Academic literature on the topic 'Id2 inhibiteur de E2A'

Abstract Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which overcomes the differentiation arrest and induces terminal granulocytic differentiation of the leukemic blasts. In 98% of the cases of APL, the leukemic cells express a promyelocytic leukemia (PML)- retinoic acid receptor a (RARa) fusion protein as a result of a t(15;17) chromosome translocation. Previously, we have identified Id1 and Id2 as direct retinoic acid target genes being upregulated after ATRA stimulation in the APL cell line NB4 as well as in primary leukemic cells from APL patients. Id (inhibitor of DNA-binding) proteins act as antagonists of basic helix-loop-helix (bHLH) transcription factors by trapping them in heterodimeric complexes, thereby inhibiting DNA-binding and gene transactivation. Various bHLH proteins are pivotal in the control of differentiation and proliferation in various tissues (like muscle and nerve). We have studied the expression pattern of E2A, an ubiquitously expressed bHLH protein, which is generally considered as a promiscuous heterodimerization partner of other, more tissue restricted bHLH proteins. The expression of E2A was high in untreated APL cells and strongly downregulated upon ATRA stimulation. The simultaneous upregulation of Id1 and Id2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells upon treatment with ATRA. To assess the importance of Id1 and Id2 induction for the neutrophilic differentiation of the cells, we have overexpressed both proteins in the APL cell line NB4 using amphotropic retroviral transduction. Ectopic expression of Id1 and Id2 resulted in respectively 27% (n=3, SD= 9%) and 48% (n=3, SD=25%) inhibition of clonogenic growth in semi-solid medium, compared to vector-transduced control cells. Apart from the reduction in the number of colonies, overexpression of Id1 and Id2 did not alter the ATRA sensitivity of APL cells. NB4 liquid cultures revealed that Id1 and Id2 overexpression resulted in inhibition of proliferation and an increase of the percentage of cells in G0/G1, without having an effect on differentiation or apoptosis. These results indicate that Id1 and Id2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.

Pax5 activity is enhanced in activated B cells and is essential for class switch recombination (CSR). We show that inhibitor of differentiation (Id)2 suppresses CSR by repressing the gene expression of activation-induced cytidine deaminase (AID), which has been shown to be indispensable for CSR. Furthermore, a putative regulatory region of AID contains E2A- and Pax5-binding sites, and the latter site is indispensable for AID gene expression. Moreover, the DNA-binding activity of Pax5 is decreased in Id2-overexpressing B cells and enhanced in Id2−/− B cells. The kinetics of Pax5, but not E2A, occupancy to AID locus is the same as AID expression in primary B cells. Finally, enforced expression of Pax5 induces AID transcription in pro–B cell lines. Our results provide evidence that the balance between Pax5 and Id2 activities has a key role in AID gene expression.

