SUMMARY PARAGRAPHDisease-causing mutations in genes encoding transcription factors (TFs) are a re... more SUMMARY PARAGRAPHDisease-causing mutations in genes encoding transcription factors (TFs) are a recurrent finding in hematopoietic malignancies and might involve key regulators of lineage adherence and cellular differentiation1–3. Such mutations can affect TF-interactions with their cognate DNA-binding motifs4, 5. Whether and how TF-mutations impact upon the nature of binding to TF composite elements (CE) and influence their interaction with other TFs is unclear. Here, we report a new mechanism of TF alteration in human lymphomas with perturbed B cell identity. It is caused by a recurrent somatic missense mutation c.295T>C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cell-differentiation and -activation6, 7. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF composite elements (CEs). Further...
Hematopoietic stem cells (HSCs) continually decide to either self-renew or differentiate. In this... more Hematopoietic stem cells (HSCs) continually decide to either self-renew or differentiate. In this study we explore the role of lineage-specific transcription factors in HSC cell fate decisions, in particular Early B cell Factor 1 (EBF1) which orchestrates the B cell lineage program and is essential for B cell differentiation. Here, we use Ebf1flox/floxTie2Cre (Ebf1KO) mice to examine its potential role in hematopoietic stem and progenitor cells (HSPCs). We find that Ebf1KO mice display an increased number of HSCs that are less quiescent. Upon chronic 5-fluorouracil treatment, Ebf1KO mice show reduced survival as compared to wild type (WT) mice, suggesting that Ebf1 helps to maintain HSC quiescence. Additionally, competitive adoptive transfers of Ebf1KO HSCs into irradiated WT mice show a decrease in chimerism in both primary and secondary recipients compared to WT HSCs, indicating that Ebf1 is required for HSC self-renewal capacity. Ebf1KO mice also reveal a significant expansion of myeloid-biased (CD41+) HSCs, myeloid-biased multipotent progenitors (MPP2/3) and mature myeloid cells. B cell-deficient IgM-/- mice do not show an increase in HSC number, loss of quiescence nor loss of chimerism in adoptive transfer experiments, therefore the observed phenotypes in Ebf1KO mice are specific to Ebf1 and a secondary effect of the loss of B cells. Additionally, we find that Ebf1KO HSPCs are not marked by DJ recombination demonstrating that the phenotypes of Ebf1 KO mice are not due to the dedifferentiation of B cells. These data indicate that the loss of Ebf1 in HSPCs results in a myeloid-biased output and that Ebf1 has an unexpected additional function in the regulation of HSC self-renewal and differentiation.
Recent breakthroughs in single cell technology have delivered maps of cell states together with t... more Recent breakthroughs in single cell technology have delivered maps of cell states together with their differentiation journeys at unprecedented resolution and scale. However, despite great progress in network inference methods, our understanding of molecular networks governing differentiation is limited due to the complexity and lack of systematic, functional data. In this study, we demonstrate a scalable approach to functionally define gene networks using genetic perturbations (CRISPR/Cas9) followed by transcriptional profiling. We utilise a lympho-myeloid progenitor model (Hoxb8-FL) to investigate mechanisms maintaining the propensity to differentiate into multiple lineages. In these cells, we built a network spanning ∼17,000 interactions between 39 transcriptions factors (TFs) and their target genes. As functional interpretation of gene perturbations is challenging, we provide a new method (DoT score) which visualises state transitions using scRNA-Seq landscapes as references, offering an alternative to Gene Ontology analysis. Furthermore, in-depth analysis of the network revealed how several lineage-associated TFs cooperate to establish lymphoid and myeloid transcription programmes. We find that myeloid gene expression is activated by Cebpa, but is kept in check by enforced expression of Hoxb8 and its downstream regulators Meis1 and Hoxa9. In parallel, Ebf1 is essential for a proper balance of cell growth and proliferation and, consistently with its established role, promotes the B cell expression programme. Conversely, B-cell differentiation is counteracted by Gata3, which efficiently blocks Pax5 expression. Altogether, our work unravels a core TF circuit, simultaneously activating several lineage programmes but wired specifically to prevent any lineage progression. Molecularly defined regulatory networks such as the one reported here may be utilised for targeted modulation of cell states, with broad applicability for the production of desired cells through targeted differentiation, as well as resetting abnormal cellular states in haematopoietic malignancies.
