Genome-wide binding map of FoxO1 and Pax5 in pre-B cells
ABSTRACT: Genome-wide analysis of FoxO1 and Pax5 binding in pre-B cells following attenuation of IL-7 signaling. Transcription factor FoxO1 has been shown to be an essential factor for Ig light chain rearrangement. Results demonstrate that FoxO1 and Pax5 co-target genes that are activated during pre-B cell differentiation. Examination of FoxO1 and/or Pax5 binding under withdrawal of IL-7R signaling.
Project description:Genome-wide analysis of FoxO1 and Pax5 binding in pre-B cells following attenuation of IL-7 signaling. Transcription factor FoxO1 has been shown to be an essential factor for Ig light chain rearrangement. Results demonstrate that FoxO1 and Pax5 co-target genes that are activated during pre-B cell differentiation. Examination of FoxO1 and/or Pax5 binding under withdrawal of IL-7R signaling.
Project description:The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.
Project description:Pre-B cell receptor (pre-BCR) signaling and migration from IL-7-rich environments cooperate to drive pre-B cell differentiation via transcriptional programs that remain unclear. We show that the Ikaros transcription factor is required for the differentiation of large pre-B to small pre-B cells. Mice deleted for Ikaros in pro/pre-B cells show a complete block of differentiation at the fraction C' stage, and Ikaros-null pre-B cells cannot differentiate upon withdrawal of IL-7 in vitro. Restoration of Ikaros function rescues pre-B cell differentiation in vitro and in vivo and depends on DNA binding. Ikaros is required for the down-regulation of the pre-BCR, Ig? germline transcription, and Ig L chain recombination. Furthermore, Ikaros antagonizes the IL-7-dependent regulation of >3,000 genes, many of which are up- or down-regulated between fractions C' and D. Affected genes include those important for survival, metabolism, B cell signaling, and function, as well as transcriptional regulators like Ebf1, Pax5, and the Foxo1 family. Our data thus identify Ikaros as a central regulator of IL-7 signaling and pre-B cell development.
Project description:Interleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemia stage in which B-cell precursors display self-renewal ability, initiating precursor B-ALL that resembles PAX5 P80R or Ph-like human leukemia. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving both JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are novel treatment avenues for IL-7R-related cases. Our model, a unique resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy.
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.
Project description:The Ig? locus undergoes a variety of different molecular processes during B cell development, including V(D)J rearrangement and somatic hypermutations (SHM), which are influenced by cis regulatory regions (RRs) within the locus. The Ig? locus includes three characterized RRs termed the intronic (iE?), 3'E?, and Ed enhancers. We had previously noted that a region of DNA upstream of the iE? and matrix attachment region (MAR) was necessary for demethylation of the locus in cell culture. In this study, we further characterized this region, which we have termed Dm, for demethylation element. Pre-rearranged Ig? transgenes containing a deletion of the entire Dm region, or of a Pax5-binding site within the region, fail to undergo efficient CpG demethylation in mature B cells in vivo. Furthermore, we generated mice with a deletion of the full Dm region at the endogenous Ig? locus. The most prominent phenotype of these mice is reduced SHM in germinal center B cells in Peyer's patches. In conclusion, we propose the Dm element as a novel Pax5-binding cis regulatory element, which works in concert with the known enhancers, and plays a role in Ig? demethylation and SHM.
Project description:To date, the trout B-cell is poorly defined, as many essential molecular markers are not yet available for this species. In mammalian systems, the transcription factor Pax5, expressed from pre-B through plasmablast stages, provides an important marker for B-cell differentiation. In a previous study we showed that Pax5 is expressed in the trout. Here we identify trout B-cell populations that vary in expression of Pax5, membrane and secreted Ig. Immune tissues were separated based on concentration of surface IgM, and analyzed by qPCR and flow cytometry. Results suggest that spleen and PBL contain mostly resting B cells, which lack secreted Ig. While the great majority of splenic B cells become strongly activated upon LPS stimulation, PBLs do not. Additionally, anterior kidney contains both developing B and Ig-secreting B-cell populations, but few resting, mature B cells. Lastly, posterior kidney contains multiple B-cell populations in various states of activation. We conclude that trout immune tissues contain multiple, developmentally diverse and tissue-specific B-cell populations as defined by their relative expression of Pax5, surface IgM, and secreted IgM.
Project description:The usage of >100 functional murine Ig heavy chain V(H) genes, when rearranged to D(H)J(H) genes, generates a diverse antibody repertoire. The V(H) locus encompasses 2.5 Mb, and rearrangement of V(H) genes in the D(H)-distal half of the locus are controlled very differently from the V(H) genes in the proximal end of the locus. The rearrangement of distal but not proximal V(H) genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5, or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal V(H) rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal V(H) segments and the repressive histone modification H3K27me3 is exclusively present on proximal V(H) segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal V(H) genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the V(H) locus by differentially modulating two reciprocal histone modifications during B lymphocyte development.
Project description:The temporal control of RAG (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the IL-7R and precursor B cell Ag receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful Ig H chain gene rearrangement. Whereas the mechanism of RAG2 protein downregulation is well defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson murine leukemia virus-transformed B cells to model this stage of development, we identified early B cell factor 1 (Ebf1) as a strong repressor of Rag transcription. Short hairpin RNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb.
Project description:PAX5 is one of the most frequently mutated genes in B-cell acute lymphoblastic leukemia (B-ALL), and children with inherited preleukemic PAX5 mutations are at a higher risk of developing the disease. Abnormal profiles of inflammatory markers have been detected in neonatal blood spot samples of children who later developed B-ALL. However, how inflammatory signals contribute to B-ALL development is unclear. Here, we demonstrate that Pax5 heterozygosis, in the presence of infections, results in the enhanced production of the inflammatory cytokine interleukin-6 (IL-6), which appears to act in an autocrine fashion to promote leukemia growth. Furthermore, in vivo genetic downregulation of IL-6 in these Pax5 heterozygous mice retards B-cell leukemogenesis, and in vivo pharmacologic inhibition of IL-6 with a neutralizing antibody in Pax5 mutant mice with B-ALL clears leukemic cells. Additionally, this novel IL-6 signaling paradigm identified in mice was also substantiated in humans. Altogether, our studies establish aberrant IL6 expression caused by Pax5 loss as a hallmark of Pax5-dependent B-ALL and the IL6 as a therapeutic vulnerability for B-ALL characterized by PAX5 loss.