Abstract The survival, self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC) are tightly regulated by extrinsic signals, and intrinsically by transcription factors and their regulatory networks. The molecular and cellular mechanisms, which regulate the complex process of hematopoiesis, depend upon the correct expression of transcription factors and their regulators. One such family of regulators is the inhibitor of DNA binding/differentiation (Id), which is helix-loop-helix proteins that function by acting as dominant negative regulators of transcription factors such as E proteins, ETS, Pax, and retinoblastoma proteins. Expression of Id2, one of the Id family proteins, is regulated by growth factor independence-1 (Gfi-1) encoding a transcriptional repressor. Gfi-1 is required for the development of multiple cell lineages including HSPC and ultimately differentiated blood cells. Although genes have been identified to mediate hematopoietic defects observed in Gfi-1 knockout (Gfi-1 KO) mice including the maturational and developmental defects in granulocyte (CSF-1, RasGRP1, and PU.1) and B cell (PU.1 or Id2), and myeloid hyperplasia (Id2 or HoxA9), Gfi-1-target genes that mediate the defects in radioprotection, maintenance of HSC, and erythroid hyperplasia in Gfi-1 KO mice are unknown. Since Id2 expression is elevated in HSPC of Gfi-1 KO mice and Id2 promotes cell proliferation, we hypothesized that lowering Id2 expression could rescue the HSPC defects in the Gfi-1 KO mice. By transplanting Gfi-1 KO mouse bone marrow cells (BMC) into lethally-irradiated recipient mice, we observed that short-term reconstituting cell (STRC) activity in Gfi-1 KO BMC is rescued by transplanting Gfi-1 KO; Id2 Het (heterozygosity at the Id2 locus) BMC, while the long-term reconstitution defect of HSC was not. Interestingly, lineage- Sca-1- c-Kithi HPC, which enriched for megakaryocyte-erythroid progenitor (MEP) as one of the STRC, were fully restored in mice transplanted with Gfi-1 KO; Id2 Het BMC, in contrast to lack of the HPC in Gfi-1 KO BM-transplanted mice. The restoration of donor c-Kithi HPC was directly correlated with increased red blood cell (RBC) levels in recipient mice, which was produced after donor BM engraftment. Furthermore, we identified that reduced Id2 levels restore erythroid cell development by rescuing short-term hematopoietic stem cell, common myeloid progenitor and MEP in the Gfi-1 KO mice. In addition, burst forming unit-erythroid (BFU-E) colony assay showed that hemoglobinized BFU-E development was restored in Gfi-1 KO BM and spleen by lowering Id2 levels. Unlike Id2 reduction, reducing other Id family (Id1 or Id3) levels in Gfi-1 KO mice does not rescue the impaired development of erythroid and other hematopoietic lineages including myeloid, T and B cells. Abnormal expansion of CD71+ Ter119-/low erythroid progenitor cells was rescued in Gfi-1 KO; Id2 Het BMC compared to those in Gfi-1 KO mice. Thus, we hypothesized that erythroid development was blocked at the early stage of erythropoiesis due to the ectopic expression of Id2 in Gfi-1 KO mice. Using Id2 promoter-driven YFP reporter mice, we found that Id2 is highly expressed in the CD71+ Ter119-/low erythroid progenitors, and decreases as the cells mature to pro-erythroblasts and erythroblasts, suggesting that repression of Id2 expression is required for proper erythroid differentiation in the later stages. The dramatic changes of Id2 expression during erythroid development support our findings that the overexpression of Id2 in the absence of Gfi-1-mediated transcriptional repression causes impaired erythropoiesis at the early stage. To identify the molecular mechanisms that could account for how reduced Id2 levels rescue erythropoiesis in Gfi-1 KO mice, we compared the expression of genes and proteins in Gfi-1 KO; Id2 Het and Gfi-1 KO BMC. Using microarray, qRT-PCR and western blot, we discovered that reduction of Id2 expression in Gfi-1 KO BMC results in increased expression of Gata1, EKlf, and EpoR genes, which are required for erythropoiesis. However, the expression levels of cell cycle regulators were not altered by lowering Id2 expression in Gfi-1 KO mice. These data suggest a novel molecular mechanism in which Gfi-1 modulates erythropoiesis by repressing the expression of Id2 that reduce the levels of Id2 protein, binding to E2A and inhibiting the formation of E2A/Scl transcription enhancer complex. Disclosures: No relevant conflicts of interest to declare.

