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Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of β-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1., (© 2022. The Author(s).)

The BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient's cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in β-hemoglobinopathy patients. Our data strongly support this approach., (© 2021 by The American Society of Hematology.)

Haploinsufficiency for transcription factor KLF1 causes a variety of human erythroid phenotypes, such as the In(Lu) blood type, increased HbA2 levels, and hereditary persistence of fetal hemoglobin. Severe dominant congenital dyserythropoietic anemia IV (OMIM 613673) is associated with the KLF1 p.E325K variant. CDA-IV patients display ineffective erythropoiesis and hemolysis resulting in anemia, accompanied by persistent high levels of embryonic and fetal hemoglobin. The mouse Nan strain carries a variant in the orthologous residue, KLF1 p.E339D. Klf1Nan causes dominant hemolytic anemia with many similarities to CDA-IV. Here we investigated the impact of Klf1Nan on the developmental expression patterns of the endogenous beta-like and alpha-like globins, and the human beta-like globins carried on a HBB locus transgene. We observe that the switch from primitive, yolk sac-derived, erythropoiesis to definitive, fetal liver-derived, erythropoiesis is delayed in Klf1wt/Nan embryos. This is reflected in globin expression patterns measured between E12.5 and E14.5. Cultured Klf1wt/Nan E12.5 fetal liver cells display growth- and differentiation defects. These defects likely contribute to the delayed appearance of definitive erythrocytes in the circulation of Klf1wt/Nan embryos. After E14.5, expression of the embryonic/fetal globin genes is silenced rapidly. In adult Klf1wt/Nan animals, silencing of the embryonic/fetal globin genes is impeded, but only minute amounts are expressed. Thus, in contrast to human KLF1 p.E325K, mouse KLF1 p.E339D does not lead to persistent high levels of embryonic/fetal globins. Our results support the notion that KLF1 affects gene expression in a variant-specific manner, highlighting the necessity to characterize KLF1 variant-specific phenotypes of patients in detail.

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells., Competing Interests: The authors have declared that no competing interests exist.

The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood-derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34 + cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies.

Context: Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis., Objective: To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients., Design, Setting, and Patients: Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points., Results: After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient-derived HEPs., Conclusions: Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells during erythropoiesis, which may contribute to the mild anemia seen in most RTHα patients.

Currently, bone marrow transplantation is the only curative treatment for β-thalassemia and sickle cell disease. In rare cases, sustained and full fetal hemoglobin production was observed in patients after failure of bone marrow transplantation. This rendered the patients transfusion-free, despite genetic disease and transplant rejection. The mechanisms underlying this phenomenon remain unexplored. We have studied a trio (father-mother-child) in which the affected child became transfusion-independent after rejection of an allogeneic bone marrow graft. Remarkably, we found that his non-thalassemic mother also expressed unusually high levels of γ-globin. High HbF in one of the parents may therefore be of prognostic value in these rare cases. Genotyping of the HBB locus and the HbF quantitative trait loci HBS1L-MYB, KLF1 and BCL11A, and protein expression analysis of KLF1 and BCL11A, failed to explain the increased HbF levels, indicating that an as yet unidentified HbF modifier locus may be involved. We hypothesize that epigenetic events brought about by the transplantation procedure allow therapeutic levels of HbF expression in the child. Potential implications of our observations for reactivation of γ-globin expression and interpretation of the French globin gene therapy case are discussed.

The TREX complex couples nuclear pre-mRNA processing with mRNA export and contains multiple protein components, including Uap56, Alyref, Cip29 and the multi-subunit THO complex. Here, we have identified Chtop as a novel TREX component. We show that both Chtop and Alyref activate the ATPase and RNA helicase activities of Uap56 and that Uap56 functions to recruit both Alyref and Chtop onto mRNA. As observed with the THO complex subunit Thoc5, Chtop binds to the NTF2-like domain of Nxf1, and this interaction requires arginine methylation of Chtop. Using RNAi, we show that co-knockdown of Alyref and Chtop results in a potent mRNA export block. Chtop binds to Uap56 in a mutually exclusive manner with Alyref, and Chtop binds to Nxf1 in a mutually exclusive manner with Thoc5. However, Chtop, Thoc5 and Nxf1 exist in a single complex in vivo. Together, our data indicate that TREX and Nxf1 undergo dynamic remodelling, driven by the ATPase cycle of Uap56 and post-translational modifications of Chtop.

Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity.

