Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

Cryptic activation of an Irf8 enhancer governs cDC1 fate specification.

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.

Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41-/-Bhlhe40-/- B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

Lineage-inappropriate PAX5 expression in t(8;21) acute myeloid leukemia requires signaling-mediated abrogation of polycomb repression.

The activation of B-cell-specific genes, such as CD19 and PAX5, is a hallmark of t(8;21) acute myeloid leukemia (AML) which expresses the translocation product RUNX1/ETO. PAX5 is an important regulator of B-lymphoid development and blocks myeloid differentiation when ectopically expressed. To understand the molecular mechanism of PAX5 deregulation, we examined its chromatin structure and regulation in t(8;21) AML cells, non-t(8;21) myeloid precursor control cells, and pre-B cells. In non-t(8;21) myeloid precursors, PAX5 is poised for transcription, but is repressed by polycomb complexes. In t(8;21) AML, PAX5 is not directly activated by RUNX1/ETO, but expression requires constitutive mitogen-activated protein (MAP) kinase signaling. Using a model of t(8;21) carrying an activating KIT mutation, we demonstrate that deregulated MAP kinase signaling in t(8;21) AML abrogates the association of polycomb complexes to PAX5 and leads to aberrant gene activation. Our findings therefore suggest a novel role of activating tyrosine kinase mutations in lineage-inappropriate gene expression in AML.

The function of the conserved regulatory element within the second intron of the mammalian Csf1r locus.

The gene encoding the receptor for macrophage colony-stimulating factor (CSF-1R) is expressed exclusively in cells of the myeloid lineages as well as trophoblasts. A conserved element in the second intron, Fms-Intronic Regulatory Element (FIRE), is essential for macrophage-specific transcription of the gene. However, the molecular details of how FIRE activity is regulated and how it impacts the Csf1r promoter have not been characterised. Here we show that agents that down-modulate Csf1r mRNA transcription regulated promoter activity altered the occupancy of key FIRE cis-acting elements including RUNX1, AP1, and Sp1 binding sites. We demonstrate that FIRE acts as an anti-sense promoter in macrophages and reversal of FIRE orientation within its native context greatly reduced enhancer activity in macrophages. Mutation of transcription initiation sites within FIRE also reduced transcription. These results demonstrate that FIRE is an orientation-specific transcribed enhancer element.

Pax5: a master regulator of B cell development and leukemogenesis.

The B cell lineage of the hematopoietic system is responsible for the generation of high-affinity antibodies, which provide humoral immunity for protection against foreign pathogens. B cell commitment and development depend on many transcription factors including Pax5. Here, we review the different functions of Pax5 in regulating various aspects of B lymphopoiesis. At B cell commitment, Pax5 restricts the developmental potential of lymphoid progenitors to the B cell pathway by repressing B-lineage-inappropriate genes, while it simultaneously promotes B cell development by activating B-lymphoid-specific genes. Pax5 thereby controls gene transcription by recruiting chromatin-remodeling, histone-modifying, and basal transcription factor complexes to its target genes. Moreover, Pax5 contributes to the diversity of the antibody repertoire by controlling V(H)-DJ(H) recombination by inducing contraction of the immunoglobulin heavy-chain locus in pro-B cells, which is likely mediated by PAIR elements in the 5' region of the V(H) gene cluster. Importantly, all mature B cell types depend on Pax5 for their differentiation and function. Pax5 thus controls the identity of B lymphocytes throughout B cell development. Consequently, conditional loss of Pax5 allows mature B cells from peripheral lymphoid organs to develop into functional T cells in the thymus via dedifferentiation to uncommitted progenitors in the bone marrow. Pax5 has also been implicated in human B cell malignancies because it can function as a haploinsufficient tumor suppressor or oncogenic translocation fusion protein in B cell precursor acute lymphoblastic leukemia.

Differential regulation of sense and antisense promoter activity at the Csf1R locus in B cells by the transcription factor PAX5.

