TGF-β–induced Phosphorylation of Smad3 Regulates Its Interaction with Coactivator p300/CREB-binding Protein

Smads are intermediate effector proteins that transduce the TGF-β signal from the plasma membrane to the nucleus, where they participate in transactivation of downstream target genes. We have shown previously that coactivators p300/CREB-binding protein are involved in TGF-β–mediated transactivation of two Cdk inhibitor genes, p21 and p15. Here we examined the possibility that Smads function to regulate transcription by directly interacting with p300/CREB-binding protein. We show that Smad3 can interact with a C-terminal fragment of p300 in a temporal and phosphorylation-dependent manner. TGF-β–mediated phosphorylation of Smad3 potentiates the association between Smad3 and p300, likely because of an induced conformational change that removes the autoinhibitory interaction between the N- and C-terminal domains of Smad3. Consistent with a role for p300 in the transcription regulation of multiple genes, overexpression of a Smad3 C-terminal fragment causes a general squelching effect on multiple TGF-β–responsive reporter constructs. The adenoviral oncoprotein E1A can partially block Smad-dependent transcriptional activation by directly competing for binding to p300. Taken together, these findings define a new role for phosphorylation of Smad3: in addition to facilitating complex formation with Smad4 and promoting nuclear translocation, the phosphorylation-induced conformational change of Smad3 modulates its interaction with coactivators, leading to transcriptional regulation.

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Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1616-1625.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1616-1625 ◽

Cited By ~ 21

Author(s):

Yang Chen ◽

R. H. Goodman ◽

Sarah M. Smolik

Keyword(s):

Transcriptional Activation ◽

Target Genes ◽

Binding Protein ◽

Point Mutations ◽

Creb Binding Protein ◽

Wild Type ◽

Interaction Domain ◽

Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

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Histone Binding Protein RbAp48 Interacts with a Complex of CREB Binding Protein and Phosphorylated CREB

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.4970-4978.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 4970-4978 ◽

Cited By ~ 46

Author(s):

Qinghong Zhang ◽

Ngan Vo ◽

Richard H. Goodman

Keyword(s):

Transcriptional Activation ◽

Binding Protein ◽

In Vitro Transcription ◽

Dependent Manner ◽

Creb Binding Protein ◽

Histone Binding ◽

Nuclear Extracts ◽

Core Histones ◽

Phosphorylated Creb

ABSTRACT A CREB-CREB binding protein (CBP) complex was used as bait to screen a mouse embryo cDNA library in yeast. One of the strongest interactions identified the histone binding protein RbAp48. RbAp48 also interacted weakly with CBP alone but did not interact with phosphorylated or nonphosphorylated CREB. CBP (or its homologue p300) from HeLa cell nuclear extracts coimmunoprecipitated with RbAp48 and its homologue RbAp46 and bound to a glutathioneS-transferase–RbAp48 fusion protein. This interaction was stimulated by the addition of phosphorylated CREB and allowed the association of core histones and mononucleosomes in an acetylation-dependent manner. RbAp48 lowered theKm of CBP histone acetylase activity and facilitated p300-mediated in vitro transcription of a chromatinized template in the presence of acetylcoenzyme A. These data indicate that the association of phosphorylated CREB with CBP promotes the binding of RbAp48 and its homologue RbAp46, allowing the formation of a complex that facilitates histone acetylation during transcriptional activation.

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The transcriptional activity of Pygopus is enhanced by its interaction with cAMP-response-element-binding protein (CREB)-binding protein

Biochemical Journal ◽

10.1042/bj20090134 ◽

2009 ◽

Vol 422 (3) ◽

pp. 493-501 ◽

Cited By ~ 24

Author(s):

Phillip G. P. Andrews ◽

Zhijian He ◽

Cathy Popadiuk ◽

Kenneth R. Kao

Keyword(s):

Histone Acetylation ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Target Genes ◽

Histone Acetyltransferase ◽

Binding Protein ◽

Camp Response Element Binding ◽

Creb Binding Protein ◽

Hek 293 ◽

Element Binding Protein

Pygopus is a core component of the β-catenin/TCF (T-cell factor) transcriptional activation complex required for the expression of canonical Wnt target genes. Recent evidence suggests that Pygopus could interpret histone methylation associated with target genes and it was shown to be required for histone acetylation. The involvement of a specific acetyltransferase, however, was not determined. In this report, we demonstrate that Pygopus can interact with the HAT (histone acetyltransferase) CBP [CREB (cAMP-responsive-element-binding protein)-binding protein]. The interaction is via the NHD (N-terminal homology domain) of Pygopus, which binds to two regions in the vicinity of the HAT domain of CBP. Transfected and endogenous hPygo2 (human Pygopus2) and CBP proteins co-immunoprecipitate in HEK-293 (human embryonic kidney 293) cells and both proteins co-localize in SW480 colorectal cancer cells. The interaction with CBP also enhances both DNA-tethered and TCF/LEF1 (lymphoid enhancing factor 1)-dependent transcriptional activity of Pygopus. Furthermore, immunoprecipitated Pygopus protein complexes displayed CBP-dependent histone acetyltransferase activity. Our data support a model in which the NHD region of Pygopus is required to augment TCF/β-catenin-mediated transcriptional activation by a mechanism that includes both transcriptional activation and histone acetylation resulting from the recruitment of the CBP histone acetyltransferase.

