Protein partners of C/EBPε

Chih, Doris Y.; Park, Dorothy J.; Gross, Mitchell E.; Idos, Gregory; Vuong, Peter T.; Hirama, Toshiyasu; Chumakov, Alexey M.; Said, Jonathan; Koeffler, H. Phillip

doi:10.1016/j.exphem.2004.08.014

Cited by 18 publications

(18 citation statements)

References 51 publications

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“…15,16 Furthermore, we find that C/EBP-⑀-DNA interaction is highly up-regulated by interaction with the activated NFkappaB pathway protein p65RelA. This interaction depends on the T75 phosphorylation status of C/EBP-⑀ and can be enhanced by introduction of phosphomimetic Thr to Asp mutation.…”

Section: Introductionmentioning

confidence: 78%

“…15,19,24 Therefore, we attempted to identify DNA sites that are preferentially bound by C/EBP-⑀ homodimers and C/EBP-⑀-ATF4 heterodimers.…”

Section: Selection Of C/ebp-⑀ Binding Sitesmentioning

confidence: 99%

“…For luciferase or ␤-galactosidase reporter assays, cells were transfected with 3 g of either pMim-luciferase or C/EBP-LacZ reporter and 0.3 g of the C/EBP expression vectors, wild type or mutants, as previously described. 14,15 The internal control for transfection efficiency was 1 g pCMV-␤-galactosidase or 0.1 g pCMVrenilla-luciferase. At 12 hours after transfection, the Jurkat cells were activated with TPA (12-Otetradecanoyl phorbol 13-acetate; 2 ng/mL) and the calcium ionophore A23187 (250 ng/mL) for 24 hours.…”

Section: Transient Transfections and Reporter Assaysmentioning

confidence: 99%

“…on May 10, 2018. by guest www.bloodjournal.org From family of transcription factors. 10,15,16 To define the consensus DNA binding site for C/EBP-⑀-ATF4 heterodimers, we used CASTing with nuclear lysates derived from COS1 cells cotransfected with ATF4 and C/EBP-⑀ expression plasmids. Target sites were identified by immunoselection of C/EBP-⑀-bound sites for 5 cycles of CASTing and then selection with anti-ATF4 antibodies at cycle 6, anti-C/EBP-⑀ antibodies at cycle 7, and, again, anti-ATF4 antibodies at cycle 8, thus enriching for DNA bound to heterodimeric ATF4-C/EBP-⑀ complexes.…”

Section: Definition Of Dna Sites Bound By Heterodimeric C/ebp-⑀-atf4mentioning

confidence: 99%

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Modulation of DNA binding properties of CCAAT/enhancer binding protein epsilon by heterodimer formation and interactions with NFkappaB pathway

Chumakov

Silla

Williamson

et al. 2007

Blood

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IntroductionSpecific regulation of gene expression is achieved through interaction of multiple transcriptional activators and repressors with their cognate DNA sites. This activity is subject to several levels of control, including posttranslational modification by phosphorylation, acetylation, selective degradation, and interaction with coactivators and corepressors.Previous studies of CCAAT/enhancer binding protein family of transcriptional activators had identified domains responsible for dimerization, specific DNA recognition, transcriptional activation, and intramolecular repression. [1][2][3][4][5][6] Several members of the C/EBP protein family (C/EBP-␣, C/EBP-␤, C/EBP-␦, C/EBP-⑀) contain a transactivation domain and a basic "leucine zipper" domain that serve for DNA recognition and dimer formation. Others like C/EBP-␥ and C/EBP-(CHOP, GADD153) are involved in transcriptional regulation through dimerization with other basic leucine zipper proteins from the C/EBP, ATF/CREB, and Fos/Jun families.Recent studies directed at identification of C/EBP-responsive genes resulted in identification of a diverse and degenerate collection of putative C/EBP binding sites. At the same time, in vitro experiments with site selection typically produce very common short consensus sequences. Identification of binding sites for each individual member of the C/EBP family is complicated by common coexpression of several of these proteins in the same cell and their ability to form heterodimeric complexes with each other and with structurally related ATF/CREB proteins. [7][8][9][10][11][12] Activity of C/EBP proteins is regulated by phosphorylation mediated through several major kinase pathways that affect DNA binding, subcellular localization in the nucleus, and interaction with cell-cycle proteins. Our recent studies of C/EBP-⑀ phosphorylation 13,14 identified several sites, including 1 for p38 MAPK within the transactivation domain of C/EBP-⑀. As a result of phosphorylation at threonine 75 (T75), C/EBP-⑀ DNA binding and specific transcriptional activity was significantly enhanced.In this study, we identified consensus binding sequences for C/EBP-⑀ and for heterodimers of C/EBP-⑀ with its most common dimerization partner ATF4. 15,16 Furthermore, we find that C/EBP-⑀-DNA interaction is highly up-regulated by interaction with the activated NFkappaB pathway protein p65RelA. This interaction depends on the T75 phosphorylation status of C/EBP-⑀ and can be enhanced by introduction of phosphomimetic Thr to Asp mutation. By using selective removal of p65RelA-and siRNA-mediated p65 knock-down, we demonstrate that the activated NFkappaB pathway is required for high-level expression of the C/EBP-⑀-responsive promoter. This novel interaction may play an important role in regulation of C/EBP-⑀-dependent genes in myeloid cells. Materials and methodsCMV-C/EBP-⑀, ATF4, and siRNA expression constructs C/EBP-⑀, C/EBP-⑀T75A, and C/EBP-⑀T75D expression and pMimluciferase reporter plasmids were described previously. 6,13,14 ATF4 (CREB2) expression pl...

