Microarray transfection analysis of conserved genomic sequences from three immediate early genes

Abstract

In an effort to define novel transcriptional regulatory elements, microarray cotransfection was used to functionally characterize conserved non-coding sequences (CNSs) of three immediate early genes: c-fos, JunB and EGR-1. Cotransfection of fluorescent CNS reporter constructs and expression vectors for constitutively active signaling proteins demonstrated that many of the CNSs alter both the basal and regulated expression of reporter constructs, but the effects of these CNSs were usually specific for their homologous promoter. One CNS located in the first intron of the c-fos gene conferred regulation by cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and Raf. Mutagenesis and cotransfection experiments showed that PKA regulation of this c-fos intronic element was mediated by two adjacent CRE-like sequences and the transcription factor CREB. In the context of a reporter containing previously characterized regulatory elements, the novel intronic sequence contributed 50% of the transcriptional response to PKA. These studies suggest that microarray transfection studies may be useful in functional characterization of conserved genomic sequences on a larger scale.

A Novel Mouse c-fos Intronic Promoter That Responds to CREB and AP-1 Is Developmentally Regulated In Vivo

“…In contrast, several recent reports suggest that c- fos first intron improves reporter induction by calcium-mobilizing agents when appended to the upstream promoter ( [36] – [39] and our unpublished observations). While this effect does not seem to be related to elongation control, it has not been established by which mechanisms this region affects overall trancription.…”