Physical and Functional Interaction of Rnf2 with Af9 Regulates Basal and Aldosterone-stimulated Transcription of the α-ENaC Gene in a Renal Collecting Duct Cell Line.

The physical and functional interaction of RING finger protein 2 (Rnf2), a central component of the Polycomb repressive complex (PRC) 1, and ALL1-fused gene from chromosome 9 protein (Af9), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in ...
mIMCD3 collecting duct cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST-Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knockdown enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/qPCR assays demonstrated enrichment of Rnf2, mono-ubiquitinated histone H2A lysine 119 (H2AK119), and trimethylated histone H3 lysine 27 (H3K27), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2-Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.
Biosci. Rep.
Date: Sep. 27, 2013
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