Numerous nuclear proteins bind the long control region of human papillomavirus type 16: a subset of 6 of 23 DNase I-protected segments coincides with the location of the cell-type-specific enhancer.

The long control region of the human papillomavirus type 16 genome is 856 base pairs (bp) long. It contains a cell-type-specific enhancer, a glucocorticoid response element, and sequences mediating the response to the viral gene products of open reading frame E2; all three regulate the promoter P97. We mapped binding sites of trans-acting proteins relevant for the cell-type-specific enhancer and other cis-acting elements by DNase I footprint experiments with nuclear extracts from HeLa cells. Throughout the human papillomavirus type 16 long control region 23 footprints protect 557 of 900 bp. Nine footprints fall into a 400-bp segment that was previously identified to contain the cell-type-specific enhancer. Variations of the protein concentration in the footprint reaction do not affect six of these nine footprints. At high protein concentrations, three footprints fuse to a 106-bp protected region, suggesting that this segment specifically binds several proteins of lower affinity or abundance. Unexpectedly, extracts from human MCF7 and mouse 3T3 cells, in which the enhancer is inactive, give footprints identical to those obtained with HeLa extracts. Seven footprints contain the sequence 5'-TTGGC-3'. Footprint competition experiments suggest that factor NFI binds to these seven motifs. Competition with cloned oligonucleotides in transfections suggests that these elements contribute to the enhancer function. Subcloning identifies a 232-bp fragment between positions 7524 and 7755 as sufficient for full enhancer activity. Several of the six footprinted elements on this segment may cooperate functionally, since subclones of this region show decreased or no cell-type-specific enhancer function.     

Evidence for cooperativity between E2 binding sites in E2 trans-regulation of bovine papillomavirus type 1.

The long control region of bovine papillomavirus type 1 (BPV-1) can function in an orientation- and position-independent manner as an E2-dependent enhancer. Dissection of the long control region has revealed two E2-responsive elements, E2RE1 and E2RE2, which map, respectively, between nucleotides 7611 and 7806 and between nucleotides 7200 and 7386 of the BPV-1 genome. In this study, we have carried out a detailed analysis of E2RE1, which has previously been shown to be involved in the regulation of the BPV-1 promoters P89 and P7940. One characteristic of E2RE1 is the presence of a pair of ACCN6GGT motifs (E2 binding sites) at each end of the element. To determine the contribution of these sites, as well as other sequences within E2RE1, to enhancer function, specific mutations and deletions were generated by oligonucleotide reconstruction. The functional analysis of these mutations confirmed that a pair of E2 binding sites was essential for E2-dependent enhancer activity but also indicated that cooperativity between the motifs at each end of E2RE1 creates a highly responsive element. Isolated ACCN6GGT motif pairs could also act as E2-dependent enhancers but at a significantly reduced level in comparison to the intact element. The sequences between the E2 binding sites in E2RE1 were not required for enhancer function and could actually block the enhancer activity of an isolated pair of E2 binding sites when positioned between the binding sites and the enhancer-deleted simian virus 40 early promoter.     

Episodic activation of the rat GnRH promoter: role of the homeoprotein oct-1

Recent reports demonstrate that the rat GnRH promoter is activated in an episodic fashion in immortalized GnRH neurons, but little information is available on molecular processes that contribute to this phenomenon. In this study, we dissected the regions of the rat GnRH promoter that mediate these effects by testing a series of 5' deletion luciferase reporter constructs on the pattern of photonic emissions from single, living GT1-7 GnRH neuronal cells. Deletion analysis revealed that the region -2012/-1597 that contains the neuron-specific enhancer (NSE) was required for the elaboration of pulses of GnRH promoter activity. The importance of this region was supported by observations that episodic reporter activity could be transferred to a neutral nonpulsatile promoter (Rous sarcoma virus, RSV(180)). Immunoneutralization of Oct-1 as well as mutation of an octamer binding site located at -1787/-1783 (AT-a site) blocked the pulsatile GnRH promoter activity in GT1-7 neuronal cells. Taken together, our findings indicate that episodic GnRH gene expression is a promoter-dependent phenomenon, which is mediated by Oct-1 interaction with regulatory elements in the NSE region.     

Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester.

Several viral trans-activators and a tumor promoter were examined for the ability to activate human papillomavirus type 18 (HPV-18) gene expression. A plasmid containing the HPV-18 noncoding region placed upstream of the chloramphenicol acetyltransferase reporter gene was cotransfected with different herpes simplex virus type 1 (HSV-1) genes into several cell lines. Both HSV-1 TIF and ICP0 activated HPV-18 expression; however, activation by TIF was observed only in epithelial cells, while ICP0 stimulated expression in a wide variety of cells. The element activated by both TIF and ICP0 was mapped to a 229-base-pair fragment which also contains an HPV-18 epithelial cell-preferred enhancer. The inclusion of a papillomavirus E2 trans-activator with TIF and ICP0 further increased HPV-18 expression. In contrast, the HSV-1 ICP4 and ICP27 genes, as well as the human T-cell lymphotropic virus type I and human immunodeficiency virus type 1 tat genes, were found to have no effect on HPV-18 expression. In transient assays, the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated HPV-18 expression. The region of HPV-18 activated by TPA was localized to a sequence which is homologous to other TPA-responsive elements.