An NF1-related vitellogenin activator element mediates transcription from the estrogen-regulated Xenopus laevis vitellogenin promoter

Abstract

We have used a homologous transfection system employing cell lines derived from Xenopus hepatocytes and fibroblasts and cloned Xenopus estrogen receptor to identify a DNA sequence essential for efficient transcription of the powerful estrogen-regulated Xenopus laevis vitellogenin B1 promoter. Although deletion of the CAAT box, the conserved nematode box, and several other sequences had little effect on vitellogenin promoter activity, deletion or mutation of a short sequence related to the NF1 transcription activator reduced estrogen-dependent transcription 10-20-fold in both cell lines. The activity of the vitellogenin activator (VA) element, which is located at positions -48 to -41, is strongly position dependent. Insertion of even five copies of the VA element at -445 does not restore activity to a VA deletion, while insertion of a single VA element at -62 largely restores promoter activity. The VA element is not liver-specific, since VA deletions, mutations, and insertions elicited similar effects on vitellogenin promoter activity in the cell lines derived from hepatocytes and fibroblasts. Gel mobility shift assays using nuclear extracts from the Xenopus hepatocyte cell line demonstrate the presence of a protein which binds with high affinity and specificity to the VA sequence. The NF1 consensus sequence, but not a functional AP1 consensus sequence, which actually exhibits higher homology to the VA element than the NF1 sequence, competes for binding to the VA element. In transfections using simplified promoters in which single copies of the VA region or NF1 sequence are linked to a TATA box, the VA region, which does not contain a palindrome, is 2.4-fold more active than the consensus NF1 palindrome. The VA-binding protein therefore appears to be related to, but not identical to, the NF1 transcription activator.