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Microarray Transfection Analysis of cAMP and cGMP Regulation of Gene Expression
Author(s) -
Uhler Michael D,
Ren Xiaomei,
Kubish Ginger,
Redmond Tanya M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a930
Subject(s) - promoter , transcription factor , biology , transfection , gene , transcriptional regulation , regulation of gene expression , microbiology and biotechnology , gene expression , kinase , genetics
Transcription factor activation by phosphorylation is a common mechanism for both developmental and hormonal control of gene expression. Over 2000 transcription factors and 600 protein kinases are predicted from the human genome sequence but only a small fraction of the potential regulatory interactions have been experimentally tested. We have defined transfection conditions to functionally assess these interactions in a microarray transfection format. The transfection method employed is termed Surface Transfection and Expression Protocol (STEP) and relies on the production of recombinant polyfunctional proteins to mediate the high efficiency in situ transfection of different cell types. Cis‐acting transcriptional regulatory sequences driving the expression of GFP and other reporters were used to probe the activity of specific transcription factor families in the presence or absence of transfected protein kinases. We find that regulation of native promoters such as the c‐fos promoter by cAMP‐ and cGMP‐dependent protein kinases is controlled by conserved non‐genic (CNG) sequences surrounding the c‐fos gene. In particular, an evolutionarily conserved sequence within the first intron of the c‐fos gene plays a key role in transcriptional regulation by these two protein kinases. The regulation by the CNG sequences was promoter specific in most cases. This microarray transfection approach should be useful in defining novel pathways by which protein phosphorylation regulates transcription factor activity as well as other functional interactions in vivo. This work was supported by NIH grants HG002367 and DK063340.