Daxx Is a Transcriptional Repressor of CCAAT/Enhancer-binding Protein β

Abstract

CCAAT/enhancer-binding Protein ␤ (C/EBP␤) is a member of the bZIP transcription factor family that is expressed in various tissues, including cells of the hematopoietic system. C/EBP␤ 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/EBP␤ is negatively regulated by the transcriptional co-repressor Daxx. C/EBP␤ 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/EBP␤. Coexpression of C/EBP␤ changed the sub-nuclear Daxx distribution pattern from predominantly POD-localized to nucleoplasmic. Daxx suppressed basal and p300-enhanced transcriptional activity of C/EBP␤. Furthermore, Daxx decreased the C/EBP␤-dependent phosphorylation of p300, which in turn was associated with a diminished level of p300-mediated C/EBP␤ acetylation. Co-expression of promyelocytic leukemia protein abrogated the repressive effect of Daxx on C/EBP␤ as well as the direct interaction of Daxx and C/EBP␤, presumably by re-recruiting Daxx to PML-oncogenic domains. In acute promyelocytic leukemia (APL) cells, C/EBP␤ 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/EBP␤ fraction associated with Daxx suggesting a relief of Daxx-dependent C/EBP␤ repression as an important molecular event leading to APL cell differentiation. Overall, our data identify Daxx as a new negative regulator of C/EBP␤ and provide first clues for a link between abrogation of Daxx-C/EBP␤ complex formation and APL remission.

Death Domain-associated Protein DAXX Promotes Ovarian Cancer Development and Chemoresistance

“…Plasmids and RNA Interference-Expression constructs encoding for full-length Daxx (pLEGFP-Daxx and HA-Daxx) were kindly provided by Dr. K. N. Klempnauer (9). Two duplex oligonucleotides encoding for human Daxx-specific short hairpin RNAs (shRNA2, 5Ј-GATCCTCCCCTCCCACACAC-CTCTCCTTGGGGAGAGGTGTGTGGGAGGGTTTTTG-3Ј, and shRNA3, 5Ј-TCGACAAAAAGGAGTTGGATCTCTCA-GAACCAATTCTGAGAGATCCAACTCCGAG-3Ј) were ligated into pSRG (pSUPERretro-IRES-hrGFP) vector plasmids.…”

“…Daxx-overexpressing and Daxx RNAi Ovarian Cancer Cell Lines-Ovarian cancer cells (ES-2, A2780, and SKOV-3) that stably expressed EGFP-DAXX and Daxx shRNA (siDaxx) were established as described previously (9). Briefly, cells were seeded in 60-mm dishes and transfected with 4 g of pLEGFPDaxx or pSuper-shDaxx plasmids using Lipofectamine 2000.…”

“…DAXX can interact with several crucial proteins involved in transcriptional silencing, such as histone deacetylases HDAC1 and HDAC2 and DNA methyltransferase DNMT1. DAXX represses the activity of several transcriptional factors, including C/EBP␤, c-Met, Pax3, Ets1, p53, and its family members p73 and p63, glucocorticoid receptor, androgen receptor, and SMAD4 (7)(8)(9)(10)(11).…”

Tumor Suppressor Protein Pdcd4 Inhibits Translation of p53 mRNA

“…The expression vector encoding GFP-p53 has been described (24). The human Pdcd4 expression vector pcDNA4-hPdcd4 (encoding full-length human Pdcd4) has been described (25).…”

Daxx-β and Daxx-γ, Two Novel Splice Variants of the Transcriptional Co-repressor Daxx

“…An another important role for Daxx is its function as a transcriptional repressor of CCAAT/enhancerbinding protein ␤ (25), which is involved in tissue-specific gene * This work was supported in part by a grant from the Dr. Mildred-ScheelStiftung fü r Krebsforschung. 1 The results of this work are in partial fulfillment of the Ph.D. thesis at the Heinrich Heine University.…”