Characterization of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

The chicken riboflavin carrier protein (RCP) is an estrogen induced egg yol k and white protein. Eggs from hens which have a splice mutation in RCP gen e fail to hatch, indicating an absolute requirement of RCP for the transpor t of riboflavin to the oocyte. In order to understand the mechanism of regu lation of this gene by estrogen, the chicken RCP gene including 1 kb of the 5' flanking region has been isolated. Characterization of the gene structu re shows that it contains six exons and five introns, including an intron i n the 5' untranslated region. Sequence analysis of the 5' flanking region d oes not show the presence of any classical, palindromic estrogen response e lement (ERE). However, there are six half site ERE consensus elements. Four deletion constructs of the 5' flanking region with varying number of ERE h alf sires were made in pGL3 basic vector upstream of the luciferase-coding region. Transient transfection of these RCP promoter deletion constructs in to a chicken hepatoma cell line (LMH2A) showed 6-12-fold transcriptional in duction by a stable estrogen analogue, moxesterol. This suggests that the R CP gene is induced by estrogen even in the absence of a classical ERE and t he half sites of ERE in this promoter may be important for estrogen inducti on.