Background: In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established., Design and Methods: To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene., Results: We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic β-like globins and the phospholipase HRAS-like suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts., Conclusions: We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the down-regulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic β-like globins.

An estimated 6% to 7% of the earth's population carries a mutation affecting red blood cell function. The β-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of γ-globin ameliorate the severity of these diseases because fetal hemoglobin (HbF; α2γ2) can effectively replace adult hemoglobin (HbA; α2β2) and counteract polymerization of sickle hemoglobin (HbS; α2β(S)2). Therefore, understanding the molecular mechanism of globin switching is of biologic and clinical importance. Here, we show that the recently identified chromatin factor Friend of Prmt1 (FOP) is a critical modulator of γ-globin gene expression. Knockdown of FOP in adult erythroid progenitors strongly induces HbF. Importantly, γ-globin expression can be elevated in cells from β-thalassemic patients by reducing FOP levels. These observations identify FOP as a novel therapeutic target in β-hemoglobinopathies.

We describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified by both asymmetric and symmetric arginine methylation in vivo. Furthermore, Fop plays an important role in the ligand-dependent activation of estrogen receptor target genes, including TFF1 (pS2). Fop depletion results in an almost complete block of estradiol-induced promoter occupancy by the estrogen receptor. Our data indicate that Fop recruitment to the promoter is an early critical event in the activation of estradiol-dependent transcription.

The human monocarboxylate transporter 8 (hMCT8) protein mediates transport of thyroid hormone across the plasma membrane. Association of hMCT8 mutations with severe psychomotor retardation and disturbed thyroid hormone levels has established its physiological relevance, but little is still known about the basic properties of hMCT8. In this study we present evidence that hMCT8 does not form heterodimers with the ancillary proteins basigin, embigin, or neuroplastin, unlike other MCTs. In contrast, it is suggested that MCT8 exists as monomer and homodimer in transiently and stably transfected cells. Apparently hMCT8 forms stable dimers because the complex is resistant to denaturing conditions and dithiothreitol. Cotransfection of wild-type hMCT8 with a mutant lacking amino acids 267-360 resulted in formation of homo-and heterodimers of the variants, indicating that transmembrane domains 4-6 are not involved in the dimerization process. Furthermore, we explored the structural and functional role of the 10 Cys residues in hMCT8. All possible Cys>Ala mutants did not behave differently from wild-type hMCT8 in protein expression, cross-linking experiments with HgCl(2) and transport function. Our findings indicate that individual Cys residues are not important for the function of hMCT8 or suggest that hMCT8 has other yet-undiscovered functions in which cysteines play an essential role.

Dendritic cells are key initiators and regulators of the immune response. Dendritic cell commitment and function require orchestrated regulation of transcription. Gata1 is a transcription factor expressed in several hematopoietic lineages. However, Gata1 function has not been explored in the monocytic or dendritic cell compartment. Here, we show that Gata1 is expressed in myeloid and plasmacytoid dendritic cells and that Gata1 ablation affects the survival of dendritic cells. Furthermore, lipopolysaccharide (LPS) stimulation of dendritic cells prompts Gata1 up-regulation, which is accompanied by increased levels of BclX and Ifng. Our findings show that Gata1 is a transcriptional regulator of dendritic cell differentiation and suggest that Gata1 is involved in the dendritic cell and macrophage lineage separation.

The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5. Both Foxo3a and Stat5 promote erythroid differentiation. To understand the interplay of SCF and Epo in maintaining the balance between renewal and differentiation during erythroid development, we investigated differential Foxo3a target regulation by Epo and SCF. Expression profiling revealed that a subset of Foxo3a targets was not inhibited but was activated by Epo. One of these genes was Cited2. Transcriptional control of Epo/Foxo3a-induced Cited2 was studied and compared with that of the Epo-repressed Foxo3a target Btg1. We show that in response to Epo, the allegedly growth-inhibitory factor Foxo3a associates with the allegedly growth-stimulatory factor Stat5 in the nucleus, which is required for Epo-induced Cited2 expression. In contrast, Btg1 expression is controlled by the cooperation of Foxo3a with cyclic AMP- and Jun kinase-dependent Creb family members. Thus, Foxo3a not only is an effector of PKB but also integrates distinct signals to regulate gene expression in erythropoiesis.

