cis-acting elements and trans-acting proteins in the transcription of chorionic gonadotropin/luteinizing hormone receptor gene in human choriocarcinoma cells and placenta

We investigated the cis-acting elements and trans-acting proteins responsible for a higher basal rate of transcription of hCG/LH receptor gene in human choriocarcinoma JEG-3 cells compared with normal term pregnancy placenta. Sequential deletion of the 5'-flanking region of the gene revealed that there are three negative control regions (NCRs) designated NCR1 (-1457 to -1373 bp), NCR2 (-1051 to -835 bp), and NCR3 (-480 to -184 bp), and a promoter (-184 to -1 bp). NCRS was more inhibitory than the other two; nearly 60-70% of the inhibitory activity resides in a sequence between -480 to -276 bp, and the rest resides in the sequence between -276 to -184 bp. Gel mobility shift assays showed that the nuclear extracts from JEG-3 cells contained proteins that form three complexes with NCR1, two with NCR2, and six with NCR3. Many of the proteins that form the complexes in NCR3 are shared with the other two NCRs. Most of the proteins that form these complexes are less abundant in nuclear extracts from JEG-3 cells than in those from placenta. The JEG-3 cell nuclear extracts also contained proteins that form three complexes with the proximal promoter of the hCG/LH receptor gene. These proteins were identified as Ap2, Ap2-like I, and Sp1 from the competition studies with synthetic excess unlabeled Ap2, Sp1, and CTF/NF1 consensus oligodeoxynucleotides and/or supershift in gel mobility assays with anti-Ap2 antibody. Although the JEG-3 cell nuclear extracts contained abundant Ap2-like protein I and low levels of Ap2 and Sp1 proteins, the placental nuclear extracts contained low levels of Ap2-like protein I and very low to nondetectable levels of Ap2 and Sp1 proteins. Deoxyribonuclease I footprinting revealed that the nuclear extracts from JEG-3 cells and placenta protected the -116 to -93 bp and -65 to -45 bp regions in the proximal promoter of the hCG/LH receptor gene that contain Sp1 and Ap2 binding sites, respectively. However, the nuclear extracts from placenta only partially protected these regions, which is consistent with lower levels of proteins that bind to the proximal promoter of the gene. In summary, we conclude that the presence of low levels of proteins that bind to the NCRs and the high levels of proteins, especially Ap2-like I, that bind to the proximal promoter can potentially explain higher transcription of the hCG/LH receptor gene in JEG-3 cells compared with that in normal term pregnancy human placenta.