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Section: Introductionmentioning

confidence: 78%

“…15,19,24 Therefore, we attempted to identify DNA sites that are preferentially bound by C/EBP-⑀ homodimers and C/EBP-⑀-ATF4 heterodimers.…”

Section: Selection Of C/ebp-⑀ Binding Sitesmentioning

confidence: 99%

Section: Transient Transfections and Reporter Assaysmentioning

confidence: 99%

Section: Definition Of Dna Sites Bound By Heterodimeric C/ebp-⑀-atf4mentioning

confidence: 99%

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Modulation of DNA binding properties of CCAAT/enhancer binding protein epsilon by heterodimer formation and interactions with NFkappaB pathway

Chumakov

Silla

Williamson

et al. 2007

Blood

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“…Also, C/EBPs are least conserved in their activation domains and vary from strong activators to dominant negative repressors [17]. Six members of C/EBP family of TFs are known, designated C/EBPα, -β, γ, -δ, -ε, and -ξ [18,21]. All C/EBPs interact with each other and with other TFs to regulate mRNA transcription [18,22].…”

Section: Introductionmentioning

confidence: 99%

Trapping of transcription factors with symmetrical DNA using thiol-disulfide exchange chemistry

Panda

Jiang

Jarrett

2008

Journal of Chromatography A

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Green fluorescent protein (GFP)fused to the C-terminal 100 amino acids of CAAT enhancer binding protein (C/EBP) also containing an N-terminal (His) 6 tag (GFP-C/EBP) was used as a transcription factor model to test whether thiol-disulfide exchange reactions could be used to successfully purify transcription factors. A symmetrical dithiol oligonucleotide with dual CAAT elements was constructed with 5′ and 3′ thiols. Upon reduction, circular dichroism confirms it spontaneously anneals with its internally complementary sequence to form the hairpin structure:The specific GFP-C/EBP protein -DNA complex, formed in solution at nM concentrations, could then be recovered (trapped) via thiol-disulfide exchange with a disulfide thiopropyl-Sepharose and eluted with dithiothreitol. GFP-C/EBP was isolated from crude bacterial extract treated with iodoacetamide; DNA binding by GFP-C/EBP was unaltered by carboxyamidomethylation. Eluted GFP-C/EBP was of high purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein, after in-gel digestion with trypsin, was also characterized by capillary reversedphase liquid chromatography-nano electrospray ionization-tandem mass spectrometry and the results analyzed using MASCOT software searching of the non-redundant protein database. A score of 1874 with a sequence coverage of 51% encompassing both termini and internal sequences for the match with GFP-C/EBP confirms its identity and sequence. The method has high potential for the identification and characterization of transcription factors and other DNA-binding proteins. KeywordsTranscription Factor; Affinity Chromatography; C/EBP; GFP-C/EBP; Symmetrical DNA INTRODUCTIONDNA-binding proteins are responsible for replicating the genome, transcribing active genes, and repairing damaged DNA [1,2]. The transcription factors (TFs), one of the largest and most diverse classes of DNA-binding proteins, regulate cell development, differentiation, and growth. TFs contain DNA-binding domains belonging to several super-families, with different *Author to whom correspondence should be addressed. Telephone: (210) 458-7053, email: harry.jarrett@utsa.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The number of TFs in humans is estimated to be over 1400 [13] and the extremely small concentrations of these proteins inside the cells makes their isolation from cellular extracts for detailed studies a major challenge. Reveiws of TFs and the methods available for purifying and studying TFs are available [14,15]. We have used the CAAT enhancer binding protein (C/ EBP) as a model ...

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CCAAT/enhancer binding protein‐α is down‐regulated by toll‐like receptor agonists in microglial cells

Ejarque-Ortíz

Tusell

Serratosa

et al. 2007

J of Neuroscience Research

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The transcription factor CCAAT/enhancer binding protein‐α (C/EBPα) can regulate the expression of important genes in the inflammatory response, but little is known about its role in glial activation. By using primary cortical murine glial cultures, we show that C/EBPα is expressed by microglial cells in vitro. Lipopolysaccharide (LPS) down‐regulates C/EBPα mRNA at 2 hr and all C/EBPα protein isoforms at 4 hr. This effect is elicited by LPS concentrations ≥100 pg/ml. LPS‐induced C/EBPα down‐regulation occurs in microglial cells both in mixed glial and in microglial‐enriched cultures. As seen with LPS, other toll‐like receptor agonists (polyinosinic‐polycytidylic acid, peptidoglycan from Staphylococcus aureus, and the oligonucleotide CpG1668) also down‐regulate C/EBPα whereas cytokines such as interleukin‐1β, interleukin‐6, macrophage‐colony stimulating factor, and interferon‐γ do not. These findings suggest that C/EBPα down‐regulation in activated microglia could play an important role in the increased expression of genes that are potentially pathogenic in a variety of neurological disorders. © 2007 Wiley‐Liss, Inc.

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Protein partners of C/EBPε

Cited by 18 publications

References 51 publications

Modulation of DNA binding properties of CCAAT/enhancer binding protein epsilon by heterodimer formation and interactions with NFkappaB pathway

Modulation of DNA binding properties of CCAAT/enhancer binding protein epsilon by heterodimer formation and interactions with NFkappaB pathway

Trapping of transcription factors with symmetrical DNA using thiol-disulfide exchange chemistry

CCAAT/enhancer binding protein‐α is down‐regulated by toll‐like receptor agonists in microglial cells

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