EID3 is a novel EID family member and an inhibitor of CBP-dependent co-activation

Abstract

EID1 (E1A-like inhibitor of differentiation 1) functions as an inhibitor of nuclear receptor-dependent gene transcription by directly binding to co-regulators. Alternative targets include the co-repressor small heterodimer partner (SHP, NR0B2) and the co-activators CBP/p300, indicating that EID1 utilizes different inhibitory strategies. Recently, EID2 was characterized as an inhibitor of muscle differentiation and as an antagonist of both CBP/p300 and HDACs. Here, we describe a third family member designated EID3 that is highly expressed in testis and shows homology to a region of EID1 implicated in binding to CBP/p300. We demonstrate that EID3 acts as a potent inhibitor of nuclear receptor transcriptional activity by a mechanism that is independent of direct interactions with nuclear receptors, including SHP. Furthermore, EID3 directly binds to and blocks the SRC-1 interacting domain of CBP, which has been implicated to act as the interaction surface for nuclear receptor co-activators. Consistent with this idea, EID3 prevents recruitment of CBP to a natural nuclear receptor-regulated promoter. Our study suggests that EID-family members EID3 and EID1 act as inhibitors of CBP/p300-dependent transcription in a tissue-specific manner.

Figure 1

Primary structure and expression analysis of EID3. (A) Schematic comparisons of the EID family members 1, 2 and 3, conserved domains are coloured grey and black. (B) Tissue distribution of EID3 mRNA. Human multiple tissue northern blots were probed with radiolabelled EID3 or β-actin cDNA.

Figure 2

Sub-cellular distribution and protein analysis of EID3. (A) Intracellular localization of EID1 and EID3. FLAG-tagged EID1 and EID3 were expressed in COS-7 cells and analysed by indirect immunofluorescence using FLAG antibody (green) in the absence or presence of LMB (5 nM for 5 h). Nuclei were stained with 7-aminoactinomycin D (7-AAD) (red). More than 50 cells were studied and the experiment was independently reproduced at least three times. (B) In vitro transcribed and translated [³⁵S]methionine-labelled EID1 and EID3 are shown.

Figure 3

Analysis of NR inhibition. HuH7 cells were grown in media containing phenol red and untreated serum. The cells were transiently transfected with plasmids encoding EID1 or EID3 and luciferase reporter assays were performed. Western blots were performed to see whether the NR expression is not altered by over expressed EID1 and EID3 (indicated with arrows). (A) A luciferase reporter plasmid containing GAL4-DBD binding sites, and a plasmid expressing GAL4-SF-1. (B) Western blot using GAL4-DBD antibody to detect GAL4-SF-1 and a FLAG antibody to detect FLAG-EID1 and FLAG-EID3. (C) A luciferase reporter plasmid containing the MMTV promoter, and a plasmid encoding GR, and ligand (10 nM dexamethasone) as indicated. (D) Western blot using GR antibody to detect GR and a FLAG antibody to detect FLAG-EID1 and FLAG-EID3. (E) A luciferase reporter plasmid containing the ERE, and a plasmid encoding ERα and ligand (10 nM 17-β oestradiol) as indicated. (F) Western blot using ERα antibody to detect ERα and a FLAG antibody to detect FLAG-EID1 and FLAG-EID3.

Figure 4

EID3 binds to the CBP SID and shows a functional interaction with the C-terminus of CBP. (A) HuH7 cells were transiently transfected with plasmids encoding EID1 or EID3, a luciferase reporter plasmid containing GAL4-DBD binding sites, and a plasmid encoding GAL4-CBP C (amino acids 1678–2441). (B) A western blot using GAL4 and FLAG antibodies show that GAL4-CBP C expression is not altered by over expressed EID1 and EID3. (C) Co-immunoprecipitation of exogenous CBP (amino acids 1678–2441) and FLAG tagged EID1 or EID3. Immune complexes from HuH7 whole-cell extracts were isolated using a GAL4-DBD antibody. Precipitates were analysed using a FLAG antibody (upper gel). The input corresponds to 20% of the material subjected to immunoprecipitation and was detected using a GAL4-DBD antibody and a FLAG antibody. Asterisk represents unspecific band. (D) GST pull-down assay using the CBP SID fused to GST as indicated together with in vitro translated EID1, EID3 and TIF-2 as positive control. I (input), GST-SID (CBP amino acids 2058–2130), GST-SID mut (CBP amino acids 2058–2130 K→P). (E) HuH7 cells were transiently transfected with plasmids encoding EID3 or TIF-2, a luciferase reporter plasmid containing GAL4-DBD binding sites, and a plasmid encoding GAL4-CBP (amino acids 1678–2441).

Figure 5

EID1 and EID3 block the recruitment of CBP to the ERα-bound pS2 promoter. MCF-7 cells cultured with oestradiol were transiently transfected with FLAG vector alone for control or with FLAG-EID1 and FLAG-EID3. Soluble chromatin was prepared and immunoprecipitated by using antibodies raised against rabbit IgG as negative control, EPα, CBP and FLAG. Immunoprecipitated DNA was PCR amplified with primers that span the –353 to –159 region of the pS2 promoter. Values were time-matched and normalized for the solvent (DMSO).

Figure 6

Schematic model of the action of EID3 and EID1 that bind to the CBP SID. The binding leads to a blocking of the interaction surface of the p160 co-activators leading to a reduced target gene transcriptional activity.