Abstract
BackgroundActivin A increases production of follicle stimulating hormone (FSH) by inducing transcription of its beta subunit (FSHB). This induction has been studied here in LbetaT2 gonadotropes using transient expression of ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to Luc). Several sequences between -169/-58 bp of the ovine FSHB proximal promoter are necessary for induction by activin A in LbetaT2 cells, but deletions between -4741/-752 bp decrease induction > 70% suggesting the existence of other important 5' sequences. Induction disappears if a minimal T81 thymidine kinase promoter replaces the ovine FSHB TATA box and 3' exon/intron. The study reported here was designed to determine if sequences outside -169/-58 bp are important for induction of ovine FSHB by activin A.MethodsProgressively longer deletions of ovine FSHBLuc were created between -4741/-195 bp. Deletions internal to this region were created also, but replaced with substitute DNA. The ovine FSHB TATA box region (-40/+3 bp) was replaced by thymidine kinase and rat prolactin minimal promoters, and substitutions were made in 3' intron/exon sequences. All constructs were tested for basal and activin A-induced expression in LbetaT2 cells.ResultsSuccessive 5' deletions progressively lowered fold-induction by activin A from 9.5 to zero, but progressively increased basal expression. Replacing deletions with substitute DNA showed no changes in basal expression or fold-induction. Induction by activin A was supported by the minimal rat prolactin promoter (TATA box) but not the thymidine kinase promoter (no TATA box). Replacement mutations in the 3' region did not decrease induction by activin A.ConclusionThe data show that specific ovine FSHB sequences 5' to -175 bp or 3' of the transcription start site are not required for induction by activin A. A minimal TATA box promoter supports induction by activin A, but the sequence between the TATA box and transcription start site seems unimportant.
Highlights
Activin A increases production of follicle stimulating hormone (FSH) by inducing transcription of its beta subunit (FSHB)
Transcription of the gene encoding FSH beta subunit (FSHB) is rate limiting for overall hormone production, and the most potent and influential direct inducer of FSH production is in the activin family [4,5]
The construct used by our laboratory to study regulation of ovine FSHB is ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to the luciferase gene; see Figure 1)
Summary
Activin A increases production of follicle stimulating hormone (FSH) by inducing transcription of its beta subunit (FSHB). This induction has been studied here in LbetaT2 gonadotropes using transient expression of ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to Luc). The study reported here was designed to determine if sequences outside -169/-58 bp are important for induction of ovine FSHB by activin A. The standard approach for these studies is to analyze transient expression of FSHB promoter/reporter gene constructs in transformed murine gonadotropes (LbetaT2 cells). The construct used by our laboratory to study regulation of ovine FSHB is ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to the luciferase gene; see Figure 1)
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