Abstract
CCAAT/enhancer-binding Protein beta (C/EBPbeta) is a member of the bZIP transcription factor family that is expressed in various tissues, including cells of the hematopoietic system. C/EBPbeta is involved in tissue-specific gene expression and thereby takes part in fundamental cellular processes such as proliferation and differentiation. Here, we show that the activity of C/EBPbeta is negatively regulated by the transcriptional co-repressor Daxx. C/EBPbeta was found to directly interact with Daxx after overexpression as well as on the endogenous level. Glutathione S-transferase pulldown assays showed that Daxx binds via amino acids 190-400 to the C-terminal part of C/EBPbeta. Co-expression of C/EBPbeta changed the sub-nuclear Daxx distribution pattern from predominantly POD-localized to nucleoplasmic. Daxx suppressed basal and p300-enhanced transcriptional activity of C/EBPbeta. Furthermore, Daxx decreased the C/EBPbeta-dependent phosphorylation of p300, which in turn was associated with a diminished level of p300-mediated C/EBPbeta acetylation. Co-expression of promyelocytic leukemia protein abrogated the repressive effect of Daxx on C/EBPbeta as well as the direct interaction of Daxx and C/EBPbeta, presumably by re-recruiting Daxx to PML-oncogenic domains. In acute promyelocytic leukemia (APL) cells, C/EBPbeta activity is known to be required for all-trans-retinoic acid-induced cell differentiation and disease remission. We show that all-trans-retinoic acid as well as arsenic trioxide treatment leads to a reduced C/EBPbeta fraction associated with Daxx suggesting a relief of Daxx-dependent C/EBPbeta repression as an important molecular event leading to APL cell differentiation. Overall, our data identify Daxx as a new negative regulator of C/EBPbeta and provide first clues for a link between abrogation of Daxx-C/EBPbeta complex formation and APL remission.
Highlights
APL, acute promyelocytic leukemia; bZIP, basic-region-leucine-zipper; all-trans-retinoic acid- (ATRA), all-trans-retinoic acid; SUMO, small ubiquitin-like modifier; CBP, CREB-binding protein; PML-oncogenic domains (PODs), PML-oncogenic domain; SIM, SUMO-interacting motif; RAR, retinoic acid receptor; GFP, green fluorescent protein; HA, hemagglutinin; TSA, C/EBP proteins is the highly conserved C-terminal basic region leucine zipper DNA binding domain, which mediates a large number of protein-protein interactions [1]
Tel.: 49-251-83-33203; Fax: 49-251-83-33206; E-mail: klempna@uni-muenster.de. 2 The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein ; APL, acute promyelocytic leukemia; bZIP, basic-region-leucine-zipper; ATRA, all-trans-retinoic acid; SUMO, small ubiquitin-like modifier; CBP, CREB-binding protein; POD, PML-oncogenic domain; SIM, SUMO-interacting motif; RAR, retinoic acid receptor; GFP, green fluorescent protein; HA, hemagglutinin; TSA, C/EBP proteins is the highly conserved C-terminal basic region leucine zipper DNA binding domain, which mediates a large number of protein-protein interactions [1]
In both cell extracts Daxx could be co-precipitated, whereas precipitation with an unrelated antibody failed to do so (Fig. 1B). These results demonstrated that C/EBP and Daxx interact as endogenous proteins
Summary
Expression Constructs—To generate the GFP-Daxx expression construct the coding sequence of human Daxx was amplified from cDNA derived from HeLa cells using specific forward (5Ј-act-tcc-tcc-gtc-gac-ggg-att-gga-tcc-c-3Ј) and reverse primers (5Ј-tcc-ggt-gga-tcg-atg-cag-cta-atc-ag-3Ј) that contain SalII and ClaI restriction sites, respectively. Luciferase and -Galactosidase Reporter Assays—QT6 cells were plated in 10-cm dishes and transfected with 3 g of the p240-Luc reporter construct together with the indicated amounts of expression vectors. The Sepharose beads were washed three times with ELB buffer and used for GST pull-down assays as follows: QT6 cells transfected with the appropriate expression vectors were lysed in ELB buffer and aliquots of the lysate were incubated under constant agitation for 1 h at 4 °C with bacterially expressed GST fusion protein coupled to glutathione-Sepharose. Cells were washed five times with PBST followed by incubation with TRITC-coupled goat-anti mouse secondary antibody (Sigma-Aldrich) diluted 1:50 (detection of endogenous protein) or 1:3000 (detection of overexpressed protein) in blocking buffer for 1 h at room temperature in the dark. Cells were washed five times with PBS, mounted in Aqua Poly/Mount (Polysciences), and analyzed by confocal laser scanning microscopy
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