OBJECTIVE: The transcription factor PAX5 is essential for the activation of B-cell-specific genes and for the silencing of myeloid-specific genes. We previously determined the molecular mechanism by which PAX5 silences the myeloid-specific colony-stimulating-factor-receptor (Csf1R) gene and showed that PAX5 directly binds to the Csf1r promoter as well as to an intronic enhancer that generates an antisense transcript in B cells. Here we examine the role of PAX5 in the regulation of sense and antisense transcription in B cells. MATERIALS AND METHODS: We performed PAX5-specific chromatin immunoprecipitation analyses across the Csfr1 locus. We investigated the role of PAX5 in regulating Csf1r sense and antisense promoter activity by transient transfections and by employing a Pax5(-/-) pro-B-cell line expressing an inducible PAX5 protein. PAX5 interacting factors were identified by pull-down experiments. The role of the transcription factor Sp3 in driving antisense promoter expression was examined in B cells from Sp3 knockout mice. RESULTS: PAX5 differentially regulates the Csf1r promoter and the promoter of the antisense transcript. PAX5 interferes with PU.1 transactivation at the sense promoter by binding to a PAX5 consensus sequence. At the antisense promoter, PAX5 does not specifically recognize DNA, but interacts with Sp3 to upregulate antisense promoter activity. Antisense promoter activation by PAX5 is dependent on the presence of its partial homeo-domain. CONCLUSIONS: We demonstrate that PAX5 regulates Csf1r in B cells by reducing the frequency of binding of the basal transcription machinery to the promoter and by activating antisense RNA expression.

The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells.

Pax5 is a critical regulator of B-cell commitment. Here, we identified direct Pax5 target genes by streptavidin-mediated ChIP-chip analysis of pro-B cells expressing in vivo biotinylated Pax5. By binding to promoters and enhancers, Pax5 directly regulates the expression of multiple transcription factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer Pax5-dependent B-cell-specific activity to the Nedd9 gene controlling B-cell trafficking. Profiling of histone modifications in Pax5-deficient and wild-type pro-B cells demonstrated that Pax5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro-B cells. Pax5 rapidly induces these chromatin and transcription changes by recruiting chromatin-remodelling, histone-modifying and basal transcription factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which Pax5 controls B-cell commitment.

Stepwise activation of enhancer and promoter regions of the B cell commitment gene Pax5 in early lymphopoiesis.

Pax5 is an essential regulator of B cell identity and function. Here, we used transgenesis and deletion mapping to identify a potent enhancer in intron 5 of the Pax5 locus. This enhancer in combination with the promoter region was sufficient to recapitulate the B lymphoid expression of Pax5. The enhancer was silenced by DNA methylation in embryonic stem cells, but became activated in multipotent hematopoietic progenitors. It contained functional binding sites for the transcription factors PU.1, IRF4, IRF8, and NF-kappaB, suggesting that these regulators contribute to sequential enhancer activation in hematopoietic progenitors and during B cell development. In contrast, the promoter region was repressed by Polycomb group proteins in non-B cells and was activated only at the onset of pro-B cell development through induction of chromatin remodeling by the transcription factor EBF1. These experiments demonstrate a stepwise activation of Pax5 in early lymphopoiesis and provide mechanistic insights into the process of B cell commitment.

How transcription factors program chromatin--lessons from studies of the regulation of myeloid-specific genes.

Hematopoietic stem cells exhibit a multi-lineage gene expression program, and this expression program is either maintained when these cells self-renew, or re-programmed when they differentiate. Both processes require the regulated expression of sequence-specific transcription factors and their interaction with the epigenetic regulatory machinery which programs the chromatin of hematopoietic genes in a cell type specific fashion. This article describes recent findings on the complexity of these molecular interactions and their consequences with respect to the regulation of cell fate decisions. We also describe recent findings from studies of genes expressed in the myeloid lineage (Pu.1 and csf1r) which highlight some of the molecular principles governing cell fate decisions at the epigenetic level.

Stem cell-specific epigenetic priming and B cell-specific transcriptional activation at the mouse Cd19 locus.

