scholarly journals CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding

2021 ◽  
Vol 22 (23) ◽  
pp. 12689
Author(s):  
Monika J. Stankiewicz ◽  
Jian Du ◽  
Dominick Martinico ◽  
Steven J. Ackerman
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Allergic Diseases ◽  
Specific Gene ◽  
Sumoylation Site ◽  
Protein Protein Interaction ◽  
Enhancer Binding Protein ◽  
N Terminus ◽  
Ccaat Enhancer Binding Protein ◽  
P2 Promoter

CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε32 and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε27) as an antagonist of GATA-1 transactivation of the eosinophil’s major basic protein-1 (MBP1) P2-promoter, showing C/EBPε27 and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε27 fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε27 is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε27 mapping its repressor domains, comparing it to C/EBPε32 and C/EBPε14, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε27 repression of GATA-1 is mediated by its unique 68aa N-terminus combined with previously identified RDI domain. This repressor activity does not require, but is enhanced by, DNA binding via the basic region of C/EBPε27 but independent of sumoylation of the RDI core “VKEEP” sumoylation site. These findings identify the N-terminus of C/EBPε27 as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε27 repression of GATA-1 occurs via a combination of both C/EBPε27-GATA-1 protein–protein interaction and C/EBPε27 binding to a C/EBP site in the MBP1 promoter. The C/EBPε27 isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation, as these genes are ultimately silenced in the mature cell. Understanding the functionality of C/EBPε27 in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.

scholarly journals Inhibition of CCAAT/Enhancer Binding Protein Family DNA Binding in Mouse Epidermis Prevents and Regresses Papillomas

2007 ◽  
Vol 67 (4) ◽  
pp. 1867-1876 ◽  
Author(s):  
Won Jun Oh ◽  
Vikas Rishi ◽  
Andras Orosz ◽  
Michael J. Gerdes ◽  
Charles Vinson
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Protein Family ◽  
Mouse Epidermis ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

scholarly journals Expression of the genes encoding CCAAT-enhancer binding protein isoforms in the mouse mammary gland during lactation and involution

1998 ◽  
Vol 334 (1) ◽  
pp. 205-210 ◽  
Author(s):  
Georgios SABATAKOS ◽  
Gareth E. DAVIES ◽  
Maria GROSSE ◽  
Anthony CRYER ◽  
Dipak P. RAMJI
Keyword(s):  
Transcription Factors ◽  
Mammary Gland ◽  
Dna Binding ◽  
Binding Protein ◽  
Mouse Mammary Gland ◽  
Binding Activity ◽  
Protein Isoforms ◽  
Dna Binding Activity ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the activation of gene expression in the mammary gland during lactation. We have therefore investigated the detailed expression profile of the C/EBP family during lactation and involution of the mouse mammary gland. The expression of C/EBPβ and C/EBPδ mRNA was low during lactation, increased dramatically at the beginning of involution and remained constant thereafter. In contrast, C/EBPα mRNA expression was relatively high during the early stages of lactation, declined to low levels during the late stages of lactation and at the start of involution, and increased again during involution. Electrophoretic mobility-shift assays showed a close correlation between the expression of the C/EBP genes and the functional C/EBP DNA-binding activity and, additionally, demonstrated the participation of heterodimers, formed from among the three proteins, in DNA–protein interactions. The DNA-binding activity of the activator protein 1 (AP1) family of transcription factors was also induced during involution. These results therefore point to potentially important regulatory roles for both the C/EBP and the AP1 family during lactation and involution of the mammary gland.

A novel myeloid-restricted zebrafish CCAAT/enhancer-binding protein with a potent transcriptional activation domain

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2611-2617 ◽  
Author(s):  
Susan E. Lyons ◽  
Bixiong C. Shue ◽  
Andrew C. Oates ◽  
Leonard I. Zon ◽  
P. Paul Liu
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Myeloid Cells ◽  
Mobility Shift ◽  
Nih3t3 Cells ◽  
Amino Terminal ◽  
Enhancer Binding Protein ◽  
Bzip Domain ◽  
Ccaat Enhancer Binding Protein

