [CRE], and E-box) indicated that E-box was critical in both
basal and cloprostenol-induced promoter activity. However, there was also
significant but less dramatic inhibition of cloprostenol stimulation by
mutation of C/EBP and CRE in the Cox-2 promoter, and mutation of all three
elements eliminated cloprostenol induction of this promoter. Electrophoretic
mobility shift assays of nuclear extracts from large luteal cells revealed
that upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box in
Cox-2. Thus, PKC directly regulates transcription of the Cox-2 gene in large
luteal cells by acting through DNA elements close to the putative
transcriptional start point, particularly an E-box region at -50 bp.
Follicle stages
Corpus luteum
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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.