Low-level expression of multiple lineage-specific genes is a hallmark of hematopoietic stem cells (HSCs). HSCs predominantly express genes specific for the myeloid or megakaryocytic-erythroid lineages, whereas the transcription of lymphoid specific genes appears to begin after lymphoid specification. It has been demonstrated for a number of genes that epigenetic priming occurs before gene expression and lineage specification; however, little is known about how epigenetic priming of lymphoid genes is regulated. To address the question of how B cell-restricted expression is established, we studied activation of the Cd19 gene during hematopoietic development. We identified a B cell-specific upstream enhancer and showed that the developmental regulation of Cd19 expression involves precisely coordinated alterations in transcription factor binding and chromatin remodeling at Cd19 cis-regulatory elements. In multipotent progenitor cells, Cd19 chromatin is first remodeled at the upstream enhancer, and this remodeling is associated with binding of E2A. This is followed by the binding of EBF and PAX5 during B-cell differentiation. The Cd19 promoter is transcriptionally activated only after PAX5 binding. Our experiments give important mechanistic insights into how widely expressed and B lineage-specific transcription factors cooperate to mediate the developmental regulation of lymphoid genes during hematopoiesis.

The Pu.1 locus is differentially regulated at the level of chromatin structure and noncoding transcription by alternate mechanisms at distinct developmental stages of hematopoiesis.

The Ets family transcription factor PU.1 is crucial for the regulation of hematopoietic development. Pu.1 is activated in hematopoietic stem cells and is expressed in mast cells, B cells, granulocytes, and macrophages but is switched off in T cells. Many of the transcription factors regulating Pu.1 have been identified, but little is known about how they organize Pu.1 chromatin in development. We analyzed the Pu.1 promoter and the upstream regulatory element (URE) using in vivo footprinting and chromatin immunoprecipitation assays. In B cells, Pu.1 was bound by a set of transcription factors different from that in myeloid cells and adopted alternative chromatin architectures. In T cells, Pu.1 chromatin at the URE was open and the same transcription factor binding sites were occupied as in B cells. The transcription factor RUNX1 was bound to the URE in precursor cells, but binding was down-regulated in maturing cells. In PU.1 knockout precursor cells, the Ets factor Fli-1 compensated for the lack of PU.1, and both proteins could occupy a subset of Pu.1 cis elements in PU.1-expressing cells. In addition, we identified novel URE-derived noncoding transcripts subject to tissue-specific regulation. Our results provide important insights into how overlapping, but different, sets of transcription factors program tissue-specific chromatin structures in the hematopoietic system.

Regulation of the interleukin-7 receptor alpha promoter by the Ets transcription factors PU.1 and GA-binding protein in developing B cells.

Signaling through the IL-7 receptor (IL-7R) is required for development and maintenance of the immune system. The receptor for IL-7 is heterodimeric, consisting of a common gamma chain (gammac, encoded by Il2rg) and an alpha subunit (IL-7Ralpha, encoded by Il7r). The Il7r gene is expressed specifically in the immune system in a developmental stage-specific manner. It is not known how the Il7r gene is transcriptionally regulated during B cell development. The goal of this study is to elucidate the function of the Il7r promoter region in developing B cells. Using a combination of 5' rapid amplification of cDNA ends analysis, transient transfection assays, and DNase I hypersensitivity mapping, we identified the location of the Il7r promoter. Using a combination of electrophoretic mobility shift analysis, chromatin immunoprecipitation experiments, and RNA interference experiments, we found that the Ets transcription factors PU.1 and GA-binding protein (GABP) activate the Il7r promoter by interacting with a highly conserved Ets binding site. In committed B lineage cells, GABP can promote Il7r transcription in the absence of PU.1. However, the results of retroviral gene transfer experiments suggest that PU.1 is uniquely required to initiate transcription of the Il7r locus at the earliest stages of progenitor B cell generation. In summary, these results suggest that Il7r transcription is regulated by both PU.1 and GABP in developing B cells.