Abstract The CCAAT/enhancer-binding protein (C/EBP) family consists of transcription factors essential for hematopoiesis. The defining feature of the C/EBPs is a highly conserved carboxy-terminal bZIP domain that is necessary and sufficient for dimerization and DNA binding, whereas their amino-terminal domains are unique. This study reports a novelc/ebp gene (c/ebp1) from zebrafish that encodes a protein homologous to mammalian C/EBPs within the bZIP domain, but with an amino terminus lacking homology to any C/EBP or to any known sequence. In zebrafish embryos, c/ebp1 expression was initially observed in cells within the yolk sac circulation valley at approximately the 16-to 18-somite stage, and at 24 hours postfertilization (hpf), also in circulating cells. Mostc/ebp1+cells also expressed a known early macrophage marker, leukocyte-specific plastin (l-plastin). Expression of both markers was lost in cloche, a mutant affecting hematopoiesis at the level of the hemangioblast. Expression of both markers was retained in m683 andspadetail, mutants affecting erythropoiesis, but not myelopoiesis. Further, c/ebp1 expression was lost in a mutant with defective myelopoiesis, but intact erythropoiesis. These data suggest that c/ebp1 is expressed exclusively in myeloid cells. In electrophoretic mobility shift assays, c/ebp1 was able to bind a C/EBP consensus DNA site. Further, a chimeric protein containing the amino-terminal domain of c/ebp1 fused to the DNA-binding domain of GAL4 induced a GAL4 reporter 4000-fold in NIH3T3 cells. These results suggest that c/ebp1 is a novel member of the C/EBP family that may function as a potent transcriptional activator in myeloid cells.

CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation, and Does Not Require but Is Enhanced by DNA-Binding.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1468-1468 ◽  
Author(s):  
Monika J. Stankiewicz ◽  
Jian Du ◽  
Steven J. Ackerman
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Terminal Differentiation ◽  
Developmentally Regulated ◽  
Protein Protein Interaction ◽  
Enhancer Binding Protein ◽  
N Terminus ◽  
Repressor Activity ◽  
Secondary Granule

Abstract The CCAAT/enhancer binding protein epsilon (C/EBPε) is critical for the terminal differentiation and lineage-specific gene expression of granulocytes, and expression of C/EBPε32 and its shorter 27 kD and 14 kD isoforms is developmentally regulated during neutrophil granulocyte differentiation. We have defined a novel role for the unique 27 kD isoform (C/EBPε27) as a potent antagonist of GATA-1-mediated transactivation of the promoter of the gene encoding the eosinophil secondary granule protein, major basic protein (MBP) (Du et al, J. Biol. Chem.2002; 277:43481–43394). We also showed that these two transcription factors physically interact in eosinophil cell lines in vivo. In the present studies, we performed the first structure-function analyses of the C/EBPε27 isoform to map its potent repressor domains, with comparisons to the C/EBPε32 and C/EBPε14 isoforms, using transactivation assays of the MBP P2 promoter in the presence of GATA-1. Our results show that the repression of GATA-1 is mediated in part by the unique N-terminus of C/EBPε27 (not shared with other C/EBPε isoforms) in combination with part of a previously identified RDI domain (shared with full length C/EBPε32). We show further that this repressor activity is independent of DNA binding (via deletion of the basic region of C/EBPε27) as well as of sumoylation of the RDI “VKEEP” sumoylation consensus site present in both the C/EBPε32 and C/EBPε27 isoforms, and conserved in the C/EBPε proteins of many other species. Thus, our findings identify the unique N-terminus of the C/EBPε27 isoform, a distinct 68 amino acid sequence not shared with any other C/EBPε isoforms or other C/EBP family members, as the minimum repressor domain required for potent antagonism of GATA-1 activity. Of interest, fusion of this novel 68 amino acid sequence to the N-terminus of full length C/EBPε32 converted it into a partial repressor of GATA-1, but did not alter the transactivation potential of the C/EBPε32 isoform itself. The mechanism for maximal C/EBPε27 attenuation of GATA-1 activity requires a combination of both GATA-1-C/EBPε27 protein-protein interaction and C/EBPε27 binding to the proximal C/EBP consensus site immediately upstream in the target promoter. Neither C/EBPε32 nor C/EBPε14 inhibited C/EBPε27 antagonism of GATA-1, supporting a protein-protein interaction mechanism for its repressor activity that is enhanced by, but does not require, DNA binding to a proximal C/EBP site. Expression of the C/EBPε27 isoform likely serves to titrate and/or turn off expression of secondary granule protein genes such as MBP during eosinophil terminal differentiation, when these genes are ultimately silenced in the mature cell. These studies illustrate the unique regulatory (activating versus repressor) activities for the various C/EBPε isoforms, activities consistent with their developmentally regulated expression and lineage-specific activities during granulocyte (both neutrophil and eosinophil) differentiation.

scholarly journals CCAAT-enhancer-binding protein α (C/EBPα) is required for the thyroid hormone but not the retinoic acid induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription

1997 ◽  
Vol 322 (1) ◽  
pp. 343-349 ◽  
Author(s):  
Edwards A. PARK ◽  
Shulan SONG ◽  
Michelle OLIVE ◽  
William J. ROESLER
Keyword(s):  
Retinoic Acid ◽  
Thyroid Hormone ◽  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Binding Protein ◽  
Dominant Negative ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by cAMP, the thyroid hormone tri-iodothyronine (T3) and retinoic acid (RA). Regulation of PEPCK transcription by T3 involves two sites in the promoter including a thyroid-hormone-response element (TRE) and a CCAAT-enhancer-binding protein (C/EBP) binding site called P3(I). Mutation of either the TRE or P3(I) eliminates the T3 response. In this study, we examined the role of C/EBPs in the induction of PEPCK transcription by T3 and RA. PEPCK-CAT vectors were transfected into HepG2 cells. Co-transfection of a dominant negative C/EBP eliminated the T3 stimulation indicating that a member of the C/EBP family is required. To determine which C/EBP isoform was required, Gal4 fusion proteins were created that contained the Gal4 DNA-binding domain ligated to the transcriptional activation domain of C/EBPα, C/EBPβ or the cAMP-responsive-element-binding protein. A Gal4 DNA-binding site was introduced into the P3(I) site of the PEPCK-CAT vector. Only co-transfection of the Gal4-C/EBPα vector was able to restore T3 responsiveness to the PEPCK-CAT vector. The T3 and RA receptors are members of the nuclear receptor superfamily and bind to repeats of the AGGTCA motif. We found that the RA receptor can bind to sequences within the PEPCK-TRE and contribute to RA responsiveness of the PEPCK gene. However, the RA induction of PEPCK transcription was found to be independent of C/EBPs, further demonstrating the specificity of the involvement of C/EBPα in the T3 effect.

scholarly journals Insulin and dexamethasone induce GLUT4 gene expression in foetal brown adipocytes: synergistic effect through CCAAT/enhancer-binding protein alpha

2003 ◽  
Vol 372 (2) ◽  
pp. 617-624 ◽  
Author(s):  
Rosario HERNANDEZ ◽  
Teresa TERUEL ◽  
Margarita LORENZO
Keyword(s):  
Dna Binding ◽  
Synergistic Effect ◽  
Glucose Uptake ◽  
Binding Protein ◽  
Fold Increase ◽  
Binding Activity ◽  
Brown Adipocytes ◽  
Dna Binding Activity ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Treatment of foetal brown adipocytes in primary culture with either dexamethasone or insulin, at physiological concentrations, for 24 h up-regulates the expression of the GLUT4 gene, producing a synergistic effect on mRNA accumulation (20-fold increase), in the amount of protein in the total membrane fraction (8-fold increase) and in the transactivation of a full-promoter GLUT4-chloramphenicol acetyltransferase gene (CAT) construct (7-fold increase). However, GLUT1 expression remains essentially unmodified regardless of the presence of the hormones. As a consequence, exposure of brown adipocytes to dexamethasone and insulin results in a dramatic increase of glucose uptake (12-fold). Dexamethasone induces the expression of CCAAT/enhancer-binding protein (C/EBP) α, insulin promotes myocyte enhancer factor-2 DNA-binding activity and both combined produces a significant increase in C/EBPα DNA-binding activity. Moreover, co-transfection with a wild-type C/EBPα construct transactivates a full-promoter GLUT4-CAT fusion gene, whereas a dominant-negative C/EBPα expression vector impairs the hormonal effects. Our results show that the synergism between insulin and glucocorticoids on glucose uptake is a consequence of the activation of the GLUT4 promoter by the transcription factor C/EBPα.

scholarly journals Role of CCAAT/Enhancer-Binding Protein Alpha (C/EBPα) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP

2003 ◽  
Vol 77 (1) ◽  
pp. 600-623 ◽  
Author(s):  
Shizhen Emily Wang ◽  
Frederick Y. Wu ◽  
Masahiro Fujimuro ◽  
Jianchao Zong ◽  
S. Diane Hayward ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Kaposi's Sarcoma ◽  
Binding Protein ◽  
Lytic Cycle ◽  
Transcription Activator ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT/enhancer binding protein alpha (C/EBPα) and p21CIP-1 expression, resulting in G0/G1 cell cycle arrest during the lytic cycle. RAP and C/EBPα are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C/EBPα; this analysis showed that one of these sites has a very high affinity for C/EBPα. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPα can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C/EBPα together resulted in a threefold increase in activity over that obtained with C/EBPα alone. Importantly, the introduction of exogenous Flag-tagged C/EBPα triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C/EBPα and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C/EBPα and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPα, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPα or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPα-mediated transactivation of the RAP promoter. However, the C/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C/EBPα and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C/EBPα in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C/EBPα was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C/EBPα and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPα occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C/EBPα and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPα. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPα but that full activity requires more than just the core C/EBP binding site.

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