Enhanced Cell-Based Detection of Parvovirus B19V Infectious Units According to Cell Cycle Status.

Human parvovirus B19 (B19V) causes various human diseases, ranging from childhood benign infection to arthropathies, severe anemia and fetal hydrops, depending on the health state and hematological status of the patient. To counteract B19V blood-borne contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing does not correctly reflect B19V infectivity. There is currently no appropriate cellular model for detection of infectious units of B19V. We describe here an improved cell-based method for detecting B19V infectious units by evaluating its host transcription. We evaluated the ability of various cell lines to support B19V infection. Of all tested, UT7/Epo cell line, UT7/Epo-STI, showed the greatest sensitivity to B19 infection combined with ease of performance. We generated stable clones by limiting dilution on the UT7/Epo-STI cell line with graduated permissiveness for B19V and demonstrated a direct correlation between infectivity and S/G2/M cell cycle stage. Two of the clones tested, B12 and E2, reached sensitivity levels higher than those of UT7/Epo-S1 and CD36+ erythroid progenitor cells. These findings highlight the importance of cell cycle status for sensitivity to B19V, and we propose a promising new straightforward cell-based method for quantifying B19V infectious units.

Mild dyserythropoiesis and ß-like globin gene expression imbalance due to the loss of histone chaperone ASF1B.

The expression of the human ß-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to ß). The γ- to ß-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (ß-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to ß-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to ß-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

The integrity of the FOG-2 LXCXE pRb-binding motif is required for small intestine homeostasis.

NEW FINDINGS: What is the central question of this study? Do Fog2Rb-/Rb- mice present a defect of small intestine homeostasis? What is the main finding and its importance? The importance of interactions between FOG-2 and pRb in adipose tissue physiology has previously been demonstrated. Here it is shown that this interaction is also intrinsic to small intestine homeostasis and exerts extrinsic control over mouse metabolism. Thus, this association is involved in maintaining small intestine morphology, and regulating crypt proliferation and lineage differentiation. It therefore affects mouse growth and adaptation to a high-fat diet. ABSTRACT: GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. We have shown that GATA-1 and FOG-2 contain an LXCXE pRb-binding motif. Interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation, whereas the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Fog2-knock-in mice have defective pRb binding and are resistant to obesity, due to efficient white-into-brown fat conversion. Our aim was to investigate the pathophysiological impact of FOG-2-pRb interaction on the small intestine and mouse growth. Histological analysis of the small intestine revealed architectural changes in Fog2Rb-/Rb- mice, including villus shortening, with crypt expansion and a change in muscularis propria thickness. These differences were more marked in the proximo-distal part of the small intestine and were associated with an increase in crypt cell proliferation and disruption of the goblet and Paneth cell lineage. The small intestine of the mutants was unable to adapt to a high-fat diet, and had significantly lower plasma lipid levels on such a diet. Fog2Rb-/Rb- mice displayed higher levels of glucose-dependent insulinotropic peptide release, and lower levels of insulin-like growth factor I release on a regular diet. Their intestinal lipid absorption was impaired, resulting in restricted weight gain. In addition to the intrinsic effects of the mutation on adipose tissue, we show here an extrinsic relationship between the intestine and the effect of FOG-2 mutation on mouse metabolism. In conclusion, the interaction of FOG-2 with pRb coordinates the crypt-villus axis and controls small intestine homeostasis.

The LXCXE Retinoblastoma Protein-Binding Motif of FOG-2 Regulates Adipogenesis.

GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. Mammals have six GATA and two FOG factors. We recently demonstrated that interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation. We show here that the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Unlike GATA-1, which inhibits cell division, FOG-2 promotes proliferation. Mice with a knockin of a Fog2 gene bearing a mutated LXCXE pRb-binding site are resistant to obesity and display higher rates of white-to-brown fat conversion. Thus, each component of the GATA/FOG complex (GATA-1 and FOG-2) is involved in pRb/E2F regulation, but these molecules have markedly different roles in the control of tissue homeostasis.

Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis.

The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors.

Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis.

Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.

Parallel assessment of globin lentiviral transfer in induced pluripotent stem cells and adult hematopoietic stem cells derived from the same transplanted ß-thalassemia patient.

A patient with ß(E)/ß(0) -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, ß-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSC-derived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the ß-hemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with ß-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration.

BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7.

Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

Direct binding of pRb/E2F-2 to GATA-1 regulates maturation and terminal cell division during erythropoiesis.

How cell proliferation subsides as cells terminally differentiate remains largely enigmatic, although this phenomenon is central to the existence of multicellular organisms. Here, we show that GATA-1, the master transcription factor of erythropoiesis, forms a tricomplex with the retinoblastoma protein (pRb) and E2F-2. This interaction requires a LXCXE motif that is evolutionary conserved among GATA-1 orthologs yet absent from the other GATA family members. GATA-1/pRb/E2F-2 complex formation stalls cell proliferation and steers erythroid precursors towards terminal differentiation. This process can be disrupted in vitro by FOG-1, which displaces pRb/E2F-2 from GATA-1. A GATA-1 mutant unable to bind pRb fails to inhibit cell proliferation and results in mouse embryonic lethality by anemia. These findings clarify the previously suspected cell-autonomous role of pRb during erythropoiesis and may provide a unifying molecular mechanism for several mouse phenotypes and human diseases associated with GATA-1 mutations.

Gfi-1B promoter remains associated with active chromatin marks throughout erythroid differentiation of human primary progenitor cells.