<jats:title>Abstract</jats:title> <jats:p>Hematopoietic stem and progenitor cel... more <jats:title>Abstract</jats:title> <jats:p>Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis.</jats:p> <jats:p>Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec>
The establishment of de novo chromatin accessibility in lymphoid progenitors requires the “pionee... more The establishment of de novo chromatin accessibility in lymphoid progenitors requires the “pioneering” function of transcription factor (TF) early B cell factor 1 (EBF1), which binds to naïve chromatin and induces accessibility by recruiting the BRG1 chromatin remodeler subunit. However, it remains unclear whether the function of EBF1 is continuously required for stabilizing local chromatin accessibility. To this end, we replaced EBF1 by EBF1-FKBP F36V in pro-B cells, allowing the rapid degradation by adding the degradation TAG13 (dTAG13) dimerizer. EBF1 degradation results in a loss of genome-wide EBF1 occupancy and EBF1-targeted BRG1 binding. Chromatin accessibility was rapidly diminished at EBF1-binding sites with a preference for sites whose occupancy requires the pioneering activity of the C-terminal domain of EBF1. Diminished chromatin accessibility correlated with altered gene expression. Thus, continuous activity of EBF1 is required for the stable maintenance of the transcri...
MZB1 is an endoplasmic reticulum (ER)-resident protein that plays an important role in the humora... more MZB1 is an endoplasmic reticulum (ER)-resident protein that plays an important role in the humoral immune response by enhancing the interaction of the μ immunoglobulin (Ig) heavy chain with the chaperone GRP94 and by augmenting the secretion of IgM. Here, we show that MZB1 is also expressed in plasmacytoid dendritic cells (pDCs). Mzb1−/− pDCs have a defect in the secretion of interferon (IFN) α upon Toll-like receptor (TLR) 9 stimulation and a reduced ability to enhance B cell differentiation towards plasma cells. Mzb1−/− pDCs do not properly expand the ER upon TLR9 stimulation, which may be accounted for by an impaired activation of ATF6, a regulator of the unfolded protein response (UPR). Pharmacological inhibition of ATF6 cleavage in stimulated wild type pDCs mimics the diminished IFNα secretion by Mzb1−/− pDCs. Thus, MZB1 enables pDCs to secrete high amounts of IFNα by mitigating ER stress via the ATF6-mediated UPR.
The establishment of cell fates involves alterations of transcription factor repertoires and repu... more The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451−/− ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog. The differentiation defec...
Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) generate all cells of the bloo... more Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) generate all cells of the blood system. Despite their multipotency, MPPs display poorly understood lineage bias. Here, we examine whether lineage-specifying transcription factors, such as the B-lineage determinant EBF1, regulate lineage preference in early progenitors. We detect low-level EBF1 expression in myeloid-biased MPP3 and lymphoid-biased MPP4 cells, coinciding with expression of the myeloid determinant C/EBPα. Hematopoietic deletion of Ebf1 results in enhanced myelopoiesis and reduced HSC repopulation capacity. Ebf1-deficient MPP3 and MPP4 cells exhibit an augmented myeloid differentiation potential and a transcriptome with an enriched C/EBPα signature. Correspondingly, EBF1 binds the Cebpa enhancer, and the deficiency and overexpression of Ebf1 in MPP3 and MPP4 cells lead to an up- and downregulation of Cebpa expression, respectively. In addition, EBF1 primes the chromatin of B-lymphoid enhancers specifical...
Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification... more Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of Ebf1−/− progenitors. By RNA-seq analysis of EBF1E271A-expressing progenitors, we found an up-regulation of T lineage determinants and down-regulation of early B genes, although similar chromatin binding of EBF1E271A and EBF1wt was detected in pro-B cells expressing activated Notch1. B lineage-specific inactivation of Tnpo3 in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions.
Marginal zone (MZ) B cells represent innate-like B cells that mediate a fast immune response. The... more Marginal zone (MZ) B cells represent innate-like B cells that mediate a fast immune response. The adhesion of MZ B cells to the marginal sinus of the spleen is governed by integrins. Here, we address the question of whether β1-integrin has additional functions by analyzing Itgb1fl/flCD21Cre mice in which the β1-integrin gene is deleted in mature B cells. We find that integrin β1–deficient mice have a defect in the differentiation of MZ B cells and plasma cells. We show that integrin β1–deficient transitional B cells, representing the precursors of MZ B cells, have enhanced B cell receptor (BCR) signaling, altered PI3K and Ras/ERK pathways, and an enhanced interaction of integrin-linked kinase (ILK) with the adaptor protein Grb2. Moreover, the MZ B cell defect of integrin β1–deficient mice could, at least in part, be restored by a pharmacological inhibition of the PI3K pathway. Thus, β1-integrin has an unexpected function in the differentiation and function of MZ B cells.
