MOZ-TIF2 interacts with CBP via the AD1 domain in vitro and in vivo. (A) Schematic representations of domains in TIF2, MOZ-TIF2, MOZ, and derivatives. bHLH-PAS, basic helix-loop-helix/Per-ARNT-Sim domain; NID, nuclear receptor interaction domain; Q, glutamine-rich domain; H1, histone H1/5-like domain, PHD, plant homeodomain; Zn, C2HC zinc finger. Residue-rich domains, acidic, proline and glutamine, and methionine. Residue numbers 1117 and 869 in MOZ-TIF2 indicate the amino acid breakpoints in MOZ and TIF2, respectively. Numbers 1290 and 1363 in MOZ-TIF2ΔAD1 indicate amino acid boundaries of the deleted AD1 domain. (B) In vitro-translated TIF2, MOZ, and MOZ-TIF2were tested for interaction with GST proteins or GST fused to the LBDs ERα or RXRα in the presence (+) or absence (−) of ligand. One-tenth of the total input of labeled protein is shown for comparison (10%). (C) Interactions of TIF2, MOZ, MOZ-N, MOZ-TIF2, and MOZ-TIF2ΔAD1 with GST, GST-CBP-SID (residues 2058 to 2130), and full-length GST-CBP as in (B). (D) Coimmunoprecipitation data showing in vivo interactions between ectopically expressed CBP and FLAG fusion proteins in HEK293 cells. (E) FRET analyses between CFP-CBP and YFP-MOZ-TIF2 or YFP-MOZ-TIF2ΔAD1. Representative nuclei showing fluorescence images pre- and post-YFP bleach are shown. (F) Percent FRET efficiencies (E%) for nuclei (15 and 10 nuclei, respectively). The line indicates the average E% value for each group. The differences in E% between the two groups were analyzed by using a two-tailed type 2 t test, and the P value is indicated.

MOZ-TIF2 inhibits transcription activation by nuclear receptors and by p53. (A to D) GAL4-NR- and full-length-ERα-mediated luciferase assays from transiently transfected COS-1 cells. Cognate ligand (shaded bars) (9-cis-retinoic acid [9-cis RA], all-trans-retinoic acid [AT-RA], or 17β-estradiol [E2]) or an equal volume of vehicle (black bars) was added. Reporter activation is represented as fold induction over the control value (reporters in the absence of NRs, coactivators, or ligand), and results from representative experiments are shown. (E) p53-mediated luciferase assays from transiently transfected SaOs-2 cells with different p53-responsive reporter plasmids. The reporter activation is shown as a percentage relative to the activation of the reporter gene in the presence of p53, which is set to 100%. The quantity of DNA (nanograms) transfected is indicated, with + indicating 100 ng and − indicating no DNA. The data are presented as averages and standard errors of the means from triplicate samples.

Role of CBP in the inhibition of NR-mediated and p53-mediated transcription activation by MOZ-TIF2. (A) GAL4-RARα/TIF2-mediated reporter assays in COS-1 cells expressing MOZ and MOZ-TIF2 fusion proteins tagged with YFP or CFP. (B) GAL4-RARα/TIF2-dependent activation of a (Gal4)₅-1bΔ-luc reporter gene in COS-1 cells in the presence of various MOZ-containing proteins. (C) Rescue experiment showing the effect of overexpressed CBP on MOZ-TIF2-mediated inhibition of GAL4-RARα/TIF2 reporter activation in COS-1 cells. (D) Dose-dependent inhibition of endogenous PPAR- and RAR-mediated transcription activation by MOZ-TIF2 is dependent on the AD1 domain in U937 cells. (E) p53-dependent activation of the IRGC-luc and the GADD45-luc reporter genes in SaOs-2 cells in the presence of various MOZ-containing proteins. (F) Rescue experiment showing the effect of overexpressed CBP on MOZ-TIF2-mediated inhibition of p53-dependent reporter activation of the IRGC-luc reporter gene in SaOs-2 cells. Error bars indicate standard errors of the means.

Subcellular localization of endogenous and exogenously expressed MOZ proteins. (A) Epifluorescence microscope images of endogenous MOZ proteins for COS-1, U-2Os, HEK293, HL60, and U937 cells. (B) FLAG-MOZ proteins detected with anti-FLAG (green) and anti-MOZ (red) antibodies in COS-1 cells. Yellow indicates colocalization. (C) Distinct nuclear localization of epitope-tagged MOZ (upper panels) and MOZ-TIF2 proteins (lower panels), showing punctate and mesh-like staining, respectively. (D) Representative time lapse images of YFP-MOZ-TIF2 proteins expressed in HEK293 cells. Images i and ii are still pictures of the same cells captured 25 min apart. The arrows indicate perinucleolar regions of interest highlighted at higher magnification in the insets. Bars, 1 μm.

Subcellular localization of MOZ, MOZ-TIF2, MOZ-N, and MOZ-TIF2ΔAD1 and their effect on the nuclear distribution of endogenous CBP and PML in COS-1 cells. (A) Staining of COS-1 cells for endogenous CBP and PML. (B) Detection of the indicated FLAG-MOZ fusion proteins and endogenous CBP. (C) Costaining of FLAG-tagged MOZ proteins with endogenous PML. (D) Costaining of FLAG-MOZ-TIF2 for endogenous SP100. (E) Detection of YFP-MOZ and YFP-MOZ-TIF2 proteins with staining for endogenous CBP. (F) Staining of cells expressing YFP-MOZ-TIF2 for endogenous PML. Red and green channels are shown in the Merge panels; yellow indicates colocalization. Bar, 1 μm.

Effect of MOZ-TIF2 on cellular CBP protein levels and on the proliferation of hematopoietic stem cells in vitro. (A) Quantitative analysis of CBP-containing speckles (PML bodies) in control cells (n = 300) and YFP-MOZ-TIF2- and YFP-MOZ-TIF2ΔAD1-expressing cells (n = 50). (B) Representative images of COS-1 cells, showing the effect of YFP-MOZ-TIF2 and YFP-MOZ-TIF2ΔAD1 expression on detection of endogenous CBP speckles. (C) Western blot analysis of whole-cell extracts from YFP⁺ sorted HEK293 cells expressing YFP, YFP-MOZ-TIF2,and YFP-MOZ-TIF2ΔAD1 cultured in the absence (upper panels) or presence (lower panels) of lactacystin. The band corresponding to full-length CBP is indicated. The loading controls α-tubulin and vinculin are also indicated. (D) Serial replating assays to assess the proliferative potential of GFP⁺ Lin⁻ cells transduced with the indicated vectors in methylcellulose medium containing SCF, IL-3, and Il-6. Error bars indicate standard errors of the means. (E) Anti-CBP staining of sorted GFP⁺ Lin⁻ cells transduced with the indicated retroviral vectors. Images were taken at identical exposure times. DNA was counterstained with 0.1 μg of DAPI per ml.