LFM-A13, or alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide, was shown to inhibit Bruton's tyrosine kinase (Btk). Here we show that LFM-A13 efficiently inhibits erythropoietin (Epo)-induced phosphorylation of the erythropoietin receptor, Janus kinase 2 (Jak2) and downstream signalling molecules. However, the tyrosine kinase activity of immunoprecipitated or in vitro translated Btk and Jak2 was equally inhibited by LFM-A13 in in vitro kinase assays. Finally, Epo-induced signal transduction was also inhibited in cells lacking Btk. Taken together, we conclude that LFM-A13 is a potent inhibitor of Jak2 and cannot be used as a specific tyrosine kinase inhibitor to study the role of Btk in Jak2-dependent cytokine signalling.

Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor-induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes. We identified B cell translocation gene 1 (BTG1)/antiproliferative protein 2 as a FoxO3a target gene in erythroid progenitors. Promoter studies indicated BTG1 as a direct target of FoxO3a. Expression of BTG1 in primary mouse bone marrow cells blocked the outgrowth of erythroid colonies, which required a domain of BTG1 that binds protein arginine methyl transferase 1. During erythroid differentiation, increased arginine methylation coincided with BTG1 expression. Concordantly, inhibition of methyl transferase activity blocked erythroid maturation without affecting expansion of progenitor cells. We propose FoxO3a-controlled expression of BTG1 and subsequent regulation of protein arginine methyl transferase activity as a novel mechanism controlling erythroid expansion and differentiation.

Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.

The eosinophil-derived neurotoxin (EDN), a member of the mammalian ribonuclease family, is found in the large specific granules of human eosinophilic leukocytes. We have investigated the role of the C/EBP transcription factor family in the regulation of EDN promoter activity. Here we show that the C/EBP family is involved in intrinsic regulation of EDN promoter activity. We have identified a C/EBP binding site located at -124 in the proximal promoter of the EDN gene. Mutation of this C/EBP site results in a decrease of promoter activity in HL-60-eos cells as well as in eosinophils differentiated in vitro from CD34+ cells. Different C/EBP proteins are able to bind to the C/EBP site as shown by gel shift assay. Our results indicate the importance of the C/EBP family in the regulation of the EDN gene in eosinophils.

The common beta-chain (beta c) is the main signaling component of the heterodimeric receptors for IL-3, IL-5, and GM-CSF and is primarily expressed on myeloid cells. The proximal beta c promoter is regulated by GGAA binding proteins, including PU.1, a hemopoietic specific member of the Ets family. However, it is not likely that PU.1 alone accounts for the myeloid-restricted expression of the beta c subunit. Here we describe the identification of a C/EBP binding enhancer that is located 2 kb upstream of the transcription start site. The enhancer contains two elements that bind C/EBP alpha and -beta in U937 cells, while C/EBP epsilon is also bound in extracts of HL-60 cells. Importantly, deletion of the enhancer or mutation of either of one of the C/EBP sites results in a complete loss of promoter activity in cell lines as well as in primary cells, showing the importance of C/EBP members in beta c gene activation. We further show that PU.1 has to cooperate with C/EBP proteins to induce beta c transcription. Since the beta c is already expressed on CD34+ cells, these results demonstrate that both C/EBP and PU.1 are not only important for the myeloid-specific gene regulation at later stages of myeloid differentiation.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) regulate proliferation, differentiation and apoptosis of target cells. Receptors for these cytokines consist of a cytokine-specific alpha subunit and a common shared beta c subunit. Tyrosine phosphorylation of the beta c is thought to play a critical role in mediating signal transduction events. We have examined the effect of mutation of beta c tyrosines on the activation of multiple signal transduction pathways. Activation of protein kinase B (PKB) required JAK2 and was inhibited by dominant-negative phosphatidylinositol 3-kinase (P13K). Overexpression of JAK2 was sufficient to activate both protein kinase B (PKB) and extracellular regulated kinase-1 (ERK1). Tyrosine 577 and 612 were found to be critical for the activation of PKB and ERK1, but not activation of STAT transcription factors. Activation of both PKB and ERK have been implicated in the regulation of proliferation and apoptosis. We generated GM-CSFR stable cell lines expressing receptor mutants to evaluate their effect on these processes. Activation of both PKB and ERK was perturbed, while STAT activation remained unaffected. Tyrosines 577 and 612 were necessary for optimal proliferation, however, mutation of these tyrosine residues did not affect GM-CSF mediated rescue from apoptosis. These data demonstrate that while phosphorylation of beta c tyrosine residues 577 and 612 are important for optimal cell proliferation, rescue from apoptosis can be mediated by alternative signalling routes apparently independent of PKB or ERK activation.