A two-step, PU.1-dependent mechanism for developmentally regulated chromatin remodeling and transcription of the c-fms gene.

Hematopoietic stem cells and multipotent progenitors exhibit low-level transcription and partial chromatin reorganization of myeloid cell-specific genes including the c-fms (csf1R) locus. Expression of the c-fms gene is dependent on the Ets family transcription factor PU.1 and is upregulated during myeloid differentiation, enabling committed macrophage precursors to respond to colony-stimulating factor 1. To analyze molecular mechanisms underlying the transcriptional priming and developmental upregulation of the c-fms gene, we have utilized myeloid progenitors lacking the transcription factor PU.1. PU.1 can bind to sites in both the c-fms promoter and the c-fms intronic regulatory element (FIRE enhancer). Unlike wild-type progenitors, the PU.1(-/-) cells are unable to express c-fms or initiate macrophage differentiation. When PU.1 was reexpressed in mutant progenitors, the chromatin structure of the c-fms promoter was rapidly reorganized. In contrast, assembly of transcription factors at FIRE, acquisition of active histone marks, and high levels of c-fms transcription occurred with significantly slower kinetics. We demonstrate that the reason for this differential activation was that PU.1 was required to promote induction and binding of a secondary transcription factor, Egr-2, which is important for FIRE enhancer activity. These data suggest that the c-fms promoter is maintained in a primed state by PU.1 in progenitor cells and that at FIRE PU.1 functions with another transcription factor to direct full activation of the c-fms locus in differentiated myeloid cells. The two-step mechanism of developmental gene activation that we describe here may be utilized to regulate gene activity in a variety of developmental pathways.

The mechanism of repression of the myeloid-specific c-fms gene by Pax5 during B lineage restriction.

The transcription factor Pax5 (BSAP) is required for the expression of a B-cell-specific genetic program and for B-cell differentiation, and also to suppress genes of alternative lineages. The molecular mechanism by which repression of myeloid genes occurs during early B-lineage restriction is unknown and in this study we addressed this question. One of the genes repressed by Pax5 in B cells is the colony-stimulating factor receptor 1 gene (csf1r or c-fms). We examined the changes in chromatin caused by Pax5 activity, and we show that Pax5 is directly recruited to c-fms resulting in the rapid loss of RNA polymerase II binding, followed by loss of transcription factor binding and DNaseI hypersensitivity at all cis-regulatory elements. We also show that Pax5 targets the basal transcription machinery of c-fms by interacting with a binding site within the major transcription start sites. Our results support a model by which Pax5 does not lead to major alterations in chromatin modification, but inhibits transcription by interfering with the action of myeloid transcription factors.

Differentiation-dependent alterations in histone methylation and chromatin architecture at the inducible chicken lysozyme gene.

It is now well established that locus-wide chromatin remodeling and dynamic alterations of histone modifications are required for the developmentally regulated activation of tissue-specific genes. However, little is known about the dynamics of these events during cell differentiation and how chromatin of an entire gene locus responds to signal transduction processes. To address this issue we investigated chromatin accessibility, linker histone distribution, and the histone methylation status at the macrophage-specific chicken lysozyme locus and the ubiquitously expressed gas41 locus in multipotent precursor cell lines and BM2 monoblast cells. The latter can be induced to go through macrophage maturation by treatment with phorbol-12-myristate acetate and can be further stimulated with bacterial lipopolysaccharide. We show that expression of the lysozyme gene in undifferentiated monoblasts is low and that a high level of gene expression requires both cell differentiation and lipopolysaccharide stimulation. However, depletion of the linker histone H1 is observed already in lysozyme non-expressing multipotent precursor cells. In undifferentiated monoblasts, the lysozyme regulatory regions are marked by the presence of monomethylated histone H3 lysine 4, which becomes increasingly converted into trimethylated H3 lysine K4 during cell differentiation. We also present evidence for extensive, differentiation-dependent alterations in nuclease accessibility at the lysozyme promoter without alterations of nucleosome and transcription factor occupancy.

c-FMS chromatin structure and expression in normal and leukaemic myelopoiesis.

