FIG. 6.

Localization of cAMP response elements within CNS1-A. (A) Luciferase reporter assay of UAMS-32P cells stably transfected with the CNS1-A-97 reporter construct or reporter constructs derived from CNS1-A in which different regions of CNS1-A were deleted (Delta 1 through Delta 5). Open boxes indicate the region of CNS1-A retained in each construct. veh, vehicle. (B) Sequence comparison of a portion of DNA contained in the region of CNS1-A retained in the Delta 5 construct shown in panel A. Sequences for rat, dog, human, and chimp CNS1-A were obtained by BLAST search of Ensembl genome databases with mouse CNS1-A. For each search, only a single high-homology region was obtained, and in each case the region was located a similar distance upstream from the RANKL gene. Potential binding sites for Runx2 (OSE2) and CREB (dCRE and pCRE) are highlighted in gray. Nucleotides differing from consensus binding sites are shown in bold. (C) Luciferase reporter assay of UAMS-32P cells stably transfected with CNS1-A-97 or CNS1-A-97 containing point mutations within the indicated consensus sites (mutation indicated by an X over the site). In all reporter assays, cells were treated with vehicle or 1.5 mM db-cAMP for 24 h and relative light units (RLU) were determined.