Myeloid transformation by MLL-ENL depends strictly on C/EBP.

Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL-transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL-transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPß deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPß expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPß, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

C/EBPα regulates osteoclast lineage commitment.

Despite recent insights gained from the effects of targeted deletion of the Finkel-Biskis-Jinkins osteosarcoma oncogene (c-fos), Spleen focus-forming virus (SFFV) proviral integration 1 (PU.1), microphthalmia-associated transcription factor, NF-κB, and nuclear factor of activated cells cytoplasmic 1 (NFATc1) transcription factor genes, the mechanism underlying transcription factors specifying osteoclast (OC) lineage commitment from monocyte/macrophage remains unclear. To characterize the mechanism by which transcription factors regulate OC lineage commitment, we mapped the critical cis-regulatory element in the promoter of cathepsin K (Ctsk), which is expressed specifically in OCs, and found that CCAAT/enhancer binding protein α (C/EBPα) is the critical cis-regulatory element binding protein. Our results indicate that C/EBPα is highly expressed in pre- OCs and OCs. The combined presence of macrophage colony-stimulating factor and receptor activator of NF-κB ligand significantly induces high C/EBPα expression. Furthermore, C/EBPα(-/-) newborn mice exhibited impaired osteoclastogenesis, and a severe osteopetrotic phenotype, but unaffected monocyte/macrophage development. Impaired osteoclastogenesis of C/EBPα(-/-) mouse bone marrow cells can be rescued by c-fos overexpression. Ectopic expression of C/EBPα in mouse bone marrow cells and monocyte/macrophage cells, in the absence of receptor activator of NF-κB ligand, induces expression of receptor activator of NF-κB, c-fos, Nfatc1, and Ctsk, and it reprograms monocyte/macrophage cells to OC-like cells. Our results demonstrate that C/EBPα directly up-regulates c-fos expression. C/EBPα(+/-) mice exhibit an increase in bone density compared with C/EBPα(+/+) controls. These discoveries establish C/EBPα as the key transcriptional regulator of OC lineage commitment, providing a unique therapeutic target for diseases of excessive bone resorption, such as osteoporosis and arthritis.

Down regulation of a matrix degrading cysteine protease cathepsin L, by acetaldehyde: role of C/EBPα.

BACKGROUND: The imbalance between extra cellular matrix (ECM) synthesis and degradation is critical aspect of various hepatic pathologies including alcohol induced liver fibrosis. This study was carried out to investigate the effect of acetaldehyde on expression of an extra cellular matrix degrading protease cathepsin L (CTSL) in HepG2 cells. METHODOLOGY AND RESULTS: We measured the enzymatic activity, protein and, mRNA levels of CTSL in acetaldehyde treated and untreated cells. The binding of CAAT enhancer binding protein α (C/EBP α) to CTSL promoter and its key role in the transcription from this promoter and conferring responsiveness to acetaldehyde was established by site directed mutagenesis, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assays and siRNA technology. Acetaldehyde treatment significantly decreased CTSL activity and protein levels in HepG2 cells. A similar decrease in the mRNA levels and promoter activity was also observed. This decrease by acetaldehyde was attributed to the fall in the liver enriched transcription factor C/EBP α levels and it's binding to the CTSL promoter. Mutagenesis of C/EBP α binding motifs revealed the key role of this factor in CTSL transcription as well as conferring responsiveness to acetaldehyde. The siRNA mediated silencing of the C/EBP α expression mimicked the effect of acetaldehyde on CTSL levels and its promoter activity. It also abolished the responsiveness of this promoter to acetaldehyde. CONCLUSION: Acetaldehyde down regulates the C/EBP α mediated CTSL expression in hepatic cell lines. The decreased expression of CTSL may at least in part contribute to ECM deposition in liver which is a hallmark of alcoholic liver fibrosis.

C/EBPalpha in leukemogenesis: a matter of being in the right place with the right signals.

Leukemia-initiating cells can originate from hematopoietic progenitor cells that have acquired self-renewal capacity upon transformation with leukemic fusion genes. In this issue of Cancer Cell, Kirstetter and colleagues describe a mouse model for the frequent CEBPA mutations in human acute myeloid leukemia that result in the synthesis of only the 30kDa isoform, but not the 42kDa isoform of C/EBPalpha. This mutation uncouples C/EBPalpha's roles in myeloid differentiation and proliferation control. Furthermore, this mutation activates self-renewal in committed myeloid progenitor cells and induces myeloid malignancy with complete penetrance that is sustained by leukemia-initiating cells with a committed myeloid molecular signature.

Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells.

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.

SIRT1 regulates adiponectin gene expression through Foxo1-C/enhancer-binding protein alpha transcriptional complex.

Adiponectin is an adipose-derived hormone that plays an important role in maintaining energy homeostasis. Adiponectin gene expression is diminished in both obesity and type 2 diabetes. However, the mechanism underlying the impaired adiponectin gene expression remains poorly understood. Recent studies have indicated that forkhead transcription factor O1 (Foxo1) and silent information regulator 2 mammalian ortholog SIRT1 are involved in adipogenesis. Here we have shown that Foxo1 up-regulates adiponectin gene transcription through a Foxo1-responsive region in the mouse adiponectin promoter that contains two adjacent Foxo1 binding sites. Foxo1 interacts with CCAAT/enhancer-binding protein alpha (C/EBPalpha) to form a transcription complex at the mouse adiponectin promoter and up-regulates adiponectin gene transcription. Our study has revealed that C/EBPalpha accesses the adiponectin promoter through two Foxo1 binding sites and acts as a co-activator. Further, SIRT1 increases adiponectin transcription in adipocytes by activating Foxo1 and enhancing Foxo1 and C/EBPalpha interaction. Importantly, both Foxo1 and SIRT1 protein levels were significantly lower in epididymal fat tissues from db/db and high fat diet-induced obese mice compared with normal mice. We propose that low expression of SIRT1 and Foxo1 leads to impaired Foxo1-C/EBPalpha complex formation, which contributes to the diminished adiponectin expression in obesity and type 2 diabetes.

Mitogen-activated protein kinase-mediated disruption of enhancer-promoter communication inhibits hepatocyte nuclear factor 4alpha expression.

Hepatocyte nuclear factor 4 (HNF-4) is a key member of the transcription factor network regulating hepatocyte differentiation and function. Activation of the HNF-4 gene involves physical interaction between a distant enhancer and the proximal promoter region, bound by distinct sets of transcription factors. Here we report that, upon mitogen-activated protein (MAP) kinase activation, HNF-4 expression is downregulated in human hepatoma cells. This effect is mediated by the loss of CEBPalpha expression. During MAP kinase signaling, the recruitment of HNF-3beta and HNF-1alpha to the HNF-4 enhancer and RNA polymerase II to the proximal HNF-4 promoter was compromised. CBP, Brg1, and TFIIB were also dissociated from the HNF-4 regulatory regions, and the enhancer-promoter complex was disrupted. Interestingly, the extent of nucleosome acetylation did not decrease at either regulatory region, and HNF-6 and HNF-1alpha, as well as components of the TFIID, remained associated with the proximal promoter during the repressed state. The results point to an absolute requirement of enhancer-promoter communication for maintaining the active state of the HNF-4 gene and provide evidence for a molecular bookmarking mechanism, which may contribute to the prevention of permanent silencing of the locus during the repressed state.

C/EBPbeta is required for 'emergency' granulopoiesis.

During 'emergency' situations such as infections, host defense requires rapid mobilization of bone marrow granulocyte progenitors. 'Steady-state' granulopoiesis is absolutely dependent on the C/EBPalpha transcription factor, but the transcriptional mechanisms underlying emergency granulopoiesis remain unclear. Here we show that large numbers of granulocytes were generated from C/EBPalpha-deficient progenitors after cytokine stimulation in vivo. Cytokine treatment or fungal infection induced upregulation of C/EBPbeta but not C/EBPalpha or C/EBPepsilon transcripts in granulocyte progenitors, and C/EBPbeta-deficient progenitors showed decreased emergency-induced granulopoiesis in vitro and in vivo. C/EBPbeta inhibited proliferation less severely than did C/EBPalpha. These data suggest a critical function for C/EBPbeta in emergency granulopoiesis, which demands both differentiation and proliferation of granulocyte precursors.

Absence of the transcription factor CCAAT enhancer binding protein alpha results in loss of myeloid identity in bcr/abl-induced malignancy.

