Differential Transactivation Activities of Sumoylated CCAAT Enhancer Binding Protein alpha (C/EBPα) p42 Versus p30 May Contribute in Part, to Aberrant C/EBPα Activity in Acute Leukemias.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1231-1231
Author(s):  
Arati Khanna-Gupta ◽  
Matthew Silver ◽  
William Hankey ◽  
Hong Sun ◽  
Nancy Berliner
Keyword(s):  
Leucine Zipper ◽  
Binding Protein ◽  
Myeloid Cells ◽  
Leukemic Cell ◽  
Dominant Negative ◽  
Acute Leukemias ◽  
Leukemic Cell Lines ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Immature Myeloid Cells

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 genes at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for C/EBPα, including a selective early block in the differentiation of granulocytes. Recently mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting normal karyotypes. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. Since the molecular basis for this observation remains unknown, a complete understanding of the regulation of this key transcription factor during myelopoiesis is critical. C/EBPα was recently shown to be post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) which lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. Sumoylation at K159 in the C/EBPα protein is thought to prevent association of the SWI/SNF chromatin remodeling complex with C/EBPα, thereby hampering transactivation. In order to demonstrate the role of sumoylation in mediating C/EBPα activity, we examined the effect of both the full length (p42) and the dominant negative (p30) isoforms of the C/EBPα protein on myeloid gene expression. We demonstrate that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation, and that transactivation of the myeloid-specific lactoferrin 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 while loss of sumoylation correlates with loss of p42C/EBPα binding and LF expression in more mature cells. Based on these observations we conclude that sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid 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. We are now in the process of confirming this finding in leukemic cell lines. This observation leads us to hypothesize that the differential transactivation potential of sumoylated p30 versus the p42 C/EBPα proteins may contribute, in part, to the aberrant activity of C/EBPα in acute leukemias.

Mutations in the gene encoding the transcription factor CCAAT/enhancer binding protein α in myelodysplastic syndromes and acute myeloid leukemias

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1332-1340 ◽  
Author(s):  
Adrian F. Gombart ◽  
Wolf-K. Hofmann ◽  
Seiji Kawano ◽  
Seisho Takeuchi ◽  
Utz Krug ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Binding Protein ◽  
Nonsmall Cell Lung Cancer ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Nonsense Codons ◽  
Basic Region

The CCAAT/enhancer binding protein α (C/EBPα) protein is essential for proper lung and liver function and granulocytic and adipose tissue differentation. It was hypothesized that abnormalties in C/EBPα function contribute to the development of malignancies in a variety of tissues. To test this, genomic DNA from 408 patient samples and 5 cell lines representing 11 different cancers was screened for mutations in the C/EBPα gene. Two silent polymorphisms termed P1 and P2 were present at frequencies of 13.5% and 2.2%, respectively. Of the12 mutations detected in 10 patients, silent changes were identified in one nonsmall cell lung cancer, one prostate cancer, and one acute myelogenous leukemia (AML) subtype M4. The 9 remaining mutations were detected in 1 of 92 (1.1%) myelodysplastic syndrome (MDS) samples and 6 of 78 (7.7%) AML (AML-M2 and AML-M4) samples. Some mutations truncated the predicted protein with loss of the DNA-binding (basic region) and dimerization (leucine zipper [ZIP]) domains by either deletions or nonsense codons. Also, inframe deletions or insertions in the fork region located between the leucine zipper and basic region, or within the leucine zipper, disrupted the α-helical phase of the bZIP domain. The inframe deletion and insertion mutations abrogated the transcriptional activation function of C/EBPα on the granulocyte colony-stimulating factor receptor promoter. These mutants localized properly to the nucleus, but were unable to bind to the C/EBP site in the promoter and did not possess dominant-negative activity. The mutations in the MDS patient and one AML-M2 patient were biallelic, indicating a loss of C/EBPα function. These results suggest that mutation of C/EBPα is involved in specific subtypes of AML and in MDS, but may occur rarely in other types of leukemias or nonhematologic malignancies.

DDRE-30. CELL-PENETRATING PEPTIDE ANTAGONIST OF CCAAT/ENHANCER BINDING PROTEIN ß DISPLAYS POTENT ANTI-GLIOBLASTOMA ACTIVITY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi80-vi81
Author(s):  
Jim Rotolo ◽  
Lila Ghamsari ◽  
Ricardo Ramierez ◽  
Mark Koester ◽  
Siok Leong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Leucine Zipper ◽  
Binding Protein ◽  
Xenograft Model ◽  
Mesenchymal Transition ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Subcutaneous Xenograft ◽  
Peptide Antagonist ◽  
Anti Tumor Activity

Abstract CCAAT/Enhancer Binding Protein Beta (C/EBPß) is a transcription factor overexpressed in glioblastoma (GBM). Mechanistically, C/EBPß is a master regulator of mesenchymal transition in GBM, and its increased expression correlates with mesenchymal differentiation and predicts poor clinical outcome. C/EBPß activity requires dimerization with co-factors such as CREB/ATF family members via leucine zipper interactions. ST101 is a novel peptide antagonist of C/EBPß currently being evaluated in a Phase 1/2 clinical study in patients with advanced unresectable and metastatic solid tumors. ST101 binds to the C/EBPß leucine zipper, thereby preventing dimer formation and inhibiting its transcriptional activity, resulting in selective tumor cell cytotoxicity. Here, we describe ST101 non-clinical anti-tumor activity against GBM. In vitro studies in T98G and U251 cells demonstrate ST101 dose-dependent impact of cell viability (EC50 of 2.2 and 1.2 μM, respectively), accompanied by significant impact on C/EBPß-mediated gene expression as determined by qPCR analysis. In contrast, normal human mononuclear and epithelial cells were not sensitive to ST101 (EC50 > 80 μM). In vivo, ST101 displayed significant anti-tumor activity in a U251 GBM subcutaneous xenograft model, resulting in 81.4% tumor growth inhibition (TGI) vs. control and undetectable tumors in 50% of animals. Following ST101 exposure tumors displayed reduced BIRC3 and ID2 gene expression, and significantly increased cleaved caspase 3 immunostaining indicative of cell death induction. In U251 tumors, subtherapeutic ST101 (< 5% TGI) in combination with temozolomide (< 5% TGI) resulted in 52.8% TGI, significantly greater than either single-agent alone. Similarly, in a temozolomide-refractory T98G GBM subcutaneous xenograft model, ST101 (41.6% TGI) in combination with TMZ (< 5% TGI) resulted in significant anti-GBM response (72.4% TGI). These data emphasize the potential of ST101 as a potent peptide therapeutic for GBM.

scholarly journals A CCAAT/Enhancer Binding Protein β Isoform, Liver-Enriched Inhibitory Protein, Regulates Commitment of Osteoblasts and Adipocytes

2005 ◽  
Vol 25 (5) ◽  
pp. 1971-1979 ◽  
Author(s):  
Kenji Hata ◽  
Riko Nishimura ◽  
Mio Ueda ◽  
Fumiyo Ikeda ◽  
Takuma Matsubara ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Osteoblast Differentiation ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Mesenchymal Cells ◽  
Dominant Negative ◽  
Inhibitory Protein ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

ABSTRACT Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein β (C/EBPβ), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPβ, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPβ, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPβ isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.

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.

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.

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