Characterization of RAP1A and RAP1B expression across cell types and identification of their core promoter elements (946.1)

Abstract

The RAP1A and RAP1B genes are members of the RAS oncogene family of GTPases and are implicated in many processes, including cell adhesion, growth, and signal transduction. Cell type‐specific expression of these genes and their regulatory elements, however, have been poorly investigated. Our lab has worked to define the levels of RAP1A and RAP1B native expression in a variety of human cell lines and to characterize the cis‐regulatory elements controlling this expression. In investigating native expression, RNA was isolated from eight different lines and analyzed using qRT‐PCR. Our findings suggest that RAP1A is expressed at statistically similar levels in all lines tested, while RAP1B shows more variable expression. Putative promoter fragments for both genes were identified using the UCSC genome browser, cloned by PCR, and inserted directly upstream of the Green Fluorescent Protein (GFP) gene. Plasmids were transiently transfected into a variety of cell lines, and GFP expression was analyzed by fluorescence microscopy. Activity of the core promoter for RAP1A appears to explain the observed native expression, and a possible repressor element has been identified. For RAP1B, the identified core promoter fails to explain cell type‐specific native expression, requiring further analysis of potential enhancer regions. Additionally, lack of RAP1B core promoter function in an osteosarcoma line may be SP1‐mediated.