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-seq, ChIP-seq and reporter gene assays, we identified six EBF responsive enhancer elements 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, ChIP-seq 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-ALL cell line indicate that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development.

The EBNA2-EBF1 complex promotes oncogenic MYC expression levels and metabolic processes required for cell cycle progression of Epstein-Barr virus-infected B cells

“…Thus, the protruding α1-helix within the EBNA2 N-terminal transactivation (END) domain is the structural element of EBNA2 that confers B cell specific cellular activation and is a critical structure. Our study also highlights the role of EBF1 in controlling and regulating proliferation of mature human B cells extending the finding of Somasundaram et al in mouse pro-B cells (Somasundaram et al, 2021).…”

“…As a pioneer factor, EBF1 alters the chromatin signature of B cell precursors allowing additional transcription factors and transactivators the access to chromatin (Boller et al, 2016). Furthermore, EBF1 directly activates MYC in murine pro-B cells to promote proliferation of these cells (Somasundaram et al, 2021). Here, we show that EBNA2 not only requires the pioneer activity of EBF1 to gain access to open chromatin but it tackles and usurps EBF1 in B cells to access its response elements and to activate direct target genes of EBNA2 such as MYC and LMP1.…”

Chromodomain helicase DNA‐binding 4 (CHD4) regulates early B cell identity and V(D)J recombination*

“…Maintenance of the B lineage program not only prepares B cell progenitors for additional differentiation to produce antibody-secreting cells but may also serve as a barrier against malignant transformation of B cells. [47][48][49] Expression of early B cell markers is coupled with the onset of immunoglobulin (Ig) gene assembly by V(D)J recombination. The generation of functional Ig loci proceeds stepwise, resulting in expression of the B cell-specific receptor for antigen (BCR) and functional antibody proteins for secretion in response to infection (reviewed in 50).…”

“…74 PAX5 physically interacts with NuRD components, as detected following pull-down and mass spectrometry experiments. 75 PAX5 negatively regulates expression of c-Myc, 48,49 which may be important for prevention of B cell leukemias. In this regard, previous studies on the induction of pluripotency suggest a potential role of NuRD in the maintenance of the B cell fate.…”

Enhancing B-Cell Malignancies—On Repurposing Enhancer Activity towards Cancer

“…Both EBF1 and PAX5 are bound to MYC enhancers in mouse pro-B cells as well as pro-B ALL NALM6 cells. While EBF1 promoted MYC expression, PAX5 negatively regulated MYC levels in normal B-cell progenitors [283]. Although it is not clear how this regulation looks in malignant cells, another report suggested that EBF1 and PAX5 prevent malignant transformation by limiting MYC levels [284].…”