Interleukin-5 (IL-5), IL-3, and granulocyte macrophage colony-stimulating factor (GM-CSF) are hematopoietic cytokines which signal through a common beta subunit (betac) of a heterodimeric receptor. Among the intracellular signaling pathways activated via betac is the JAK/STAT pathway. We show that different STAT5 isoforms are activated by IL-5 and GM-CSF in eosinophils, neutrophils, and differentiated eosinophilic HL-60 cells. Whereas IL-5 activated the wild-type STAT5A and STAT5B proteins in HL60-eos cells, a carboxyl-terminally truncated 80-kDa STAT5 isoform was activated in mature eosinophils and neutrophils. Surprisingly, while both isoforms bind strongly to an element from the beta-casein promoter, only p80 STAT5 binds to the ICAM1-IRE. Consequently, a carboxyl-terminal truncated STAT5 is capable of blocking STAT3-mediated transcription of an IREtkCAT reporter construct. The cell type-specific expression of these functionally distinct STAT5 isoforms might contribute to the pleiotropic effects of IL-5 and GM-CSF on different target cells.

Binding of interferon-gamma (IFN-gamma) to its heterodimeric receptor induces activation of the tyrosine kinases JAK1 and JAK2 followed by tyrosine phosphorylation of STAT1alpha. Selective activation of STAT1alpha at the IFN-gamma receptor is achieved by specific interaction between a cytosolic tyrosine motif including Y440 in the IFN-gamma receptor alpha-chain and the SH2 domain of STAT1alpha. We demonstrate that, in addition to STAT1alpha, STAT3 is also activated by IFN-gamma in human neutrophils. The activation of STAT3 was not found in human eosinophils, monocytes, and HL-60 cells, although the STAT3 protein was expressed in these cells. The cell type-specific activation of STAT3 by IFN-gamma was also observed in neutrophils that are differentiated in vitro from human CD34+ hematopoietic stem cells. These results indicate that a single cytokine receptor can activate different STAT family members in a cell-specific manner, which might result in cell-specific gene transcription.

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common beta chain (betac). Whereas the mouse has two homologous beta subunits (betac and betaIL-3), in humans, only a single beta chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor beta subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine betac/betaIL-3 genes. Surprisingly, we also found the remnants of a second betac chain gene directly downstream of betac. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from -103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5' element specifically binds PU.1, whereas the 3' element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances betac promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.

Interleukin-5 (IL-5) plays a crucial role in the proliferation, differentiation and activation of eosinophils. The IL-5 receptor is composed of an IL-5-specific alpha subunit, which is expressed by eosinophils and basophils, and a beta c-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5R alpha promoter activity in eosinophilic HL60 cells. The AP-1 site and the previously identified EOS1 site cooperate, since single mutation of either of the sites decreased promoter activity. We show that the AP-1 site of the IL-5R alpha promoter binds multiple proteins, including cJun, CREB, and CREM.

Eosinophil-derived neurotoxin (EDN) found in the granules of human eosinophils is a cationic ribonuclease toxin. Expression of the EDN gene (RNS2) in eosinophils is dependent on proximal promoter sequences in combination with an enhancer located in the first intron. We further define here the active region of the intron using transfections in differentiated eosinophilic HL60 cells. We show that a region containing a tandem PU.I binding site is important for intronic enhancer activity. This region binds multiple forms of transcription factor PU.I as judged by gel-shift analysis and DNA affinity precipitation. Importantly, introducing point mutations in the PU.I site drastically reduces the intronic enhancer activity, showing the importance of PU.I for expression of EDN in cells of the eosinophilic lineage.

Interleukin-5 (IL-5) and granulocyte macrophage-colony stimulating factor (GM-CSF) are important cytokines for the proliferation, differentiation, and activation of myeloid lineages. The JAK/STAT pathway is one of the signaling pathways implicated in mediating biological responses induced by these cytokines. Previous studies have demonstrated that these cytokines predominantly activate an 80 kDa STAT5 isoform in mature granulocytes. To better understand the role of STAT proteins during growth and differentiation of granulocytes, we evaluated differentiation of human CD34+ hematopoietic stem cells ex vivo toward eosinophils and neutrophils. Bandshift experiments showed that in an early stage of both differentiation pathways (14 days), the 94 kDa STAT5B protein was activated by both IL-5 (eosinophil lineage) and GM-CSF (neutrophil lineage). However, during maturation of both lineages (days 21 and 28), increased expression of a functionally distinct 80 kDa STAT5 isoform was observed, resulting in heterodimer DNA-binding complexes containing both the 94 and 80 kDa STAT5 proteins. The finding that functionally distinct isoforms of STAT5 are activated during the early and late differentiation stages of granulocytes suggests that they might be involved in regulating different biological functions in these cells.

