CLIM and RLIM are co-localized with ERα in the nucleus of breast cancer cells. Immunohistochemical staining of breast tumor samples was performed on cryosections using an anti-ERα monoclonal antibody together with anti-CLIM (A) or anti-RLIM (B) polyclonal antibodies. CLIM and RLIM expression is observed almost exclusively in the nucleus and is co-localized with ERα expression. CLIM, RLIM and ERα are also co-localized in the nucleus of MCF7 breast cancer cells (C). (D) Interaction of endogenous RLIM, CLIM and ERα in MCF7 cells. Total cell extracts of MCF7 cells were immunoprecipitated with specific anti-RLIM, -CLIM, -ERα or non-specific IgG polyclonal antibodies. Western blot analysis was performed with each antibody. Twenty percent input is shown.

RLIM targets ERα for ubiquitination. (A) ERα is an in vitro substrate for ubiquitination by RLIM. In vitro 35S-labelled full-length ERα protein was incubated alone or together with various combinations of the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) UbcH5 or bacterially expressed and purified full-length GST-RLIM. The brackets indicate higher molecular weight ubiquitinated ERα. Ub-ERα, ubiquitinated ERα. (B) Overexpression of RLIM increases the formation of high molecular weight, detergent insoluble forms of ERα. H1299 cells were transfected with ERα and ubiquitin expression vectors as indicated. Whole extracts were prepared and detergent soluble and insoluble fractions were separately analyzed by western blot analysis with specific anti-RLIM polyclonal or anti-ERα monoclonal antibodies. Note that there is a noticeable increase in a high molecular weight form of ERα upon overexpression of RLIM in the detergent insoluble fraction which is not apparent in the detergent soluble fraction. Ubiq, ubiquitin. (C) Overexpression of RLIM results in increased ERα ubiquitination in cells. H1299 cells were transfected with a His-tagged ubiquitin (His-Ub) expression vector with or without RLIM overexpression as in (B) and the ubiquitinated proteins were isolated using a Ni-NTA affinity matrix and analyzed by western blot using a specific anti-ERα monoclonal antibody. The brackets indicate the increase in high molecular weight ubiquitinated ERα upon RLIM overexpression.

CLIM2 and RLIM regulate endogenous estrogen receptor activity. (A) Endogenous CLIM2 regulates ERα activity in cells. MCF7 breast cancer cells were transfected with control siRNA (A and B), CLIM2 siRNA (A) or RLIM siRNA (B). Cells were grown for 48 hours and treated for 2h with estrogen (10⁻⁸ M ethinyl estradiol) or untreated. Gene expression was measured by quantitative RT-PCR using primers specific for PS2-hnRNA, Cathepsin D, WISP2 or CLIM2 mRNA. mRNA levels were normalized to an unregulated gene (36B4) and expressed relative to the uninduced control siRNA transfected cells. CLIM2 mRNA levels are shown to verify efficient knock-down. (B) RLIM is necessary for optimal induction of gene expression by the endogenous estrogen receptor. MCF7 cells were transfected with RLIM siRNA and treated as in (A). In opposition to CLIM2 siRNA, RLIM siRNA significantly decreases the induction of estrogen-regulated gene expression compared to the control conditions. RLIM mRNA levels are decreased approximately 90% upon siRNA transfection. (C) CLIM and RLIM are recruited to endogenous EREs. CLIM or RLIM recruitment to the PS2, Cathepsin D and WISP2 genes was analyzed by chromatin immunoprecipitation analysis using chromatin from MCF7 cells untreated or treated with estrogen (10⁻⁸ M ethinyl estradiol) for 1h. Estrogen treatment increased the recruitment of both CLIM and RLIM to the PS2 and WISP2 genes while only CLIM recruitment was also increased on the Cathepsin D gene where RLIM was also present in an estrogen-independent manner. Non-specific IgG was used to distinguish between specific and background binding. ChIP samples were normalized to input samples and expressed as fold enrichment relative to the average of all IgG ChIP samples.

CLIM and RLIM are specific ERα-interacting proteins. ³⁵S-labeled in vitro transcribed and translated full-length human ERα (A–D), isolated human ERα-LBD (A,B), full-length human ERα ERβ (C), full-length human AR (D) and full-length human ERα GR (D) were tested for their ability to interact with full-length GST-CLIM2 or GST-RLIM (A–D) or various mutants of CLIM2 (A) or RLIM (B). Equal amounts of GST and GST-fusion proteins were used and input samples of in vitro translated proteins were loaded separately. (A) CLIM contains a RID (upper panel) within the LCCD domain which is necessary for interaction with full-length (middle panel) ERα and with the isolated ERα-LBD (lower panel). DD, dimerization domain; LCCD, LDB/Chip conserved domain; NLS, nuclear localization signal; LID, LIM-interacting domain. (B) RLIM interacts with the LBD of ERα through the C-terminal RING finger-containing domain. NLS, nuclear localization signal; BD, basic domain; RING, RING-H2 zinc finger. CLIM2 and RLIM specifically interact with ERα, but not with ERβ (C), AR (D) or GR (D) in the presence of the respective ligands.

CLIM and RLIM regulate the transcriptional activity of ERα. (A) CLIM overexpression dose-dependently decreases ERE transcriptional activity. MCF7 cells were transfected with an ERE-containing luciferase reporter construct (ERE-TK-Luc) and an internal control plasmid for transfection efficiency (phRG-TK) which constitutively expresses Renilla luciferase together with the indicated amounts of a plasmid expressing CLIM2 or a CLIM2 RID mutant (AxxAA). The total amount of DNA was kept constant by adding an appropriate amount of control plasmid (pCS2). Estrogen (10⁻⁸ M ethinyl estradiol) was added 24h after transfection and cells were harvested after another 24h. All transfections were normalized and expressed relative to the average of the estrogen-treated, control transfected cells as relative activity. (B) RLIM overexpression dose-dependently increases ERE transcriptional activity. MCF7 cells were transfected with ERE-TK-Luc and phRG-TK together with the indicated amounts of a RLIM or RLIMΔRING expression vector and treated with estrogen as in (A). The total amount of plasmid DNA was kept constant by adding the control plasmid (pCS2). (C) CLIM2 and RLIM siRNA increase and decrease ERE activity, respectively. ERE-TK-Luc and phRG-TK were transfected as in (A) and (B) together with control, CLIM2 or RLIM siRNA. Cells were grown for 48h to allow for a knock-down of endogenous CLIM2 or RLIM prior to estrogen treatment for another 24h. Luciferase activity was expressed as relative activity as in (A) and (B).