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- Title
Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the a-ENaC gene in a renal collecting duct cell line.
- Authors
Zhi-Yuan YU; Qun KONG; KONE, Bruce C.
- Abstract
The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the a-ENaC (epithelial Na+ channel a- subunit) gene was explored in mIMCD3 CD (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 (glutathione transferase)-Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated a-ENaC mRNA levels and a-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple a-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2-Af9 co-occupancy of the a-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the a-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the a-ENaC promoter to constrain basal a-ENaC transcription in collecting duct cells, and aldosterone reverses this process.
- Subjects
MOLECULAR interactions; CHROMOSOMAL proteins; ALDOSTERONE; TRANSCRIPTION factors; NEURAL stimulation; DNA-protein interactions; PROMOTERS (Genetics)
- Publication
Bioscience Reports, 2013, Vol 33, Issue 5, p823
- ISSN
0144-8463
- Publication type
Article
- DOI
10.1042/BSR20130086