ABSTRACT Epstein-Barr virus (EBV) is associated with the development of malignant lymphomas and lymphoproliferative disorders in immunocompromised individuals. The LMP2A protein of EBV is thought to play a central role in this process by allowing the virus to persist in latently infected B lymphocytes. We have demonstrated that LMP2A, when expressed in B cells of transgenic mice, allows normal B-cell developmental checkpoints to be bypassed. To identify cellular genes targeted by LMP2A that are involved in this process, we have utilized DNA microarrays to compare gene transcription in B cells from wild-type versus LMP2A transgenic mice. In B cells from LMP2A transgenic mice, we observed decreased expression of many genes associated with normal B-cell development as well as reduced levels of the transcription factors that regulate their expression. In particular, expression of the transcription factor E2A was down-regulated in bone marrow and splenic B cells. Furthermore, E2A activity was inhibited in these cells as determined by decreased DNA binding and reduced expression of its target genes, including the transcription factors early B-cell factor and Pax-5. Expression of two E2A inhibitors, Id2 and SCL, was up-regulated in splenic B cells expressing LMP2A, suggesting a possible mechanism for E2A inhibition. These results indicate that LMP2A deregulates transcription factor expression and activity in developing B cells, and this likely allows for a bypass of normal signaling events required for proper B-cell development. The ability of LMP2A to interfere with B-cell transcription factor regulation has important implications regarding its role in EBV latency.

Abstract ZNF384 fusion (Z-fusion) genes are recently identified recurrent fusion genes of B-acute lymphoblastic leukemia (ALL) and cause differentiation block of B-cells; however, its molecular mechanisms have yet to be clarified. Common structural character of Z-fusion proteins is that fusion partners are fused to the N-terminal end of full-length ZNF384 (Figure 1A), suggesting that protein-fusions confer specific transcriptional targets on ZNF384. We searched Z-fusion-specific transcriptional targets that could cause differentiation block of B-cells by analyzing the data of gene expression profile of 54 primary B-ALL samples containing 9 Z-fusion positive ALL. We selected ID2 and SALL4 as potential targets. Both genes were expressed markedly higher in Z-fusion-positive ALL. ID2 acts as an inhibitor of E2A, B cell differentiation regulator, and SALL4 plays essential roles in maintaining pluripotency of embryonic stem cells. In the luciferase assays, EP300-ZNF384 (E-Z) and SYNRG-ZNF384 (S-Z) showed stronger transcriptional activities on the promoters of these genes than wild-type ZNF384 (Wild-Z). The introduction of E-Z or S-Z into 293T cells and THP-1 cells induced mRNA expression of these genes more strongly than that of Wild-Z (Figure 1B). We identified Z-fusion binding sites in the promoters of these genes. DNA binding abilities of Z-fusions to these sites were not stronger than that of Wild-Z in electro mobility shift assay. GST-pull down assay showed that E-Z associated with EP300 more strongly than Wild-Z (Figure 1C). Consistent with this, co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z (Figure 1D). These results indicated that ID2 and SALL4 were Z-fusion-specific transcriptional targets and that the high affinity to EP300 was responsible for the strong transcriptional activity of Z-fusions. Our results shed a new insight into the molecular mechanisms of leukemia development by Z-fusions. Figure legends Figure 1. A. Schematic presentation of structures of Wild-Z and Z-fusion proteins. B. Introduction of Z-fusion genes enhanced the mRNA expression of ID2 and SALL4. Wild-Z and Z-fusion genes were introduced to THP-1 cells by nucleofection. Twenty-four hours after gene introduction, the mRNA expression of ID2 and SALL4 were quantified by RQ-PCR and potted on bar charts as relative values to the mRNA expression in the control cells. The expression of ID2 and SALL4 were shown in the left and right bar charts, respectively. C. E-Z associated with HAT more strongly than Wild-Z. Glutathione beads attached with Glutathione S transferase (GST) or GST-fused HAT were incubated with Wild-Z or E-Z synthesized in vitro with [35S]-Methionine labeling. Wild-Z or E-Z associated with GST-HAT were visualized with autoradiography (left panel), quantified, and plotted on the bar charts (right panel). Of note, the quantified association were adjusted for the ratio of the number of methionine containing in Wild-Z and E-Z, 12 to 45, and plotted on the bar charts. D. Co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z. Luciferase assay were performed with or without co-expression of EP300 and the relative value to the control were plotted on the bar charts. Disclosures Naoe: Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding. Kiyoi:FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding.