SUMMARY PARAGRAPHDisease-causing mutations in genes encoding transcription factors (TFs) are a re... more SUMMARY PARAGRAPHDisease-causing mutations in genes encoding transcription factors (TFs) are a recurrent finding in hematopoietic malignancies and might involve key regulators of lineage adherence and cellular differentiation1–3. Such mutations can affect TF-interactions with their cognate DNA-binding motifs4, 5. Whether and how TF-mutations impact upon the nature of binding to TF composite elements (CE) and influence their interaction with other TFs is unclear. Here, we report a new mechanism of TF alteration in human lymphomas with perturbed B cell identity. It is caused by a recurrent somatic missense mutation c.295T>C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cell-differentiation and -activation6, 7. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF composite elements (CEs). Further...
Hematopoietic stem cells (HSCs) continually decide to either self-renew or differentiate. In this... more Hematopoietic stem cells (HSCs) continually decide to either self-renew or differentiate. In this study we explore the role of lineage-specific transcription factors in HSC cell fate decisions, in particular Early B cell Factor 1 (EBF1) which orchestrates the B cell lineage program and is essential for B cell differentiation. Here, we use Ebf1flox/floxTie2Cre (Ebf1KO) mice to examine its potential role in hematopoietic stem and progenitor cells (HSPCs). We find that Ebf1KO mice display an increased number of HSCs that are less quiescent. Upon chronic 5-fluorouracil treatment, Ebf1KO mice show reduced survival as compared to wild type (WT) mice, suggesting that Ebf1 helps to maintain HSC quiescence. Additionally, competitive adoptive transfers of Ebf1KO HSCs into irradiated WT mice show a decrease in chimerism in both primary and secondary recipients compared to WT HSCs, indicating that Ebf1 is required for HSC self-renewal capacity. Ebf1KO mice also reveal a significant expansion of myeloid-biased (CD41+) HSCs, myeloid-biased multipotent progenitors (MPP2/3) and mature myeloid cells. B cell-deficient IgM-/- mice do not show an increase in HSC number, loss of quiescence nor loss of chimerism in adoptive transfer experiments, therefore the observed phenotypes in Ebf1KO mice are specific to Ebf1 and a secondary effect of the loss of B cells. Additionally, we find that Ebf1KO HSPCs are not marked by DJ recombination demonstrating that the phenotypes of Ebf1 KO mice are not due to the dedifferentiation of B cells. These data indicate that the loss of Ebf1 in HSPCs results in a myeloid-biased output and that Ebf1 has an unexpected additional function in the regulation of HSC self-renewal and differentiation.
Recent breakthroughs in single cell technology have delivered maps of cell states together with t... more Recent breakthroughs in single cell technology have delivered maps of cell states together with their differentiation journeys at unprecedented resolution and scale. However, despite great progress in network inference methods, our understanding of molecular networks governing differentiation is limited due to the complexity and lack of systematic, functional data. In this study, we demonstrate a scalable approach to functionally define gene networks using genetic perturbations (CRISPR/Cas9) followed by transcriptional profiling. We utilise a lympho-myeloid progenitor model (Hoxb8-FL) to investigate mechanisms maintaining the propensity to differentiate into multiple lineages. In these cells, we built a network spanning ∼17,000 interactions between 39 transcriptions factors (TFs) and their target genes. As functional interpretation of gene perturbations is challenging, we provide a new method (DoT score) which visualises state transitions using scRNA-Seq landscapes as references, offering an alternative to Gene Ontology analysis. Furthermore, in-depth analysis of the network revealed how several lineage-associated TFs cooperate to establish lymphoid and myeloid transcription programmes. We find that myeloid gene expression is activated by Cebpa, but is kept in check by enforced expression of Hoxb8 and its downstream regulators Meis1 and Hoxa9. In parallel, Ebf1 is essential for a proper balance of cell growth and proliferation and, consistently with its established role, promotes the B cell expression programme. Conversely, B-cell differentiation is counteracted by Gata3, which efficiently blocks Pax5 expression. Altogether, our work unravels a core TF circuit, simultaneously activating several lineage programmes but wired specifically to prevent any lineage progression. Molecularly defined regulatory networks such as the one reported here may be utilised for targeted modulation of cell states, with broad applicability for the production of desired cells through targeted differentiation, as well as resetting abnormal cellular states in haematopoietic malignancies.
