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 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 α-ENaC (epithelial Na+ channel α-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 α-ENaC mRNA levels and α-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 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.
*Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, TX 77030, U.S.A.
Zhi-Yuan
ZY
Yu
*Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, TX 77030, U.S.A.
Qun
Q
Kong
*Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, TX 77030, U.S.A.
Bruce C
BC
Kone
D000450:Aldosterone / Q000502:physiology; D000818:Animals; D002460:Cell Line; D053503:Epithelial Sodium Channels / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D007685:Kidney Tubules, Collecting / Q000166:cytology; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D063150:Polycomb Repressive Complex 1 / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
D053503:Epithelial Sodium Channels; C489902:Mllt3 protein, mouse; D009687:Nuclear Proteins; D000450:Aldosterone; D063150:Polycomb Repressive Complex 1; C489842:Rnf2 protein, mouse; D044767:Ubiquitin-Protein Ligases