The macrophage colony-stimulating factor receptor is encoded by the c-FMS gene, and it has been suggested that altered regulation of c-FMS expression may contribute to leukaemic transformation. c-FMS is expressed in pluripotent haemopoietic precursor cells and is subsequently upregulated during monocytic differentiation, but downregulated during granulopoiesis. We have examined transcription factor occupancy and aspects of chromatin structure of the critical c-FMS regulatory element located within the second intron (FIRE - fms intonic regulatory element) during normal and leukaemic myelopoiesis. Granulocytic differentiation from normal and leukaemic precursors is accompanied by loss of transcription factors at FIRE and downregulated c-FMS expression. The presence of AML1-ETO in leukaemic cells does not prevent this disassembly. In nonleukaemic cells, granulocytic differentiation is accompanied by reversal to a chromatin fine structure characteristic of c-FMS-nonexpressing cells. In addition, we show that low-level expression of the gene in leukaemic blast cells and granulocytes does not associate with increased CpG methylation across the c-FMS locus.

Rapid, solid-phase based automated analysis of chromatin structure and transcription factor occupancy in living eukaryotic cells.

Transcription factors, chromatin components and chromatin modification activities are involved in many diseases including cancer. However, the means by which alterations in these factors influence the epigenotype of specific cell types is poorly understood. One problem that limits progress is that regulatory regions of eukaryotic genes sometimes extend over large regions of DNA. To improve chromatin structure-function analysis over such large regions, we have developed an automated, relatively simple procedure that uses magnetic beads and a capillary sequencer for ligation-mediated-PCR (LM-PCR). We show that the procedure can be used for the rapid examination of chromatin fine-structure, nucleosome positioning as well as changes in transcription factor binding-site occupancy during cellular differentiation.

Epigenetic silencing of the c-fms locus during B-lymphopoiesis occurs in discrete steps and is reversible.

The murine c-fms (Csf1r) gene encodes the macrophage colony-stimulating factor receptor, which is essential for macrophage development. It is expressed at a low level in haematopoietic stem cells and is switched off in all non-macrophage cell types. To examine the role of chromatin structure in this process we studied epigenetic silencing of c-fms during B-lymphopoiesis. c-fms chromatin in stem cells and multipotent progenitors is in the active conformation and bound by transcription factors. A similar result was obtained with specified common myeloid and lymphoid progenitor cells. In developing B cells, c-fms chromatin is silenced in distinct steps, whereby first the binding of transcription factors and RNA expression is lost, followed by a loss of nuclease accessibility. Interestingly, regions of de novo DNA methylation in B cells overlap with an intronic antisense transcription unit that is differently regulated during lymphopoiesis. However, even at mature B cell stages, c-fms chromatin is still in a poised conformation and c-fms expression can be re-activated by conditional deletion of the transcription factor Pax5.

Dynamic reorganization of chromatin structure and selective DNA demethylation prior to stable enhancer complex formation during differentiation of primary hematopoietic cells in vitro.

In order to gain insights in the true molecular mechanisms involved in cell fate decisions, it is important to study the molecular details of gene activation where such decisions occur, which is at the level of the chromatin structure of individual genes. In the study presented here we addressed this issue and examined the dynamic development of an active chromatin structure at the chicken lysozyme locus during the differentiation of primary myeloid cells from transgenic mouse bone marrow. Using in vivo footprinting we found that stable enhancer complex assembly and high-level gene expression are late events in cell differentiation. However, even before the onset of gene expression and stable transcription factor binding, specific chromatin alterations are observed. This includes changes in DNA topology and the selective demethylation of CpG dinucleotides located in the cores of critical transcription factor binding sites, but not in flanking DNA. These results firmly support the idea that epigenetic programs guiding blood cell differentiation are engraved into the chromatin of lineage-specific genes and that such chromatin changes are implemented before cell lineage specification.