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EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene.

ABSTRACT: Genes encoding B lineage-restricted transcription factors are frequently mutated in B-lymphoid leukemias, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is early B-cell factor 1 (EBF1), a protein of critical importance for lineage specification and survival of B-lymphoid progenitors. Here, we report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC partially rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC-sequencing, chromatin immunoprecipitation-sequencing, and reporter gene assays, six EBF1-responsive enhancer elements were identified within the Myc locus. CRISPR-Cas9-mediated targeting of EBF1-binding sites identified one element of key importance for Myc expression and pro-B cell expansion. These data provide evidence that Myc is a direct target of EBF1. Furthermore, chromatin immunoprecipitation-sequencing analysis revealed that several regulatory elements in the Myc locus are targets of PAX5. However, ectopic expression of PAX5 in EBF1-deficient cells inhibits the cell cycle and reduces Myc expression, suggesting that EBF1 and PAX5 act in an opposing manner to regulate Myc levels. This hypothesis is further substantiated by the finding that Pax5 inactivation reduces requirements for EBF1 in pro-B-cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-cell acute lymphoblastic leukemia cell line indicates that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development.

SUBMITTER: Somasundaram R

PROVIDER: S-EPMC8176764 | biostudies-literature |

REPOSITORIES: biostudies-literature

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EBF1 and PAX5 control pro-B cell expansion via oppose regulation of the Myc gene

Project description:Lineage restricted transcription factors are frequently found mutated in B-lymphoid leukemia, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is EBF1, a protein of critical importance for specification but also survival of B-lymphoid progenitors. We here report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC, ChIP-seq and reporter gene assays, identified six EBF responsive enhancer elements that could be annotated to the Myc gene. Crispr-Cas9 mediated targeting of EBF1 binding sites identified one element of importance for Myc expression and pro-B cell expansion providing evidence for that Myc is a direct target for EBF1. Furthermore, ChIP-seq analysis suggested that several regulatory elements in the Myc gene were targeted by PAX5. However, ectopic expression of PAX5 in EBF1 deficient cells was found to induce a cell cycle block and reduce Myc expression suggesting that EBF1 and PAX5 act in an opposing manner at this shared target gene. This hypothesis was further substantiated by the finding that Pax5 inactivation reduced the need for EBF1 in pro-B cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-ALL cell line indicate that this regulatory loop may be of relevance for our understanding of both normal and malignant B-cell development.

2021-03-05 | GSE159957 | GEO

EBF1 and PAX5 control pro-B cell expansion via inverse regulation of the c-Myc gene.

Project description:EBF1 and PAX5 are two transcription factors targeted in human B-ALL. Their importance for normal B-cell differentiation has led to the suggestion that impaired function of these proteins contributes to the developmental block observed in B-lineage leukemia. However, increased understanding of the function of these proteins has suggested more complex roles in malignant transformation. We here report that the loss of EBF1 function in B-cell progenitors results in reduced expression of c-MYC. Ectopic expression of c-MYC rescues B-cell expansion in the absence of EBF1 both in vivo and in vitro and ChIP-seq analysis identifies multiple binding sites for EBF1 in putative regulatory elements of the mouse c-Myc gene. On the contrary, expression of PAX5 in EBF1 deficient cells resulted in down regulation of c-Myc and inhibition of cell expansion. Since both EBF1 and PAX5 bind multiple regulatory elements in the c-Myc gene, these data suggest that EBF1 and PAX5 have opposing functions with regard to regulation of c-Myc expression. This suggests the existence of a lineage restricted regulatory loop controlling the expansion of B-lineage progenitors. The binding of EBF1 and PAX5 to multiple regulatory elements in the human c-MYC gene indicate that this regulatory loop may be of relevance for our understanding of leukemia formation.

2019-11-30 | GSE136238 | GEO

EBF1 and PAX5 control pro-B cell expansion via oppose regulation of the Myc gene

Project description:EBF1 and PAX5 control pro-B cell expansion via oppose regulation of the Myc gene

| PRJNA670949 | ENA

EBF1 and PAX5 control pro-B cell expansion via inverse regulation of the c-Myc gene.