The lineage-determining transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is required for myeloid differentiation. Decreased function or expression of C/EBPalpha is often found in human acute myeloid leukemia. However, the precise impact of C/EBPalpha deficiency on the maturation arrest in leukemogenesis is not well understood. To address this question, we used a murine transplantation model of a bcr/abl-induced myeloproliferative disease. The expression of bcr/abl in C/EBPalphapos fetal liver cells led to a chronic myeloid leukemia-like disease. Surprisingly, bcr/abl-expressing C/EBPalpha-/- fetal liver cells failed to induce a myeloid disease in transplanted mice, but caused a fatal, transplantable erythroleukemia instead. Accordingly, increased expression of the transcription factors SCL and GATA-1 in hematopoietic precursor cells of C/EBPalpha-/-R01-EY-11298 ) fetal livers was found. The mechanism for the lineage shift from myeloid to erythroid leukemia was studied in a bcr/abl-positive cell line. Consistent with findings of the transplant model, expression of C/EBPalpha and GATA-1 was inversely correlated. Id1, an inhibitor of erythroid differentiation, was identified as a critical direct target of C/EBPalpha. Down-regulation of Id1 by RNA interference impaired C/EBPalpha-induced granulocytic differentiation. Taken together, our study provides evidence that myeloid lineage identity of malignant hematopoietic progenitor cells requires the residual expression of C/EBPalpha.

Respiratory failure due to differentiation arrest and expansion of alveolar cells following lung-specific loss of the transcription factor C/EBPalpha in mice.

The leucine zipper family transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) inhibits proliferation and promotes differentiation in various cell types. In this study, we show, using a lung-specific conditional mouse model of C/EBPalpha deletion, that loss of C/EBPalpha in the respiratory epithelium leads to respiratory failure at birth due to an arrest in the type II alveolar cell differentiation program. This differentiation arrest results in the lack of type I alveolar cells and differentiated surfactant-secreting type II alveolar cells. In addition to showing a block in type II cell differentiation, the neonatal lungs display increased numbers of proliferating cells and decreased numbers of apoptotic cells, leading to epithelial expansion and loss of airspace. Consistent with the phenotype observed, genes associated with alveolar maturation, survival, and proliferation were differentially expressed. Taken together, these results identify C/EBPalpha as a master regulator of airway epithelial maturation and suggest that the loss of C/EBPalpha could also be an important event in the multistep process of lung tumorigenesis. Furthermore, this study indicates that exploring the C/EBPalpha pathway might have therapeutic benefits for patients with respiratory distress syndromes.

CCAAT/enhancer binding protein alpha maintains the ability of insulin-stimulated GLUT4 translocation in 3T3-C2 fibroblastic cells.

In 3T3-L1 preadipocytes, hormonal induction causes adipose conversion and facilitates the expression of insulin-sensitive glucose transporter, GLUT4. Evidence has accumulated that, in 3T3-L1 preadipocytes, the formation of GLUT4 storage vesicle and its translocation to plasma membrane precede both lipid accumulation and expression of GLUT4 and C/EBPalpha, a key transcription factor for adipose differentiation. On the other hand, 3T3-C2 fibroblastic cells, a subline of 3T3-L1, follow adipogenic process till mitotic clonal expansion stage (2 days after hormonal induction), but do not proceed to terminal differentiation stage (8 days after the induction), resulting in a lack of adipose conversion and GLUT4 expression. Here we show that, when myc-tagged GLUT4 was retrovirally expressed in 3T3-C2 cells, insulin-stimulated GLUT4 translocation did occur on day 2 after the induction. On day 8 after the induction, however, neither GLUT4 translocation nor the expression of C/EBPalpha was observed. We also created 3T3-C2 cells stably expressing both myc-tagged GLUT4 and C/EBPalpha, demonstrating that co-expressed cells showed insulin-stimulated GLUT4 translocation on day 8 after the induction, as well as adipose conversion coupling with PPARgamma expression. Our results provide evidence that C/EBPalpha has the potential to maintain the ability of insulin-stimulated GLUT4 translocation in C/EBPalpha-deficient 3T3-C2 fibroblastic cells.

Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha.

The transcription factor C/EBP alpha is required for granulopoiesis and frequently disrupted in human acute myeloid leukemia (AML). Here, we show disruption of C/EBP alpha blocks the transition from the common myeloid to the granulocyte/monocyte progenitor but is not required beyond this stage for terminal granulocyte maturation. C/EBP alpha-deficient hematopoietic stem cells (HSCs) have increased expression of Bmi-1 and enhanced competitive repopulating activity. Bone marrow in adult C/EBP alpha-deficient mice was filled with myeloblasts, similar to human AML, supporting the notion that disruption of C/EBP alpha cooperates with other events in the development of leukemia. Therefore, C/EBP alpha is not only essential for granulocyte development but, in addition, is a regulator of hematopoietic stem cell activity.

