CCAAT/Enhancer-binding Protein (C/EBP) β Is Acetylated at Multiple Lysines

The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-␤ (C/EBP␤) is governed by protein interactions and post-translational protein modifications. In a proteome-wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine 9-specific 3 (G9a), was found to directly interact with the C/EBP␤ transactivation domain (TAD). Binding between G9a and C/EBP␤ was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBP␤ is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBP␤ TAD served as a substrate for G9a-mediated methylation. G9a, but not a methyltransferase-defective G9a mutant, abrogated the transactivation potential of wild type C/EBP␤. A C/EBP␤ TAD mutant that contained a lysine-toalanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBP␤ target gene. Our data identify C/EBP␤ as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBP␤ during gene regulation.

Section: Discussionmentioning

confidence: 99%

“…The acetyltransferases p300 and p300/CREBbinding protein-associated factor (PCAF) catalyze acetylation of lysine 39, and mutation of lysine 39 has been shown to affect C/EBP␤-dependent reporter activity (26).…”

mentioning

confidence: 99%

G9a-mediated Lysine Methylation Alters the Function of CCAAT/Enhancer-binding Protein-β

Pleß

Kowenz‐Leutz

Knoblich

et al. 2008

“…Acetylation of NF-YA and Bcl6 Regulates RGS4 Promoter ActivityIn view of recent evidence that acetylation of NF-YA (32), C/EBP␤ (44,45), and Bcl6 (46) can modulate their transcriptional activities, we undertook studies to determine whether acetylation of these transcription factors was associated with activation of the RGS4 promoter at cellular confluency. Acetylation was assessed by immunoprecipitation of these proteins followed by anti-acetyl-lysine immunoblotting.…”

Section: Cell Density-dependent Transcriptional Activation Of Rgs4 Ismentioning

confidence: 99%

A Novel Mechanism Involving Coordinated Regulation of Nuclear Levels and Acetylation of NF-YA and Bcl6 Activates RGS4 Transcription

Yang

Huang

Chatterjee

et al. 2010

Neuronally enriched RGS4 plays a critical role attenuating G protein signaling in brain, although the mechanisms regulating RGS4 expression are unknown. Here we describe a novel mechanism for transcriptional activation of RGS4 in neuron-like PC6 cells, where RGS4 is markedly induced during confluence-induced growth arrest. Transcriptional activation of RGS4 in confluent PC6 cells was accompanied by impaired G i/o -dependent MAPK activation. In the human RGS4 gene promoter, we identified three phylogenetically conserved cis-elements: an inverted CCAAT box element (ICE), a cAMP response element, and a B-cell lymphoma 6 (Bcl6)-binding site. The ICE and the cAMP response element mediate activation, and the Bcl6 site mediates repression of RGS4 transcription. Activation of RGS4 transcription in confluent PC6 cells is accompanied by increases in NF-YA and C/EBP␤ and decreases in Bcl6 levels in the nucleus. Increases in NF-YA and C/EBP␤ lead to their increased binding to the RGS4 promoter in vivo, and dominant negative forms of these proteins repressed RGS4 promoter activity. Acetylation of NF-YA and Bcl6 were increased in postconfluent cells. Trichostatin A stimulation of RGS4 promoter activity, accompanied by increased binding of NF-YA and decreased binding of Bcl6 to the promoter, was abolished by mutation of the ICE and enhanced by mutation of the Bcl6 site. These findings demonstrate a dynamic and coordinated regulation of nuclear levels and acetylation status of trans-acting factors critical in determining the off/on state of the RGS4 promoter. Regulator of G protein signaling 4 (RGS4)2 is a member of the mammalian RGS family of proteins of which 30 members exist in humans. RGS proteins were discovered as essential negative regulators of heterotrimeric G protein signaling by genetic studies in yeast and Caenorhabditis elegans (1, 2). These proteins act as GTPase-activating proteins for heterotrimeric G ␣ subunits (3), thereby accelerating the shut-off mechanism for G protein signaling. Some RGS proteins, including RGS4, can also act as effector antagonists (4, 5) or can directly or indirectly interact with G protein-coupled receptors (6 -8), actions that contribute to their negative regulatory effects on G protein signaling in cells.Although G protein signaling is involved in virtually every known physiological process and RGS proteins are an important component of this signaling, the mechanisms regulating expression of RGS genes are largely unknown. Studies have shown that several RGS genes are induced under certain physiological conditions, for example RGS1 during mitogenic activation of lymphocytes (9), RGS2 in the early stage of 3T3-L1 differentiation to adipocytes (10), RGS16 during genotoxic stress (11), and the yeast RGS gene SST2 by pheromone (12). Regulation of RGS4 expression has also been noted in facial motoneuronal precursors during embryonic development in mice (13), in epithelial and endothelial cells during tubulogenesis (14), and in rat PC12 cells during cell confluence (15). In the study by Gril...

“…C/EBP␤ acetylation was shown to be dynamically regulated by HDAC1 leading to deacetylation, and by acetyltransferases such as p300 or P/CAF, which support acetylation (8,9), and to increase the transcriptional activity of C/EBP␤ (8). Because Daxx is known to recruit HDAC proteins (8,9) we suspected that the inhibitory activity of Daxx might be due to a suppression of the acetylation of C/EBP␤. To address this possibility, FLAG-C/EBP␤ and fulllength p300 were co-expressed without or with rising amounts of HA-Daxx.…”

Section: C/ebp␤ Interacts With Daxx Inmentioning

confidence: 99%

“…In the latter context, Duprez et al recently demonstrated that C/EBP␤ activity is required during all-trans-retinoic acid-(ATRA)-induced differentiation of APL cells (6). Transcriptional activity of C/EBP␤ was shown to involve the recruitment of co-regulatory components leading to post-translational modifications of C/EBP␤ such as phosphorylation, sumoylation, and acetylation (7)(8)(9)(10)(11). For instance, p300, CBP and GCN5 directly bind to C/EBP␤ and via their intrinsic acetyltransferase activity trigger C/EBP␤ acetylation leading to an increase in transcriptional C/EBP␤ activity (12)(13)(14).…”

Section: Ccaat/enhancer-binding Protein ␤ (C/ebp␤)mentioning

confidence: 99%

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

Wethkamp

Klempnauer

2009

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.