Neuronally enriched RGS4 plays a critical role attenuating G protein signaling in brain, although the mechanisms regulating RGS4 expression are unknown. Here we describe a novel mechanism for transcriptional activation of RGS4 in neuron-like PC6 cells, where RGS4 is markedly induced during confluence-induced growth arrest. Transcriptional activation of RGS4 in confluent PC6 cells was accompanied by impaired G i/o -dependent MAPK activation. In the human RGS4 gene promoter, we identified three phylogenetically conserved cis-elements: an inverted CCAAT box element (ICE), a cAMP response element, and a B-cell lymphoma 6 (Bcl6)-binding site. The ICE and the cAMP response element mediate activation, and the Bcl6 site mediates repression of RGS4 transcription. Activation of RGS4 transcription in confluent PC6 cells is accompanied by increases in NF-YA and C/EBP and decreases in Bcl6 levels in the nucleus. Increases in NF-YA and C/EBP lead to their increased binding to the RGS4 promoter in vivo, and dominant negative forms of these proteins repressed RGS4 promoter activity. Acetylation of NF-YA and Bcl6 were increased in postconfluent cells. Trichostatin A stimulation of RGS4 promoter activity, accompanied by increased binding of NF-YA and decreased binding of Bcl6 to the promoter, was abolished by mutation of the ICE and enhanced by mutation of the Bcl6 site. These findings demonstrate a dynamic and coordinated regulation of nuclear levels and acetylation status of trans-acting factors critical in determining the off/on state of the RGS4 promoter.
Regulator of G protein signaling 4 (RGS4)2 is a member of the mammalian RGS family of proteins of which 30 members exist in humans. RGS proteins were discovered as essential negative regulators of heterotrimeric G protein signaling by genetic studies in yeast and Caenorhabditis elegans (1, 2). These proteins act as GTPase-activating proteins for heterotrimeric G ␣ subunits (3), thereby accelerating the shut-off mechanism for G protein signaling. Some RGS proteins, including RGS4, can also act as effector antagonists (4, 5) or can directly or indirectly interact with G protein-coupled receptors (6 -8), actions that contribute to their negative regulatory effects on G protein signaling in cells.Although G protein signaling is involved in virtually every known physiological process and RGS proteins are an important component of this signaling, the mechanisms regulating expression of RGS genes are largely unknown. Studies have shown that several RGS genes are induced under certain physiological conditions, for example RGS1 during mitogenic activation of lymphocytes (9), RGS2 in the early stage of 3T3-L1 differentiation to adipocytes (10), RGS16 during genotoxic stress (11), and the yeast RGS gene SST2 by pheromone (12). Regulation of RGS4 expression has also been noted in facial motoneuronal precursors during embryonic development in mice (13), in epithelial and endothelial cells during tubulogenesis (14), and in rat PC12 cells during cell confluence (15). In the study by Gril...