The Id proteins and basic helix-loop-helix (bHLH) proteins play major roles in specifying cell fate decisions in diverse biologic settings. A potential role for Id and TAL1/E2A bHLH genes in hematopoiesis has been suggested by studies on immortalized cell lines. However, it is uncertain whether these observations reflect normal hematopoiesis. We have investigated the expression pattern of Id2 and TAL1/E2A genes in liquid suspension culture of purified hematopoietic progenitor cell (HPCs) undergoing erythroid or granulopoietic differentiation in the first culture week and maturation to terminal cells in the second week. In quiescent, freshly purified HPCs, Id2 mRNA is detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas TAL1 and E2A mRNAs are not. At the onset of erythroid differentiation, Id2 mRNA is downregulated, while E2A and TAL1 mRNAs are concomitantly upregulated: their expression is further increased at erythroblast level. Conversely, Id2 is not downmodulated in granulopoietic culture, except for a late decline at day 10 to 12, while TAL1 and E2A are only transiently induced in the first week of granulopoietic differentiation. The expression pattern of the TAL1/E2A heterodimer, as evaluated by mobility shift assay, is consistent with RT-PCR results (except for lower levels of the heterodimer in late erythroid maturation). TAL1 protein level, analyzed by Western blot, shows a pattern consistent with gelshift results. Functional experiments were performed on purified HPCs treated with phosphorothioate antisense oligodeoxynucleotides to Id2 or TAL1 mRNA. The results are strictly consistent with the expression studies: anti-Id2 oligomer (alpha-Id2) causes a significant dose-dependent increase of erythroid colony formation, whereas alpha-TAL1 induces a selective dose-related inhibitory effect on erythroid colonies, as compared with untreated or scrambled oligomer-treated control HPCs. Finally, murine and human glutathione-S-transferase (GST)-Id2 polypeptides compete the TAL1/E2A- specific DNA binding activity when added to the nuclear extracts derived from erythroid culture cells, thus indicating biochemical and suggesting functional interaction of Id2 with the TAL1/E2A complex. These novel observations indicate a coordinate expression and function of an inhibitory Id protein (Id2) and a stimulatory bHLH/bHLH heterodimer (TAL1/E2A) in normal erythroid differentiation.

The Id2 transcriptional repressor is essential for development of natural killer (NK) cells, lymphoid tissue–inducing (LTi) cells, and secondary lymphoid tissues. Id2 was proposed to regulate NK and LTi lineage specification from multipotent progenitors through suppression of E proteins. We report that NK cell progenitors are not reduced in the bone marrow (BM) of Id2−/− mice, demonstrating that Id2 is not essential for NK lineage specification. Rather, Id2 is required for development of mature (m) NK cells. We define the mechanism by which Id2 functions by showing that a reduction in E protein activity, through deletion of E2A, overcomes the need for Id2 in development of BM mNK cells, LTi cells, and secondary lymphoid tissues. However, mNK cells are not restored in the blood or spleen of Id2−/−E2A−/− mice, suggesting a role for Id2 in suppression of alternative E proteins after maturation. Interestingly, the few splenic mNK cells in Id2−/− and Id2−/−E2A−/− mice have characteristics of thymus-derived NK cells, which develop in the absence of Id2, implying a differential requirement for Id2 in BM and thymic mNK development. Our findings redefine the essential functions of Id2 in lymphoid development and provide insight into the dynamic regulation of E and Id proteins during this process.

