RNF2 promotes TGFβ signaling.

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RNF2 promotes TGFβ signaling. RNF2 promotes TGFβ signaling. A, overlap of genes with corresponding promoters occupied by RNF2 (using ChIP-Seq data) and differentially expressed genes. As many as 363 genes show overlap, of which 169 (47%, green) are downregulated and 194 (53%, red) are upregulated. B, top 5 pathways from upstream regulating factor enrichment by Ingenuity Pathway Analysis (IPA). Note that TGFβ target genes were one of the most significantly deregulated and occupied genes. C, luciferase assay showing increased TGFβ-responsive promoter activity in HEK293 cells with overexpression of RNF2WT, but not with RNF2R70C and RNF2I53S. D, representative image from a Boyden chamber Matrigel invasion experiment in HMEL–BRAFV600E cells overexpressing GFP or RNF2WT and treated with DMSO or LY2157299 (TGFβRI inhibitor). Invaded cells stained with crystal violet are shown. E, occupancy of RNF2 on the LTBP2 promoter. Two ChIP-Seq tracks are shown: top: HMEL–BRAFV600E–RNF2WT tumor cells; bottom: HMEL–BRAFV600E–RNF2WT cells. F–G, (F) qPCR validation showing enrichment of V5-RNF2 (V5 antibody) and H2AK119ub on LTBP2 promoter and (G) mRNA expression of LTBP2 in HMEL–BRAFV600E cells overexpressing GFP, RNF2 wild-type or catalytic mutant derivatives (R70C and I53S). H and I, graph shows relative occupancy enrichment of RNF2 (endogenous), H2AK119ub, H3K9ac, H3K27ac, H4TetraAc, and IgG on LTBP2 promoter as obtained by ChIP-qPCR in shGFP or shRNF2-infected 501Mel (H) and WM983B (I) cells. J, relative mRNA expression of LTBP2 in shGFP or shRNF2-infected 501Mel or WM983B cells. K, Western blot showing protein levels of TGFβ target genes ID1, ID2, and ID3 in HMEL–BRAFV600E cells with knockdown of LTBP2 using two shRNAs. L and M, representative image of invaded cells from a triplicate Boyden chamber Matrigel invasion experiment in (L) HMEL–BRAFV600E or WM115 cells with knockdown of LTBP2 using two shRNAs and (M) HMEL–BRAFV600E cells overexpressing GFP or RNF2WT along with either RFP or LTBP2. N, graph showing quantitation of experiment shown in panel M. Across all panels, *, significant change t test P < 0.05 and **, P value < 0.01. Kunal Rai et al. Cancer Discov 2015;5:1314-1327 ©2015 by American Association for Cancer Research