In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R beta chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied IL-5-induced endogenous STAT-dependent transcription. Our results indicate that: (a) tyrosine phosphorylation of the IL-5R beta chain is required for endogenous STAT5 activation, (b) multiple tyrosine residues are phosphorylated upon IL-5 stimulation, including Tyr577, Tyr612, Tyr695, and Tyr750, and (c) Tyr612, Tyr695, and Tyr750 are all capable of inducing activation of STAT5, demonstrating a high level of functional redundancy within the IL-5R beta chain.

Interleukin-5 (IL-5) is one of the major regulators of eosinophilic granulocytes in vivo. IL-5 exerts its pleiotropic effects by binding to the IL-5 receptor, which is composed of an IL-5-specific alpha chain and a common betac chain shared with the receptors for IL-3 and granulocyte-macrophage colony-stimulating factor. Previous studies have shown that binding of IL-5 to its receptor triggers the activation of multiple signaling cascades, including the Ras/mitogen-activated protein kinase, the phosphatidyl -3'-kinase, and the Janus kinase/signal transducer and activator of transcription pathways. Here we describe that IL-5 activates the serine/threonine protein kinase Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway. We show that IL-5 activates TPA response element (TRE)-dependent transcription in transfection experiments. TRE activation by IL-5 is mediated by a region of the betac (577-581) that is also responsible for activation of JNK/SAPK and for activation of dyad symmetry element (DSE)-dependent transcription. Dominant-negative SAPK or ERK kinase-1 was used to demonstrate that JNK/SAPK activation is necessary for induction of DSE- and TRE-dependent transcription by IL-5, whereas extracellular signal-regulated kinase 2 was not essential for TRE- and DSE-dependent transcription. By contrast, IL-5-induced activation of the tyrosine kinase Janus kinase 2 seems to be a prerequisite for TRE- and DSE-dependent transcription. Taken together, we show for the first time that IL-5 activates kinases of the JNK/SAPK family, and that this activation is linked to IL-5-induced TRE- and DSE-dependent transcription.

The 89-kDa STAT3 protein is a latent transcription factor which is activated in response to cytokines (interleukin (IL)-5 and -6) and growth factors (epidermal growth factor). Binding of IL-5 to its specific receptor activates JAK2 which leads to the tyrosine phosphorylation of STAT3 proteins. Here we report the cloning of a cDNA encoding a variant of the transcription factor STAT3 (named STAT3beta) which was isolated by screening an eosinophil cDNA library. Compared to wild-type STAT3, STAT3beta lacks an internal domain of 50 base pairs located near the C terminus. This splice product is a naturally occurring isoform of STAT3 and encodes a 80-kDa protein. We found by reconstitution of the human IL-5R in COS cells that like STAT3, STAT3beta is phosphorylated on tyrosine and binds to the pIRE from the ICAM-1 promoter after IL-5 stimulation. However, STAT3beta fails to activate a pIRE containing promoter in transient transfection assays. Instead, co-expression of STAT3beta inhibits the transactivation potential of STAT3. These results suggests that STAT3beta functions as a negative regulator of transcription.

In this paper we describe the cloning of the mouse Parathyroid Hormone/Parathyroid Hormone related Peptide Receptor (PTH/PTHrPR) cDNA and expression of its mRNA during mouse postimplantation development from day 5.5 until day 15.5 post coitum (p.c.). In support of a model from previous studies, in which parietal endoderm differentiation is regulated by the interaction of the PTH/PTHrPR and Parathyroid Hormone related Peptide (PTHrP), high levels of PTH/PTHrPR mRNA levels were detected in developing parietal endoderm from day 5.5 p.c. and onwards. In the embryo proper, PTH/PTHrPR mRNA expression was mainly detected at sites of epithelium/mesenchyme interactions, starting at day 9.5 p.c. in the epithelium of the intestine and later in the mesenchyme of the lung, the epithelium of meso- and metanephric tubuli, the dermis and at all sites where bone formation takes place. The complexity of the PTH/PTHrPR expression pattern suggests tight developmental regulation and indicates multiple roles in embryogenesis for the receptor and its ligands, not only in extraembryonic tissue but also in the formation of various organs.