Abstract Inhibitors of DNA binding (Id) family members are key regulators of cellular differentiation and proliferation. These activities are related to the ability of Id proteins to antagonize E proteins and other transcription factors. As negative regulators of E proteins, Id proteins have been implicated in lymphocyte development. Overexpression of Id1, Id2, or Id3 has similar effects on lymphocyte development. However, which Id protein plays a physiologic role during lymphocyte development is not clear. By analyzing Id2 knock-out mice and retroviral transduced hematopoietic progenitors, we demonstrated that Id2 is an intrinsic negative regulator of B-cell development. Hematopoietic progenitor cells overexpressing Id2 did not reconstitute B-cell development in vivo, which resembled the phenotype of E2A null mice. The B-cell population in bone marrow was significantly expanded in Id2 knock-out mice compared with their wild-type littermates. Knock-down of Id2 by shRNA in hematopoietic progenitor cells promoted B-cell differentiation and induced the expression of B-cell lineage–specific genes. These data identified Id2 as a physiologically relevant regulator of E2A during B lymphopoiesis. Furthermore, we identified a novel Id2 function in erythroid development. Overexpression of Id2 enhanced erythroid development, and decreased level of Id2 impaired normal erythroid development. Id2 regulation of erythroid development is mediated via interacting with transcription factor PU.1 and modulating PU.1 and GATA-1 activities. We conclude that Id2 regulates lymphoid and erythroid development via interaction with different target proteins.

The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators which function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. We report here that nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. We further show that disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life.

Key Points Loss of Id2 in T cells results in overexpression of IL-10 during influenza infection and GVHD and protects against GVHD immunopathology. Id2 represses the direct E2A-mediated activation of the Il10 locus in effector T cells.

L’herpèsvirus humain de type 6 (HHV-6) au caractère lymphotropique présente deux variants A et B, ce dernier étant associé au lymphome de Hodgkin (LH). Toutefois, cette association est controversée dans la littérature car elle est présente dans de rares cas et absente dans la cellule caractéristique du LH : la cellule de Reed-Sternberg (RS). Lors du premier objectif de notre travail de thèse, la PCR quantitative a démontre�� l��existence et a quantifié le virus HHV-6 au sein d’une cohorte de patients atteints de LH; ceci de façon concomitante ou non au virus d’Epstein et Barr (EBV) connu pour être associé à cette pathologie. Par la suite, l’anticorps anti-DR7B de l’HHV-6 produit par nos soins a permis de connaître la localisation cellulaire du virus HHV-6 et ainsi de détecter sa présence en grande quantité dans la cellule RS pour 73,7% des patients atteints de LH seulement positifs pour l’HHV-6. En outre, un double marquage chez les patients de la cohorte positifs pour les deux herpèsvirus a démontré la colocalisation, notamment au niveau de la cellule RS, des oncoprotéines DR7B du virus HHV-6 et LMP1 du virus EBV signifiant une probable interaction virale. Enfin, un système de transfection stable permettant de moduler l’expression de DR7B au sein de cellules B matures proches des cellules RS a été réalisé. Il a permis d’observer l’induction, en présence de DR7B, de la surexpression tant au niveau transcriptionnel que traductionnel, comme au sein des cellules RS du LH, de Id2 qui est un inhibiteur de E2A, facteur de transcription impliqué dans la reprogrammation phénotypique des cellules RS
There are two variants, A and B, of human lymphotropic herpesvirus type 6 (HHV-6). The B variant is associated with Hodgkin’s lymphoma (HL). However this association is contreversial in the literature because it only exists in rare cases and not in Reed-Sternberg cells (RS), the characteristic cells of HL. In the first part of our study quantitative PCR demonstrated the existence of HHV-6 alone or concommitantly with Epstein-Barr virus, known to be associated with HL, in a cohort of HL patients. We then used an anti-HHV-6 DR7B antibody produced in our laboratory to determine the cellular localization of HHV-6 and to detect its presence in large quantities in RS cells from 73. 7% of HL patients positive only for HHV-6. Furthermore, double labeling studies in patients from the cohort positive for both herpesviruses demonstrated the colocalization, notably in RS cells, of DR7B from HHV-6 and LMP1 from EBV oncoproteins, indicating a probable viral interaction. Finally, a stable transfection system was realised to adjust DR7B expression in mature B cells which are close to RS cells. In the presence of DR7B, a transcriptionnal and translational expression of Id2, an inhibitor of E2A wich is a transcription factor implicated in phenotypical reprogramming of RS cells, similar to RS cells in HL, was observed