C/EBPalpha deficiency results in hyperproliferation of hematopoietic progenitor cells and disrupts macrophage development in vitro and in vivo.

CCAAT enhancer binding protein-alpha (C/EBPalpha) inhibits proliferation in multiple cell types; therefore, we evaluated whether C/EBPalpha-deficient hematopoietic progenitor cells (HPCs) have an increased proliferative potential in vitro and in vivo. In this study we demonstrate that C/EBPalpha(-/-) fetal liver (FL) progenitors are hyperproliferative, show decreased differentiation potential, and show increased self-renewal capacity in response to hematopoietic growth factors (HGFs). There are fewer committed bipotential progenitors in C/EBPalpha(-/-) FL, whereas multipotential progenitors are unaffected. HGF-dependent progenitor cell lines can be derived by directly culturing C/EBPalpha(-/-) FL cells in vitro Hyperproliferative spleen colonies and myelodysplastic syndrome (MDS) are observed in mice reconstituted with C/EBPalpha(-/-) FL cells, indicating progenitor hyperproliferation in vitro and in vivo. C/EBPalpha(-/-) FL lacked macrophage progenitors in vitro and had impaired ability to generate macrophages in vivo. These findings show that C/EBPalpha deficiency results in hyperproliferation of HPCs and a block in the ability of multipotential progenitors to differentiate into bipotential granulocyte/macrophage progenitors and their progeny.

Cross-talk between regulators of myeloid development: C/EBPalpha binds and activates the promoter of the PU.1 gene.

CCAAT/enhancer-binding protein (C/EBP)alpha and PU.1 are required for myelopoiesis. Examination of the murine PU.1 promoter revealed several potential C/EBP-binding sites. Gel-shift assay demonstrated that C/EBPalpha expressed in 293T cells bound the site centered at -68 most potently. C/EBPalpha from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPbeta did so to a lesser extent, whereas these C/EBP isoforms bound the neutrophil elastase promoter with equal affinity. The -68 site in the murine PU.1 promoter is conserved in the human PU.1 promoter. Mutation of the -68 C/EBP-binding site in a -85/+152 promoter segment linked to the luciferase cDNA reduced promoter activity fourfold in 293T cells in the presence of cotransfected C/EBPalpha and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBPalpha-estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBPalpha DNA-binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBPalpha and C/EBPalpha-ER with the PU.1 promoter. Finally, PU.1 RNA is reduced several-fold in immortalized C/EBPalpha (-/-) compared with (+/-) cells. Together, these findings indicate that C/EBPalpha binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBPalpha may account for increased levels of PU.1 in myeloid as compared with B lymphoid cells and in this way, may contribute to the specification of myeloid progenitors.

Biallelic mutations in the CEBPA gene and low CEBPA expression levels as prognostic markers in intermediate-risk AML.

The CCAAT/enhancer binding protein alpha is an essential transcription factor for granulocytic differentiation. Recent studies reported N- and C-terminal CEBPA mutations in approximately 7% of acute myeloid leukaemia (AML) patients. C-terminal mutations are usually in-frame and occur in the basic-leucine zipper (bZIP) domain, resulting in deficient DNA binding. Using a rapid PCR approach, we screened for bZIP mutations and determined the prognostic value of these mutations in a cohort of 277 de novo AMLs. In addition, we set out to quantify CEBPA mRNA levels by 'real-time' PCR using TaqMan technology. In-frame insertions were observed in 12 (4.3%) cases. All cases with mutations carried an intermediate-risk karyotype and all but one belonged to M1 or M2 FAB class. Further sequence analysis revealed that CEBPA C-terminal mutations are associated with frameshift mutations in the N-terminus of CEBPA. These two mutations were always found in different alleles. Event-free survival (EFS) and overall survival (OS) of patients with CEBPA mutations were significantly increased (P=0.02 and 0.03, respectively) in comparison to the patients lacking these mutations. Mutations were associated with a significantly reduced hazard ratio for death (OS: HR=0.35, P=0.04) and failure (EFS: no CR, death in CR or relapse, HR=0.37, P=0.03). This favourable hazard ratio was maintained after adjustment for cytogenetic risk, FLT3-ITD and CEBPA expression levels in multivariable analysis. In contrast, low CEBPA expression in AML with intermediate-risk karyotype (n=6) seemed to be associated with poor prognosis (not significant). By including this newly developed PCR assay, we define a subgroup of good-risk patients within the heterogeneous intermediate-risk group of AML.