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CITED4 mediates proliferation, apoptosis and steroidogenesis of Hu sheep granulosa cells in vitro

Reproduction ◽

10.1530/rep-20-0427 ◽

2020 ◽

Author(s):

Xiaolei Yao ◽

EI-Samahy M.a. ◽

Shenhua Xiao ◽

Zhibo Wang ◽

Fanxing Meng ◽

...

Keyword(s):

Cell Cycle ◽

Mrna Expression ◽

Granulosa Cells ◽

Target Genes ◽

Binding Protein ◽

Follicular Development ◽

Creb Binding Protein ◽

Downstream Target ◽

Hu Sheep

Being a novel target of luteinizing hormone (LH), the effect of CREB-binding protein/P300-interacting trans-activator with ED-rich tail member 4 (CITED4) gene on the proliferation, apoptosis, and steroidogenesis of ovarian granulosa cells (GCs) in Hu sheep was investigated. The presence of CITED4, CREB-binding protein (CBP), CCAAT/enhancer binding protein alpha (C/EBPα) and -beta (C/EBPβ) proteins was demonstrated in GCs and luteal cells. CITED4 protein in GCs was induced by LH, and CITED4 overexpression moderately increased GC responses to LH. In contrast, CITED4 knockdown in GCs decreased prostaglandin (PGE2)-induced LH target genes levels. Moreover, PGE2-stimulated CITED4 mRNA expression was blocked by ERK1/2 inhibition (U0126), suggesting that CITED4 is a downstream target of the ERK1/2 pathway in sheep GCs. In contrast to CITED4 knockdown, CITED4 overexpression promoted GC proliferation, inhibited apoptosis, upregulated cell cycle-related genes, and downregulated apoptosis-related genes. Additionally, CITED4 overexpression induced cell cycle transition from S to G2/M phase. No effect was observed with CITED4 knockdown. CITED4 overexpression increased progesterone (P4) production levels and StAR mRNA expression, whereas CITED4 knockdown decreased P4 production and StAR and 3β-HSD mRNA expression levels. Thus, our results suggest that CITED4 is involved in regulating the expression of LH-induced genes and the ERK1/2 pathway and the proliferation, apoptosis, and steroidogenesis in Hu sheep GCs by modulating the expression of related genes. These findings will help understand the role of CITED4 in follicular development and ovulation of pre-ovulatory follicles.

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PPARδ-mediated p21/p27 induction via increased CREB-binding protein nuclear translocation in beraprost-induced antiproliferation of murine aortic smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00069.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. C321-C329 ◽

Cited By ~ 22

Author(s):

Yuh-Mou Sue ◽

Chih-Peng Chung ◽

Heng Lin ◽

Ying Chou ◽

Chih-Yu Jen ◽

...

Keyword(s):

Smooth Muscle ◽

Transcriptional Activation ◽

Nuclear Translocation ◽

Binding Protein ◽

Peroxisome Proliferator ◽

Creb Binding Protein ◽

Promoter Regions ◽

Peroxisome Proliferator Activated Receptor ◽

Aortic Smooth Muscle ◽

Responsive Element

We previously showed that an increase in the peroxisome proliferator-activated receptor-δ (PPARδ), together with subsequent induction of inducible nitric oxide synthase (iNOS) by beraprost (BPS), inhibits aortic smooth muscle cell proliferation. Herein, we delineated the mechanisms of the antiproliferative effects of BPS through the induction of p21/p27. BPS concentration dependently induced the p21/p27 promoter- and consensus cAMP-responsive element (CRE)-driven luciferase activities, which were significantly suppressed by blocking PPARδ activation. Surprisingly, other than altering the CRE-binding protein (CREB), BPS-mediated PPARδ activation increased nuclear localization of the CREB-binding protein (CBP), a coactivator, which was further confirmed by chromatin immunoprecipitation. Furthermore, novel functional PPAR-responsive elements (PPREs) next to CREs in the rat p21/p27 promoter regions were identified, where PPARδ interacted with CREB through CBP recruitment. BPS-mediated suppression of restenosis in mice with angioplasty was associated with p21/p27 induction. Herein, we demonstrate for the first time that BPS-mediated PPARδ activation enhances transcriptional activation of p21/p27 by increasing CBP nuclear translocation, which contributes to the vasoprotective action of BPS.

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PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3

Veterinary Research ◽

10.1186/s13567-020-00843-4 ◽

2020 ◽

Vol 51 (1) ◽

Author(s):

Zongyi Bo ◽

Yurun Miao ◽

Rui Xi ◽

Qiuping Zhong ◽

Chenyi Bao ◽

...

