Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells.

Abstract

The proximal rat prolactin (rPRL) promoter contains three cell-specific elements, designated footprints I, III, and IV, which restrict rPRL gene expression to anterior pituitary lactotroph cells. Footprint II (-130 to -120) binds a factor, which we have termed F2F, present in pituitary and nonpituitary cell types. Here we demonstrate that a key role of the footprint II site is to inhibit rPRL promoter activity in nonpituitary cells, specifically, by interfering with the basal activating function of a vicinal element. Gene transfer analysis revealed 20-fold activation of the rPRL promoter in nonpituitary cell types when footprint II was either deleted or specifically mutated. Similar activation of the intact rPRL promoter was obtained by in vivo F2F titration studies. In GH4 rat pituitary cells, the footprint II inhibitory activity was masked by the redundant, positively acting cell-specific elements and was inhibitory only if the two upstream sites, footprints III and IV, were deleted. Deletion of the -112 to -80 region in the footprint II site-specific mutant background resulted in complete loss of rPRL promoter activity in both pituitary and nonpituitary cell types, mapping a basal activating element that is operative irrespective of cell type to this region. While the basal activating element imparted an activating function in a heterologous promoter assay, the footprint II sequence did not display any inherent repressor function and actually induced several minimal heterologous promoters. However, the inhibitory activity of the footprint II site was detected only if it was in context with the basal activating element.These data underscore the importance of ubiquitous activating and inhibitory factors in establishing cell-specific gene expression and further emphasize the complexity of the molecular mechanisms which restrict gene expression to specific cell types. We provide a novel paradigm to study rPRL promoter function and hormone responsiveness independently of lactotroph cell-specific requirements.Eukaryotic cells have evolved a striking capacity to completely alter their program of gene expression in response to temporal, developmental, and metabolic signals. Indeed, differential activation and simultaneous repression of specific genes appears to be critical for orderly development of highly specialized tissues (19,22,37,65). While the role of nuclear factors which activate novel patterns of tissuespecific gene transcription during development have been well documented (6,13,17,22,24,25,33,38,52,59,60)

Binding of NF-Y Transcription Factor to One of the Cis-elements in the Myeloperoxidase Gene Promoter That Responds to Granulocyte Colony-stimulating Factor

“…A repressor(s) inhibiting the MPO gene expression in the IL-3-containing medium may be limiting. Since the exogenously induced gene usually integrates in the host chromosome as many copies, it is possible that such a repressor(s) may have been titrated out, as found in rat prolactin promoter (42). This may also explain the failure of several trials to localize the G-CSF-responsive element in the MPO gene by transient transfection (16,25).…”

The c-Jun δ-Domain Inhibits Neuroendocrine Promoter Activity in a DNA Sequence- and Pituitary-specific Manner

“…By contrast, mapping of the cis-acting element mediating c-Jun's inhibitory response co-localized the Jun responsive element to the FP II site, previously identified as a binding site for the putative repressor, F2F (43). Since F2F (43) and the c-Jun inhibitor both require an intact FP II site, the formal possibility remains that these two proteins might belong to the same family of transcription factors. Nevertheless, it is highly unlikely that they will be the same factor.…”

“…Plasmid Constructs-The pituitary promoter-luciferase constructs, pA 3 PRLluc-425, pA 3 rGHluc, and pA 3 h␣luc-1760 have been described (39,42,43). The Ϫ255, Ϫ189, Ϫ125, Ϫ54, and Ϫ36 rPRL promoter deletions were prepared in pG7PRL by 5Ј exonuclease digestion, subcloned into SalI/HindIII-cut pA 3 luc, and verified by dideoxy sequencing, and they will be described in detail elsewhere.…”

“…The Ϫ255, Ϫ189, Ϫ125, Ϫ54, and Ϫ36 rPRL promoter deletions were prepared in pG7PRL by 5Ј exonuclease digestion, subcloned into SalI/HindIII-cut pA 3 luc, and verified by dideoxy sequencing, and they will be described in detail elsewhere. 2 The site-specific mutants in FP I (pA 3 ⌬1luc), FP II (pA 3 ⌬2luc), and FP II together with a loop-out deletion of the Ϫ112 to Ϫ80 basal transcription element (BTE) region (pA 3 ⌬2,Dluc) were constructed in the Ϫ425 to ϩ73 rPRL promoter as described previously (39,43). The FP I/FP III double mutant (pA 3 ⌬1,3luc) was constructed in the Ϫ425 to ϩ73 rPRL promoter as described previously (45).…”

“…In the rPRL promoter, footprints (FPs) I, III, and IV bind GHF-1/Pit-1, a pituitary-specific POU-homeodomain transactivator (36,53). FP II binds a ubiquitous repressor denoted as F2F, and the Ϫ117 to Ϫ80 BTE binds a ubiquitous basal transcription-activating factor, both of which have yet to be characterized (43). Using a series of 5Ј deletions and site-specific mutations of the rPRL promoter impinging on these various regulatory cis-acting sites (shown in Fig.…”