Induction of C/EBPalpha activity alters gene expression and differentiation of human CD34+ cells.

The CCAAT/enhancer binding protein alpha (C/EBPalpha) belongs to a family of transcription factors that are involved in the differentiation process of numerous tissues, including the liver and hematopoietic cells. C/EBPalpha(-/-) mice show a block in hematopoietic differentiation, with an accumulation of myeloblasts and an absence of mature granulocytes, whereas expression of C/EBPalpha in leukemia cell lines leads to granulocytic differentiation. Recently, dominant-negative mutations in the C/EBPalpha gene and down-regulation of C/EBPalpha by AML1-ETO, an AML associated fusion protein, have been identified in acute myelogenous leukemia (AML). To better understand the role of C/EBPalpha in the lineage commitment and differentiation of hematopoietic progenitors, we transduced primary human CD34(+) cells with a retroviral construct that expresses the C/EBPalpha cDNA fused in-frame with the estrogen receptor ligand-binding domain. Induction of C/EBPalpha function in primary human CD34(+) cells, by the addition of beta-estradiol, leads to granulocytic differentiation and inhibits erythrocyte differentiation. Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we have identified C/EBPalpha target genes in primary human hematopoietic cells, including granulocyte-specific genes that are involved in hematopoietic differentiation and inhibitor of differentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.

C/EBPalpha triggers proteasome-dependent degradation of cdk4 during growth arrest.

CCAAT/enhancer binding protein alpha (C/EBPalpha) causes growth arrest via direct interaction with the cyclin-dependent kinases cdk2 and cdk4. In this paper, we present evidence showing that C/EBPalpha enhances a proteasome-dependent degradation of cdk4 during growth arrest in liver of newborn mice and in cultured cells. Overexpression of C/EBPalpha in several biological systems leads to a reduction of cdk4 protein levels, but not mRNA levels. Experiments with several tissue culture models reveal that C/EBPalpha enhances the formation of cdk4-ubiquitin conjugates and induces degradation of cdk4 through a proteasome-dependent pathway. As a result, the half-life of cdk4 is shorter and protein levels of cdk4 are reduced in cells expressing C/EBPalpha. Gel filtration analysis of cdk4 complexes shows that a chaperone complex cdk4-cdc37-Hsp90, which protects cdk4 from degradation, is abundant in proliferating livers that lack C/EBPalpha, but this complex is weak or undetectable in livers expressing C/EBPalpha. Our studies show that C/EBPalpha disrupts the cdk4-cdc37-Hsp90 complex via direct interaction with cdk4 and reduces protein levels of cdk4 by increasing proteasome-dependent degradation of cdk4.

Multipotent hematopoietic cell lines derived from C/EBPalpha(-/-) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte- colony-stimulating factor, and retinoic acid-induced granulocytic differentiation.

The transcription factor C/EBPalpha is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte-colony-stimulating factor (G-CSF) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBPalpha knockout mice display a profound block in granulocyte differentiation. To study this block in granulocytic differentiation in more detail, retroviral vector-mediated transduction of a dominant-negative retinoic acid receptor was used to establish hematopoietic growth factor-dependent, lympho-myeloid progenitor cell lines from the fetal livers of both the C/EBPalpha knockout animals (C/EBPalpha(-/-)) and their heterozygous littermates (C/EBPalpha(+/-)). Surprisingly, the C/EBPalpha(-/-) cell lines displayed significant spontaneous granulocytic differentiation, and this differentiation was markedly enhanced when the cells were stimulated with granulocyte macrophage (GM)-CSF. This GM-CSF-mediated differentiation was associated with the up-regulation of G-CSF receptor mRNA, and the combination of GM-CSF and G-CSF generated more than 95% mature neutrophils in the C/EBPalpha(-/-) cultures. The addition of all-trans retinoic acid also enhanced this granulocytic differentiation of the cultured C/EBPalpha(-/-) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBPalpha. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBPalpha-responsive genes can occur in the absence of C/EBPalpha, and they indicate the existence of multiple independent molecular pathways potentially used by primitive hematopoietic precursors that can lead to the development of mature granulocytes.