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.

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 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 CCAAT enhancer binding protein β and hepatocyte nuclear factor 3β are necessary and sufficient to mediate dexamethasone-induced up-regulation of alpha2HS-glycoprotein/fetuin-A gene expression

2006 ◽  
Vol 36 (2) ◽  
pp. 261-277 ◽  
Author(s):  
Michael Wöltje ◽  
Beate Tschöke ◽  
Verena von Bülow ◽  
Ralf Westenfeld ◽  
Bernd Denecke ◽  
...  
Keyword(s):  
Serum Protein ◽  
Binding Protein ◽  
Protein A ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Tissue Remodelling ◽  
Mobility Shift ◽  
Fetuin A ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Alpha2HS-glycoprotein/fetuin-A (Ahsg) is a serum protein preventing soft tissue calcification. In trauma and inflammation, Ahsg is down-regulated and therefore considered a negative acute phase protein. Enhancement of Ahsg expression as a protective serum protein is desirable in several diseases including tissue remodelling after trauma and infection, kidney and heart failure, and cancer. Using reporter gene assays in hepatoma cells combined with electrophoretic mobility shift assays we determined that dexamethasone up-regulates hepatic Ahsg. A steroid response unit at position −146/−119 within the mouse Ahsg promoter mediates the glucocorticoid-induced increase of Ahsg mRNA. It binds the hepatocyte nuclear factor 3β and CCAAT enhancer binding protein β (C/EBP-β). The up-regulation is mediated indirectly via glucocorticoid hormone-induced transcriptional up-regulation in C/EBP-β protein. A high degree of sequence identity in mouse, rat and human Ahsg promoters suggests that the promoter is similarly up-regulated by dexamethasone in all three species. Therefore, our findings suggest that glucocorticoids may be used to enhance the level of Ahsg protein circulating in serum.

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.

Post-Translational Modification and Differential Protein Binding Partners of CCAAT Enhancer Binding Protein Alpha (C/EBPα) p42 Versus p30 May Contribute to Aberrant C/EBPα Activity in Acute Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3577-3577
Author(s):  
Matthew Silver ◽  
Nirmalee Abayasekara ◽  
Dylan Perry ◽  
Hong Sun ◽  
Nancy Berliner ◽  
...  
Keyword(s):  
Protein Binding ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Myeloid Cells ◽  
Leukemic Cells ◽  
Myeloid Differentiation ◽  
Differential Protein ◽  
Binding Partners ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid- specific gene promoters at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for including a selective early block in the differentiation of C/EBPα, granulocytes. Mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting with a normal karyotype. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. We have sought to understand the molecular basis for this observation. We and others have demonstrated that C/EBPα is post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) that lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. We have demonstrated that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation and that transactivation of the myeloid-specific lactoferrin (LF) promoter reporter is significantly enhanced by a p42 sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, we show that sumoylated p42C/EBPα binds to the C/EBP site in the LF promoter in immature myeloid cells (which do not express LF) while loss binding and LF of sumoylation correlates with loss of p42C/EBPα expression in more mature cells. Based on these observations we is associated with the negative conclude that sumoylated p42C/EBPα regulation of LF in early myeloid cells. We further demonstrate that sumoylated p42C/EBPα remains bound to the LF promoter following ATRA induction of the leukemic NB4 cells, which do not express LF despite induction of morphologic maturation. Based on these observations we conclude that during normal myeloid differentiation, sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid cells, and that LF expression upon maturation is associated with loss of binding of sumoylated p42 C/EBPα In leukemic cells induced toward mature neutrophils, sumoylated p42C/EBPα remains bound to the LF promoter, contributing to the lack of expression of LF in these cells. We show in addition, that p30 C/EBPα can also be sumoylated. In transactivation assays, however, sumoylated p42C/EBPα suppresses LF promoter activity more efficiently than p30C/EBPα in 293 cells. In order to identify differential protein binding partners of p30 and p42C/EBPα that could account for the differential transcriptional activity of the two isoforms, we have used a one step purification method that allows isolation of biotinylated C/EBPα p30 and p42- containing complexes using magnetic-streptavidin beads. The K562 myelomonocytic cell line stably expressing a biotin ligase (BirA) plasmid was transfected with p30C/EBPα or p42C/EBPα each containing a 23 amino acid tag at the N-terminus that allows for in vivo biotinylation. Proteins complexed with the two C/EBP isofoms have been isolated and are currently being identified by LC- MS MS analysis. Their differential association with the two isofoms of C/EBPα will be confimed by coimmunoprecipitation assays in normal myeloid and in leukemic cells. The identification of differentially bound proteins to p30 and p42 C/EBPα may identify molecular targets for future drug development.

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