Keyword(s):

Transcriptional Activation ◽

Pseudorabies Virus ◽

Nuclear Translocation ◽

Economic Loss ◽

Inhibitory Effect ◽

Poly I:C ◽

Interferon Response ◽

Type I ◽

Creb Binding Protein ◽

Swine Industry

Abstract Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky’s disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-β production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-β secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS–STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS–STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-β gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS–STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-β gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3.

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The search of CAR, AhR, ESRs binding sites in promoters of intronic and intergenic microRNAs

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720017500299 ◽

2018 ◽

Vol 16 (01) ◽

pp. 1750029 ◽

Cited By ~ 6

Author(s):

Vladimir Y. Ovchinnikov ◽

Denis V. Antonets ◽

Lyudmila F. Gulyaeva

Keyword(s):

Transcriptional Activation ◽

Binding Sites ◽

In Silico ◽

Target Genes ◽

Nuclear Translocation ◽

Regulation Of Gene Expression ◽

Experimental Studies ◽

Transcriptional Level ◽

Aryl Hydrocarbon ◽

Exogenous Compounds

MicroRNAs (miRNAs) play important roles in the regulation of gene expression at the post-transcriptional level. Many exogenous compounds or xenobiotics may affect microRNA expression. It is a well-established fact that xenobiotics with planar structure like TCDD, benzo(a)pyrene (BP) can bind aryl hydrocarbon receptor (AhR) followed by its nuclear translocation and transcriptional activation of target genes. Another chemically diverse group of xenobiotics including phenobarbital, DDT, can activate the nuclear receptor CAR and in some cases estrogen receptors ESR1 and ESR2. We hypothesized that such chemicals can affect miRNA expression through the activation of AHR, CAR, and ESRs. To prove this statement, we used in silico methods to find DRE, PBEM, ERE potential binding sites for these receptors, respectively. We have predicted AhR, CAR, and ESRs binding sites in 224 rat, 201 mouse, and 232 human promoters of miRNA-coding genes. In addition, we have identified a number of miRNAs with predicted AhR, CAR, and ESRs binding sites that are known as oncogenes and as tumor suppressors. Our results, obtained in silico, open a new strategy for ongoing experimental studies and will contribute to further investigation of epigenetic mechanisms of carcinogenesis.

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Functional Domains of the TOL Plasmid Transcription Factor XylS

Journal of Bacteriology ◽

10.1128/jb.182.4.1118-1126.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 1118-1126 ◽

Cited By ~ 33

Author(s):

Niilo Kaldalu ◽

Urve Toots ◽

Victor de Lorenzo ◽

Mart Ustav

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Dependent Manner ◽

Tol Plasmid ◽

Terminal Fragment ◽

Central Regions ◽

Basic Features ◽

Regulatory Functions

ABSTRACT The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

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A Novel Transcriptional Repression Domain Mediates p21WAF1/CIP1 Induction of p300 Transactivation

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2676-2686.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2676-2686 ◽

Cited By ~ 88

Author(s):

Andrew W. Snowden ◽

Lisa A. Anderson ◽

Gill A. Webster ◽

Neil D. Perkins

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

Transcriptional Repression ◽

Kinase Inhibitor ◽

Core Promoter ◽

Dependent Manner ◽

Creb Binding Protein ◽

Cycle Dependent ◽

Repression Domain

ABSTRACT The transcriptional coactivators p300 and CREB binding protein (CBP) are important regulators of the cell cycle, differentiation, and tumorigenesis. Both p300 and CBP are targeted by viral oncoproteins, are mutated in certain forms of cancer, are phosphorylated in a cell cycle-dependent manner, interact with transcription factors such as p53 and E2F, and can be found complexed with cyclinE-Cdk2 in vivo. Moreover, p300-deficient cells show defects in proliferation. Here we demonstrate that transcriptional activation by both p300 and CBP is stimulated by coexpression of the cyclin-dependent kinase inhibitor p21WAF/CIP1. Significantly this stimulation is independent of both the inherent histone acetyltransferase (HAT) activity of p300 and CBP and of the previously reported carboxyl-terminal binding site for cyclinE-Cdk2. Rather, we describe a previously uncharacterized transcriptional repression domain (CRD1) within p300. p300 transactivation is stimulated through derepression of CRD1 by p21. Significantly p21 regulation of CRD1 is dependent on the nature of the core promoter. We suggest that CRD1 provides a novel mechanism through which p300 and CBP can switch activities between the promoters of genes that stimulate growth and those that enhance cell cycle arrest.

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Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

Journal of Experimental Medicine ◽

10.1084/jem.20051450 ◽

2005 ◽

Vol 203 (1) ◽

pp. 63-72 ◽

Cited By ~ 128

Author(s):

Chang Hoon Lee ◽

Mark Melchers ◽

Hongsheng Wang ◽

Ted A. Torrey ◽

Rebecca Slota ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Transcriptional Activation ◽

Germinal Center ◽

Target Genes ◽

Fluorescent Protein ◽

Binding Protein ◽

Consensus Sequence ◽

B Cell Lymphoma ◽

Regulatory Factor

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

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