ALBERT

Description: The “zinc-finger” transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and β-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation.

Description: Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBP mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ protein could bind to the potential C/EBP-binding site (bp −90 to −82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPβ and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (F̲riendo̲fG̲ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPβ synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.

Description: Abstract 215 Dysregulation of myeloid differentiation can result in the development of a variety of pathological conditions ranging from bone marrow failure to myelodysplastic syndromes and leukemia. Neutrophil development is tightly regulated by key transcription factors including CCAAT enhancer-binding protein-alpha (C/EBPα) and CCAAT enhancer-binding protein-epsilon (C/EBPε). In recent years it has become clear that the expression and function of such proteins is regulated by post-translational modifications. Here, we have investigated the regulation and functional role of C/EBPε acetylation. Our results demonstrate that C/EBPε is indeed acetylated and that this can be increased by the lysine acetyltransferases (KAT) TIP60 and p300, as well as the sirtuin 1(SIRT1) inhibitor nicotinamide (NAM). In agreement with this, acetylation was decreased upon co-transfection of SIRT1. Despite normal expression levels and the capacity to form homo- and heterodimers, the C/EBPε-lysine dead mutant (C/EBPε K15xR) was transcriptionally inactive in luciferase reporter assays, suggesting that acetylation of C/EBPε is functionally important. Moreover, co-transfection of SIRT1 inhibited C/EBPε transcriptional activation. In order to investigate the functional relevance of acetylaton of C/EBPε, we retrovirally transduced CD34+ hematopoietic progenitors with C/EBPε or C/EBPε K15xR and differentiated sorted progenitors towards mature neutrophils. We observed a significant decrease in the percentage of mature neutrophils after transduction with C/EBPε K15xR compared to C/EBPε or control cells. Cytospin analysis demonstrated an immature phenotype of the C/EBPε K15xR-transduced cells, suggesting a differentiation block at the promyelocytic stage. In order to determine the functionally important acetylated lysine residues, we performed mass spectometry and identified four C/EBPε acetylation sites including two sites in the repression domain and one site in the basic region, which includes the DNA-binding domain. To investigate the functional role of acetylation of these lysine residues, we designed specific C/EBPε lysine mutants. Utilizing the previously described ectopic expression system, we observed reduced levels of total C/EBPε acetylation upon transfection of two specific lysine mutants (K121R and K198R). Acetylation of these specific residues was confirmed by performing add-back experiments in the C/EBPε K15xR background. Furthermore, upon transfection of C/EBPε K121R and C/EBPε K198R in a luciferase reporter assay, we observed reduced transcriptional activation by C/EBPε, suggesting that acetylation of lysine 121 and lysine 198 is important for C/EBPε function. Currently we are investigating the functional role of acetylation of these specific lysine residues during neutrophil differentiation. There is increasing knowledge concerning the role of epigenetic modifications in the development of myeloid malignancies. However, the specific role of acetylation of non-histone proteins, including key-transcription factors involved in myelopoiesis, remains largely unknown. We demonstrate, for the first time, the functional importance of C/EBPε acetylation during neutrophil differentiation. Together our data provide new insights in the regulation of both normal and aberrant myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: The “zinc-finger” transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and β-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation.

Description: Instructive roles for both GATA-1 and PU.1 have been demonstrated in hematopoiesis, and recent studies have identified both antagonistic and synergistic interactions between them in myeloid gene transcription and lineage development. In prior studies, we reported that PU.1 synergizes with rather than antagonizes GATA-1 for transactivation of a hallmark eosinophil gene, the major basic protein P2 promoter (MBP-P2), which possesses a novel dual (double) GATA-binding site, similar to the palindromic double site in the murine GATA-1 control locus that may specify eosinophil lineage-specific expression of GATA-1 and eosinophil development. To address the transcriptional mechanism for PU.1-GATA-1 synergy through the MBP-P2 dual GATA site, we investigated GATA-1 and PU.1 physical and functonal interactions via their binding sites in the MBP-P2 promoter. DNA binding affinities of GATA-1 and its C- versus N-terminal zinc fingers were assessed for single versus double GATA sites in the presence or absence of PU.1. Our results show that the dual GATA site strongly binds full length GATA-1 with higher affinity than either of the single sites, using both zinc fingers, but that mutant GATA-1 proteins with C-finger or N-finger deletions retain their ability to bind, albeit at lower affinity, to the dual site. DNA binding activities of the two zinc fingers with the dual GATA site were confirmed using peptides containing only the C-finger or N-finger region. Of note, formation of GATA-1 complexes with the dual GATA site was not inhibited by the addition of PU.1, whereas formation of binding complexes for mutants of GATA-1 containing only the C- or N-finger region could be completely inhibited in a dose-response fashion by PU.1. These unique features of PU.1/GATA-1 interactions on a dual versus single GATA-1 site were confirmed using peptides containing only the C- or N-finger regions of GATA-1. Our findings indicate that both zinc fingers of GATA-1 are involved in formation of the high-affinity GATA-1 complex with the dual site. Importantly, we show that the higher affinity dual GATA-1 site complex is not affected by the addition of PU.1, whereas formation of the binding complex with a single GATA-1 site is eliminated by PU.1, emphasizing the different mechanisms of GATA-1/PU.1 interactions on dual versus single GATA binding sites. Functional analyses by transactivation confirmed that synergistic activation of the MBP-P2 promoter by GATA-1 and PU.1 is mediated by their protein-protein interactions through this unique high affinity dual GATA-1 binding site. We suggest two possible mechanisms for PU.1/GATA-1 synergy on dual GATA sites: (1) PU.1 may change GATA-1 conformation and its high affinity for the dual site, enhancing its availability for interaction with the basal transcriptional machinery. Alternatively, (2) PU.1 could impede interactions of GATA-1 with a co-repressor, e.g. FOG-1, which we and others have shown represses GATA-1 function in the eosinophil lineage.

Description: Stem cell factor (SCF) is an important mast cell growth, differentiation, and survival factor. We investigated whether SCF influenced the response of mouse mast cells to an IgE-independent stimulus, eosinophil-derived granule major basic protein (MBP). Mouse bone marrow cultured mast cells (BMCMC) were derived in either concanavalin-stimulated mouse spleen conditioned medium (CM) or SCF. The cloned growth, factor-independent mast cell line Cl.MC/C57.1 was also studied. BMCMC in SCF exhibited cytochemical staining properties, protease and histamine content, and increased serotonin uptake consistent with more mature differentiated mast cells as compared with BMCMC in CM or Cl.MC/ C57.1 cells. BMCMC in SCF released serotonin,14C-labeled arachidonic acid metabolites and tumor necrosis factor-α (TNF-α) on stimulation with MBP, while no response was seen from either BMCMC in CM or Cl.MC/C57.1 cells. All three mast cell populations released mediators on stimulation with the cationic MBP analog, poly-L-arginine, indicating that the cationic charge did not explain the selective response of BMCMC in SCF to eosinophil-derived granule MBP. These findings show that SCF significantly influences mast cell differentiation and the responsiveness of mast cells to eosinophil-derived granule MBP. © 1998 by The American Society of Hematology.

Description: Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify thecis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5′ deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp −117 to −67 were deleted. To identify transcription factors that bind to and transactivate through the bp −117 to −67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.