Growth Factor Independent-1B (Gfi-1B) is a transcriptional repressor that plays critical roles in the control of erythropoiesis and megakaryopoiesis. Gfi-1B expression was described to be repressed by an autoregulatory feedback control loop. Here, we show that Gfi-1 transcription is positively regulated early after induction of erythroid differentiation and remains highly active to late erythroblasts. Using chromatin immunoprecipitation assays in CD34+ cells from human cord blood, we found that Gfi-1 and GATA-2 in immature progenitors and then Gfi-1B and GATA-1 in erythroblasts are bound to the Gfi-1B promoter as well as to the promoter of c-myc, a known Gfi-1B target gene. Surprisingly, this Gfi-1/GATA-2-Gfi-1B/GATA-1 switch observed at erythroblast stages is associated to an increase in the Gfi-1B transcription whereas it triggers repression of c-myc transcription. Accordingly, analysis of chromatin modification patterns shows that HDAC, CoREST, and LSD1 are recruited to the c-myc promoter leading to appearance of repressive chromatin marks. In contrast, the Gfi-1B promoter remains associated with a transcriptionally active chromatin configuration as highlighted by an increase in histone H3 acetylation and concomitant release of the LSD1 and CoREST corepressors. The repressive function of Gfi-1B therefore depends on the nature of the proteins recruited to the target gene promoters and on chromatin modifications. We conclude that Gfi-1B behaves as a lineage-affiliated gene with an open chromatin configuration in multipotent progenitors and sustained activation as cells progress throughout erythroid differentiation.

Phosphatidylinositol 3-kinase/Akt induced by erythropoietin renders the erythroid differentiation factor GATA-1 competent for TIMP-1 gene transactivation.

The contribution of erythropoietin to the differentiation of the red blood cell lineage remains elusive, and the demonstration of a molecular link between erythropoietin and the transcription of genes associated with erythroid differentiation is lacking. In erythroid cells, expression of the tissue inhibitor of matrix metalloproteinase (TIMP-1) is strictly dependent on erythropoietin. We report here that erythropoietin regulates the transcription of the TIMP-1 gene upon binding to its receptor in erythroid cells by triggering the activation of phosphatidylinositol 3-kinase (PI3K)/Akt. We found that Akt directly phosphorylates the transcription factor GATA-1 at serine 310 and that this site-specific phosphorylation is required for the transcriptional activation of the TIMP-1 promoter. This chain of events can be recapitulated in nonerythroid cells by transfection of the implicated molecular partners, resulting in the expression of the normally silent endogenous TIMP-1 gene. Conversely, TIMP-1 secretion is profoundly decreased in erythroid cells from fetal livers of transgenic knock-in mice homozygous for a GATA(S310A) gene, which encodes a GATA-1 mutant that cannot be phosphorylated at Ser(310). Furthermore, retrovirus-mediated expression of GATA(S310A) into GATA-1(null)-derived embryonic stem cells decreases the rate of hemoglobinization by more than 50% compared to expressed wild-type GATA-1. These findings provide the first example of a chain of coupling mechanisms between the binding of erythropoietin to its receptor and GATA-1-dependent gene expression.

Both proteasomes and lysosomes degrade the activated erythropoietin receptor.

Activation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.

Tissue inhibitor of metalloproteinases-1 signalling pathway leading to erythroid cell survival.

Tissue inhibitors of metalloproteinases (TIMP) are specific inhibitors of matrix metalloproteinases (MMPs) and thus participate in maintaining the balance between extracellular matrix deposition and degradation in several physio-pathological processes. Nevertheless, TIMP must be regarded as multifunctional proteins involved in cell growth, angiogenesis and apoptosis. The molecular mechanisms induced by TIMP remain largely unknown. In the present study, we provide evidence that TIMP-1 induces a significant anti-apoptotic effect in the human erythroleukaemic cell line UT-7 and in the murine myeloid cell line 32D. Using specific kinases inhibitors, we show that TIMP-1-mediated cell survival is dependent upon Janus kinase (JAK) 2 and phosphoinositide 3-kinase (PI 3-kinase) activities. By transient transfection of dominant-negative Akt in UT-7 cells, we demonstrate that this kinase is crucial for the TIMP-1 anti-apoptotic effect. Moreover, TIMP-1 enhances specific phosphorylation of both Akt and Bad (Bcl-2/Bcl-X(L)-antagonist, causing cell death) in a PI 3-kinase-dependent manner and, besides, controls the level of the anti-apoptotic protein Bcl-X(L). We conclude that TIMP-1 induces haematopoietic cell survival via the JAK2/PI 3-kinase/Akt/Bad pathway.

Involvement of the Src kinase Lyn in phospholipase C-gamma 2 phosphorylation and phosphatidylinositol 3-kinase activation in Epo signalling.

We examined the role of the Src kinase Lyn in phospholipase C-gamma 2 (PLC-gamma 2) and phosphatidylinositol (PI) 3-kinase activation in erythropoietin (Epo)-stimulated FDC-P1 cells transfected with a wild type (WT) Epo-receptor (Epo-R). We showed that two inhibitors of Src kinases, PP1 and PP2, abolish both PLC-gamma 2 tyrosine phosphorylation and PI 3-kinase activity in WT Epo-R FDC-P1 cells. We also demonstrated that Epo-phosphorylated Lyn is associated with tyrosine phosphorylated PLC-gamma 2 and PI 3-kinase in WT Epo-R FDC-P1-stimulated cells. Moreover Epo-activated Lyn phosphorylates in vitro PLC-gamma 2 immunoprecipitated from unstimulated cells. Our results suggest that the Src kinase Lyn is involved in PLC-gamma 2 phosphorylation and PI 3-kinase activation induced by Epo.

Phosphatidylinositol 3-kinase regulates glycosylphosphatidylinositol hydrolysis through PLC-gamma(2) activation in erythropoietin-stimulated cells.

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-gamma2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-gamma2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-gamma2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-gamma2 tyrosine phosphorylation. We also present evidence that PLC-gamma2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-gamma2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-gamma2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-gamma2.