Project description:EBF1 and PAX5 control pro-B cell expansion via inverse regulation of the c-Myc gene.

| PRJNA561711 | ENA

Distinct promoters mediate the regulation of Ebf1 gene expression by interleukin-7 and Pax5.

Project description:Early differentiation of B lymphocytes requires the function of multiple transcription factors that regulate the specification and commitment of the lineage. Loss- and gain-of-function experiments have provided important insight into the transcriptional control of B lymphopoiesis, whereby E2A was suggested to act upstream of EBF1 and Pax5 downstream of EBF1. However, this simple hierarchy cannot account for all observations, and our understanding of a presumed regulatory network, in which transcription factors and signaling pathways operate, is limited. Here, we show that the expression of the Ebf1 gene involves two promoters that are differentially regulated and generate distinct protein isoforms. We find that interleukin-7 signaling, E2A, and EBF1 activate the distal Ebf1 promoter, whereas Pax5, together with Ets1 and Pu.1, regulates the stronger proximal promoter. In the absence of Pax5, the function of the proximal Ebf1 promoter and accumulation of EBF1 protein are impaired and the replication timing and subcellular localization of the Ebf1 locus are altered. Taken together, these data suggest that the regulation of Ebf1 via distinct promoters allows for the generation of several feedback loops and the coordination of multiple determinants of B lymphopoiesis in a regulatory network.

| S-EPMC1800812 | biostudies-other

EBF1 and Pax5 safeguard leukemic transformation by limiting IL-7 signaling, Myc expression, and folate metabolism.

Project description:EBF1 and PAX5 mutations are associated with the development of B progenitor acute lymphoblastic leukemia (B-ALL) in humans. To understand the molecular networks driving leukemia in the Ebf1+/-Pax5+/- (dHet) mouse model for B-ALL, we interrogated the transcriptional profiles and chromatin status of leukemic cells, preleukemic dHet pro-B, and wild-type pro-B cells with the corresponding EBF1 and Pax5 cistromes. In dHet B-ALL cells, many EBF1 and Pax5 target genes encoding pre-BCR signaling components and transcription factors were down-regulated, whereas Myc and genes downstream from IL-7 signaling or associated with the folate pathway were up-regulated. We show that blockade of IL-7 signaling in vivo and methotrexate treatment of leukemic cells in vitro attenuate the expansion of leukemic cells. Single-cell RNA-sequencing revealed heterogeneity of leukemic cells and identified a subset of wild-type pro-B cells with reduced Ebf1 and enhanced Myc expression that show hallmarks of dHet B-ALL cells. Thus, EBF1 and Pax5 may safeguard early stage B cells from transformation to B-ALL by limiting IL-7 signaling, folate metabolism and Myc expression.

| S-EPMC7608749 | biostudies-literature

Ebf1 heterozygosity results in increased DNA damage in pro-B cells and their synergistic transformation by Pax5 haploinsufficiency.

Project description:Early B-cell factor 1 (Ebf1) is a transcription factor with documented dose-dependent functions in normal and malignant B-lymphocyte development. To understand more about the roles of Ebf1 in malignant transformation, we investigated the impact of reduced functional Ebf1 dosage on mouse B-cell progenitors. Gene expression analysis suggested that Ebf1 was involved in the regulation of genes important for DNA repair and cell survival. Investigation of the DNA damage in steady state, as well as after induction of DNA damage by UV light, confirmed that pro-B cells lacking 1 functional allele of Ebf1 display signs of increased DNA damage. This correlated to reduced expression of DNA repair genes including Rad51, and chromatin immunoprecipitation data suggested that Rad51 is a direct target for Ebf1. Although reduced dosage of Ebf1 did not significantly increase tumor formation in mice, a dramatic increase in the frequency of pro-B cell leukemia was observed in mice with combined heterozygous mutations in the Ebf1 and Pax5 genes, revealing a synergistic effect of combined dose reduction of these proteins. Our data suggest that Ebf1 controls DNA repair in a dose-dependent manner providing a possible explanation to the frequent involvement of EBF1 gene loss in human leukemia.

| S-EPMC4481594 | biostudies-literature

Aryl Hydrocarbon Receptor Activation Suppresses EBF1 and PAX5 and Impairs Human B Lymphopoiesis.

