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.     

Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.     

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.     

Role of alpha-transinducing factor (VP16) in the induction of alpha genes within the context of viral genomes.

In herpes simplex virus 1, the five alpha genes are induced by alpha-transinducing factor (alpha TIF; VP16), a virion protein, acting in concert with Oct-1 and other cellular proteins on a cis-acting site in the promoter domain of alpha genes. Because alpha TIF is an essential virion protein, its function as an inducer can best be evaluated only by mutating the cis-acting site. Earlier we reported on a series of 17 mutations in and around the cis-acting site of a 275-bp alpha 27 promoter fused to a reporter gene and recombined into the viral genome. These recombinant viruses were tested in Vero cells in the presence of cycloheximide, and we demonstrated that mutations in the sequence required for Oct-1 binding abolished transactivation whereas mutations in the alpha TIF-dependent GARAT sequence decreased but did not abolish transactivation. We now report that (i) in limited-passage human embryonic lung cells, alpha gene expression from promoters mutated in the GARAT sequences is often higher and more variable than in Vero cells, (ii) in the absence of cycloheximide, the mutant viruses show less significant impairment of reporter gene expression, (iii) Oct-1 can bind either to the overlapping octamer element or to various TAATGARAT sequences with differing degrees of binding strength and these relative binding levels correlate well with levels of gene expression observed in infected cells, (iv) in the cis-acting site upstream of the alpha 4 gene, no degenerate overlapping Oct-1 sequence exists, and therefore in this instance Oct-1 must be binding directly to the TAATGARAT sequence, (v) extension of the alpha 27 promoter by an additional 1,334 bp results in much higher expression of the reporter gene as a result of additional upstream cis-acting sites, and (vi) obliteration of the most proximal Oct-1 binding element within the 275-bp promoter dramatically reduces gene expression even in the presence of the additional upstream cis-acting sites.     

Hoxb1 enhancer and control of rhombomere 4 expression: complex interplay between PREP1-PBX1-HOXB1 binding sites

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.