Mutational studies reveal a complex set of positive and negative control elements within the chicken vitellogenin II promoter.

Abstract

The endogenous chicken vitellogenin H (VTGII) gene is transcribed exclusively in hepatocytes in response to estrogen. We previously identified two estrogen response elements (EREs) upstream of this gene. We now present an analysis of the VTGH promoter activated by these EREs in response to estrogen. Chimeric VTGH-CAT genes were cotransfected into LMH chicken hepatoma cells along with an estrogen receptor expression vector, and transient CAT expression was assayed after culturing the cells in the absence or presence of estrogen. An analysis of constructs bearing deletions downstream of the more proximal ERE indicated that promoter elements relevant to transcription in LMH cells extend to between -113 and -96. The relative importance of sequences within the VTGII promoter was examined by using 10 contiguous linker scanner mutations spanning the region from -117 to -24. Although most of these mutations compromised VTGII promoter function, one dramatically increased expression in LMH cells and also rendered the VTGH promoter capable of being activated by cis-linked EREs in fibroblasts cotransfected with an estrogen receptor expression vector. Gel retardation and DNase I footprinting assays revealed four factor-binding sites within this promoter. We demonstrate that three of these sites bind C/EBP, SP1, and USF (or related factors), respectively; the fourth site binds a factor that we denote TF-Vj8. The biological relevance of these findings is suggested by the fact that three of these binding sites map to sites previously shown to be occupied in vivo in response to estrogen.The three chicken vitellogenin genes (VTGI, VTGII, and VTGIII) provide valuable models for studying hormonedependent tissue-specific gene regulation. These genes, as well as the evolutionarily unrelated apoVLDLII gene, encode the major yolk proteins. The vitellogenin and apoVL-DLII genes are normally expressed in the hepatocytes of egg-laying hens in response to elevated levels of endogenous estrogen. All four of these genes can, however, be induced in both male and female hepatocytes, in response to exogenous estrogen, beginning around day 9 of embryonic development (14, 15). The transcriptional induction of these genes occurs as a direct response to estrogen (8,21) and results in as much as a 5-order-of-magnitude increase in steady-state mRNA levels for individual yolk protein genes (16). A single injection of estrogen also induces a mechanism for selectively destabilizing these mRNAs in the absence of estrogen (17) and reprograms hepatocytes such that they respond more rapidly to a secondary injection of estrogen (1,5,16,22,40).Some insight into the transcriptional regulation of vitellogenin genes has come from the identification of estrogen response elements (EREs) flanking these genes (6,7,25,35,38). EREs constitute binding sites for estrogen receptors (27) and function as estrogen-dependent enhancers (6,25,35 (38). In contrast, only a subset of these genes contain a second ERE within 1 kb of their promoters (38). Surprisingly, we also recen...

Cis‐acting elements reinforcing the activity of the estrogen‐response element in the very‐low‐density apolipoprotein II gene promoter

“…Second, liver-specific factors might be important to overrule the inhibition by negative cis-acting elements. For instance, the chicken vitellogenin I1 promoter contains a negative element immediately upstream of the TATA box which abrogates hormone-dependent expression of chimeric vitellogenin I1 genes in CEF cells [23]. However, the finding that transfected apoVLDLII (Fig.…”

Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals

“…In fish and especially in rainbow trout the field is hardly explored and except work by Dr VaiUant [21] few metabolic activities in hepatocyte culture have been clearly demonstrated. In oviparous animals, the synthesis of VTG by hepatocytes represent a good system with which to study the mechanism of action of steroid hormones [31][32][33][34]. Most of these studies have used hepatocytes of either amphibians (especially Xenopus laevis) or birds (especially chickens), though some have been based on hepatocytes from fish [24,[35][36][37].…”

Mitogenic Up-regulation of the PRL-1 Protein-tyrosine Phosphatase Gene by Egr-1

“…Preparation of Nuclear Protein Extracts-All steps were performed at 4°C, and 2 g/ml proteinase inhibitors antipain, aprotinin, bestatin, and leupeptin were added into all buffers except phosphate-buffered saline, pH 7.4. Nuclei from regenerating liver and from cell lines HepG2, H35, and NIH 3T3 were prepared by using the described procedure (6,24,25). Nuclei were lysed by the dropwise addition of 1/9 volume of 4.2 M NaCl and shaken for 30 min before being centrifuged at 50,000 ϫ g for 50 min.…”