Identification of functional CCAAT/enhancer-binding protein and Ets protein binding sites in the human chorionic somatomammotropin enhancer sequences

Abstract

The human chorionic somatomammotropin (CS) A and B genes (listed asCSH1andCSH2in the HUGO database) are highly expressed in placenta. A 241 bp potent enhancer, nucleotides (nts) 1–241, located at the 3′ end of theCS-Bgene (CS-Benh) stimulates promoter activity specifically in placental trophoblast cellsin vitro. Strong activity is exerted by a 23 bp element within the CS-Benh (nts 117–139), shown to interact with transcription enhancer factor (TEF) members of the transcription enhancer activator (TEA) DNA-binding domain-containing family. An identical TEF element is present in the homologous (97.5%) CS-Aenh; however, a few nucleotide differences suppress its activity. Previously, we identified regulatory sequences distinct from the TEF element within an 80 bp modulatory domain (nts 1–80) in the CS-Benh. Using structural and functional assays we now show that CCAAT/enhancer-binding protein (C/EBP) binding sites exist in the 80 bp modulatory domains of both enhancers, and an Elk-1 binding site exists in the modulatory domain of the CS-Aenh. C/EBPα or C/EBPβ strongly repressedCSp.CATactivity but stimulatedCSp.CAT.CS-Benhactivity. In contrast, the equivalentCS-Aenhancer sequences were unable to relieve promoter repression. Elk-1 overexpression also resulted in differential effects on the CS-Aenh versus CS-Benh. Finally, we provide evidence for the association of C/EBPβ with theCS-AandCS-Bgenes in human placental chromatin, including differential involvement of C/EBPβ with the CS-Aenh versus the CS-Benh, and therefore consistent with the notion that these are regions of regulatory significancein vivo. We conclude that members of the C/EBP and Ets families can differentially modulate CS-Benh and CS-Aenh activity.