Project description:Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates biological responses to endogenous and environmental chemical cues. Increasing evidence shows that the AHR plays physiological roles in regulating development, homeostasis, and function of a variety of cell lineages in the immune system. However, the role of AHR in human B cell development has not been investigated. Toward this end, an in vitro feeder-free human B cell developmental model system was employed using human cord blood CD34+ hematopoietic stem/progenitor cells. Using this model, we found that AHR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly suppressed the generation of early B cells and pro-B cells from hematopoietic stem/progenitor cells, indicating the impairment of B cell lineage specification and commitment. Addition of an AHR antagonist reversed 2,3,7,8-tetrachlorodibenzo-p-dioxin-elicited suppression of early B and pro-B cells, suggesting a role of AHR in regulating B lymphopoiesis. Gene expression analysis revealed a significant decrease in the messenger RNA level of early B cell factor 1 (EBF1) and paired box 5, two critical transcription factors directing B cell lineage specification and commitment. Additionally, binding of the ligand-activated AHR to the putative dioxin response elements in the EBF1 promoter was demonstrated by EMSAs and chromatin immunoprecipitation analysis, suggesting transcriptional regulation of EBF1 by AHR. Taken together, this study demonstrates a role for the AHR in regulating human B cell development, and it suggests that transcriptional alterations of EBF1 by the AHR are involved in the underlying mechanism.

| S-EPMC5679724 | biostudies-literature

Ebf1 or Pax5 haploinsufficiency synergizes with STAT5 activation to initiate acute lymphoblastic leukemia.

Project description:As STAT5 is critical for the differentiation, proliferation, and survival of progenitor B cells, this transcription factor may play a role in acute lymphoblastic leukemia (ALL). Here, we show increased expression of activated signal transducer and activator of transcription 5 (STAT5), which is correlated with poor prognosis, in ALL patient cells. Mutations in EBF1 and PAX5, genes critical for B cell development have also been identified in human ALL. To determine whether mutations in Ebf1 or Pax5 synergize with STAT5 activation to induce ALL, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice heterozygous for Ebf1 or Pax5. Haploinsufficiency of either Pax5 or Ebf1 synergized with Stat5b-CA to rapidly induce ALL in 100% of the mice. The leukemic cells displayed reduced expression of both Pax5 and Ebf1, but this had little effect on most EBF1 or PAX5 target genes. Only a subset of target genes was deregulated; this subset included a large percentage of potential tumor suppressor genes and oncogenes. Further, most of these genes appear to be jointly regulated by both EBF1 and PAX5. Our findings suggest a model whereby small perturbations in a self-reinforcing network of transcription factors critical for B cell development, specifically PAX5 and EBF1, cooperate with STAT5 activation to initiate ALL.

| S-EPMC3173246 | biostudies-literature

EBF1 drives hallmark B cell gene expression by enabling the interaction of PAX5 with the MLL H3K4 methyltransferase complex.

Project description:PAX5 and EBF1 work synergistically to regulate genes that are involved in B lymphocyte differentiation. We used the KIS-1 diffuse large B cell lymphoma cell line, which is reported to have elevated levels of PAX5 expression, to investigate the mechanism of EBF1- and PAX5-regulated gene expression. We demonstrate the lack of expression of hallmark B cell genes, including CD19, CD79b, and EBF1, in the KIS-1 cell line. Upon restoration of EBF1 expression we observed activation of CD19, CD79b and other genes with critical roles in B cell differentiation. Mass spectrometry analyses of proteins co-immunoprecipitated with PAX5 in KIS-1 identified components of the MLL H3K4 methylation complex, which drives histone modifications associated with transcription activation. Immunoblotting showed a stronger association of this complex with PAX5 in the presence of EBF1. Silencing of KMT2A, the catalytic component of MLL, repressed the ability of exogenous EBF1 to activate transcription of both CD19 and CD79b in KIS-1 cells. We also find association of PAX5 with the MLL complex and decreased CD19 expression following silencing of KMT2A in other human B cell lines. These data support an important role for the MLL complex in PAX5-mediated transcription regulation.

| S-EPMC7810865 | biostudies-literature

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