<jats:title>Abstract</jats:title> <jats:p>Hematopoietic stem and progenitor cel... more <jats:title>Abstract</jats:title> <jats:p>Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis.</jats:p> <jats:p>Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec>
The establishment of de novo chromatin accessibility in lymphoid progenitors requires the “pionee... more The establishment of de novo chromatin accessibility in lymphoid progenitors requires the “pioneering” function of transcription factor (TF) early B cell factor 1 (EBF1), which binds to naïve chromatin and induces accessibility by recruiting the BRG1 chromatin remodeler subunit. However, it remains unclear whether the function of EBF1 is continuously required for stabilizing local chromatin accessibility. To this end, we replaced EBF1 by EBF1-FKBP F36V in pro-B cells, allowing the rapid degradation by adding the degradation TAG13 (dTAG13) dimerizer. EBF1 degradation results in a loss of genome-wide EBF1 occupancy and EBF1-targeted BRG1 binding. Chromatin accessibility was rapidly diminished at EBF1-binding sites with a preference for sites whose occupancy requires the pioneering activity of the C-terminal domain of EBF1. Diminished chromatin accessibility correlated with altered gene expression. Thus, continuous activity of EBF1 is required for the stable maintenance of the transcri...
MZB1 is an endoplasmic reticulum (ER)-resident protein that plays an important role in the humora... more MZB1 is an endoplasmic reticulum (ER)-resident protein that plays an important role in the humoral immune response by enhancing the interaction of the μ immunoglobulin (Ig) heavy chain with the chaperone GRP94 and by augmenting the secretion of IgM. Here, we show that MZB1 is also expressed in plasmacytoid dendritic cells (pDCs). Mzb1−/− pDCs have a defect in the secretion of interferon (IFN) α upon Toll-like receptor (TLR) 9 stimulation and a reduced ability to enhance B cell differentiation towards plasma cells. Mzb1−/− pDCs do not properly expand the ER upon TLR9 stimulation, which may be accounted for by an impaired activation of ATF6, a regulator of the unfolded protein response (UPR). Pharmacological inhibition of ATF6 cleavage in stimulated wild type pDCs mimics the diminished IFNα secretion by Mzb1−/− pDCs. Thus, MZB1 enables pDCs to secrete high amounts of IFNα by mitigating ER stress via the ATF6-mediated UPR.
The establishment of cell fates involves alterations of transcription factor repertoires and repu... more The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451−/− ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog. The differentiation defec...
Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) generate all cells of the bloo... more Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) generate all cells of the blood system. Despite their multipotency, MPPs display poorly understood lineage bias. Here, we examine whether lineage-specifying transcription factors, such as the B-lineage determinant EBF1, regulate lineage preference in early progenitors. We detect low-level EBF1 expression in myeloid-biased MPP3 and lymphoid-biased MPP4 cells, coinciding with expression of the myeloid determinant C/EBPα. Hematopoietic deletion of Ebf1 results in enhanced myelopoiesis and reduced HSC repopulation capacity. Ebf1-deficient MPP3 and MPP4 cells exhibit an augmented myeloid differentiation potential and a transcriptome with an enriched C/EBPα signature. Correspondingly, EBF1 binds the Cebpa enhancer, and the deficiency and overexpression of Ebf1 in MPP3 and MPP4 cells lead to an up- and downregulation of Cebpa expression, respectively. In addition, EBF1 primes the chromatin of B-lymphoid enhancers specifical...
Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification... more Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of Ebf1−/− progenitors. By RNA-seq analysis of EBF1E271A-expressing progenitors, we found an up-regulation of T lineage determinants and down-regulation of early B genes, although similar chromatin binding of EBF1E271A and EBF1wt was detected in pro-B cells expressing activated Notch1. B lineage-specific inactivation of Tnpo3 in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions.
Marginal zone (MZ) B cells represent innate-like B cells that mediate a fast immune response. The... more Marginal zone (MZ) B cells represent innate-like B cells that mediate a fast immune response. The adhesion of MZ B cells to the marginal sinus of the spleen is governed by integrins. Here, we address the question of whether β1-integrin has additional functions by analyzing Itgb1fl/flCD21Cre mice in which the β1-integrin gene is deleted in mature B cells. We find that integrin β1–deficient mice have a defect in the differentiation of MZ B cells and plasma cells. We show that integrin β1–deficient transitional B cells, representing the precursors of MZ B cells, have enhanced B cell receptor (BCR) signaling, altered PI3K and Ras/ERK pathways, and an enhanced interaction of integrin-linked kinase (ILK) with the adaptor protein Grb2. Moreover, the MZ B cell defect of integrin β1–deficient mice could, at least in part, be restored by a pharmacological inhibition of the PI3K pathway. Thus, β1-integrin has an unexpected function in the differentiation and function of MZ B cells.
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