Meinrad Busslinger

Commitment to the B-lymphoid lineage depends on the transcription factor Pax5

The Pax5 gene encoding the B-cell-specific activator protein (BSAP) is expressed within the haematopoietic system exclusively in the B-lymphoid lineage, where it is required in vivo for progression beyond the pro-B-cell stage. However, Pax5 is not essential for in vitro propagation of pro-B cells in the presence of interleukin-7 and stromal cells. Here we show that pro-B cells lacking Pax5 are also incapable of in vitro B-cell differentiation unless Pax5 expression is restored by retroviral transduction. Pax5-/- pro-B cells are not restricted in their lineage fate, as stimulation with appropriate cytokines induces them to differentiate into functional macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells. As expected for a clonogenic haematopoietic progenitor with lymphomyeloid developmental potential, the Pax5-/- pro-B cell expresses genes of different lineage-affiliated programmes, and restoration of Pax5 activity represses this lineage-promiscuous transcription. Pax5 therefore plays an essential role in B-lineage commitment by suppressing alternative lineage choices.

Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5 BSAP

The Pax5 gene, coding for the transcription factor BSAP, was mutated in the mouse germline by targeted disruption. Homozygous mutant mice were born alive, became growth retarded, and usually died within three weeks. About 5% of mutants survived to adulthood and were fertile, but severely runted. Morphogenesis of the posterior midbrain was affected as early as embryonic day 16.5, leading to a reduction of the inferior colliculus near the midline and to altered foliation of the anterior cerebellum. Moreover, all mutants failed to produce small pre-B, B, and plasma cells owing to a complete arrest of B cell development at an early precursor stage. These data define a key role for Pax5 in early B lymphopoiesis and midbrain patterning.

The Transcription Factor GATA-3 Controls Cell Fate and Maintenance of Type 2 Innate Lymphoid Cells

Innate lymphoid cells (ILCs) reside at mucosal surfaces and control immunity to intestinal infections. Type 2 innate lymphoid cells (ILC2s) produce cytokines such as IL-5 and IL-13, are required for immune defense against helminth infections, and are involved in the pathogenesis of airway hyperreactivity. Here, we have investigated the role of the transcription factor GATA-3 for ILC2 differentiation and maintenance. We showed that ILC2s and their lineage-specified bone marrow precursors (ILC2Ps), as identified here, were characterized by continuous high expression of GATA-3. Analysis of mice with temporary deletion of GATA-3 in all ILCs showed that GATA-3 was required for the differentiation and maintenance of ILC2s but not for RORγt(+) ILCs. Thus, our data demonstrate that GATA-3 is essential for ILC2 fate decisions and reveal similarities between the transcriptional programs controlling ILC and T helper cell fates.

Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors

Lineage commitment and differentiation to a mature cell type are considered to be unidirectional and irreversible processes under physiological conditions. The commitment of haematopoietic progenitors to the B-cell lineage and their development to mature B lymphocytes critically depend on the transcription factor encoded by the paired box gene 5 (Pax5). Here we show that conditional Pax5 deletion in mice allowed mature B cells from peripheral lymphoid organs to dedifferentiate in vivo back to early uncommitted progenitors in the bone marrow, which rescued T lymphopoiesis in the thymus of T-cell-deficient mice. These B-cell-derived T lymphocytes carried not only immunoglobulin heavy- and light-chain gene rearrangements but also participated as functional T cells in immune reactions. Mice lacking Pax5 in mature B cells also developed aggressive lymphomas, which were identified by their gene expression profile as progenitor cell tumours. Hence, the complete loss of Pax5 in late B cells could initiate lymphoma development and uncovered an extraordinary plasticity of mature peripheral B cells despite their advanced differentiation stage.

Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors

The mechanisms controlling the commitment of haematopoietic progenitors to the B-lymphoid lineage are poorly understood. The observations that mice deficient in E2A and EBF lack B-lineage cells have implicated these two transcription factors in the commitment process. Moreover, the expression of genes encoding components of the rearrangement machinery (RAG1, RAG2, TdT) or pre-B-cell receptor (λ5, VpreB, Igα, Igβ) has been considered to indicate B-lineage commitment. All these genes including E2A and EBF are expressed in pro-B cells lacking the transcription factor Pax5 (refs 5,6,7). Here we show that cloned Pax5-deficient pro-B cells transferred into RAG2-deficient mice provide long-term reconstitution of the thymus and give rise to mature T cells expressing α/β-T-cell receptors. The bone marrow of these mice contains a population of cells of Pax5-/- origin with the same phenotype as the donor pro-B cells. When transferred into secondary recipients, these pro-B cells again home to the bone marrow and reconstitute the thymus. Hence, B-lineage commitment is determined neither by immunoglobulin DJ rearrangement nor by the expression of E2A, EBF, λ5, VpreB, Igα and Igβ. Instead, our data implicate Pax5 in the control of B-lineage commitment.

twin of eyeless, a Second Pax-6 Gene of Drosophila, Acts Upstream of eyeless in the Control of Eye Development

The Drosophila Pax-6 gene eyeless (ey) plays a key role in eye development. Here we show tht Drosophila contains a second Pax-6 gene, twin of eyeless (toy), due to a duplication during insect evolution. Toy is more similar to vertebrate Pax-6 proteins than Ey with regard to overall sequence conservation, DNA-binding function, and early expression in the embryo, toy and ey share a similar expression pattern in the developing visual system, and targeted expression of Toy, like Ey, induces the formation of ectopic eyes. Genetic and biochemical evidence indicates, however, that Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey. Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program.

Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments.

The Pax‐5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre‐BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre‐BI cells of wild‐type and Pax‐5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax‐5‐deficient pre‐BI cells by a hormone‐inducible BSAP–estrogen receptor fusion protein. The genes coding for the B‐cell receptor component Ig‐α (mb‐1) and the transcription factors N‐myc and LEF‐1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD‐1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax‐5‐deficient pre‐BI cells with full‐length BSAP or a truncated form containing only the paired domain. IL‐7 signalling was able to efficiently induce the N‐myc gene only in the presence of full‐length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb‐1 and LEF‐1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA‐binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss‐ and gain‐of‐function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context.

The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP.

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.

DNA methylation and the regulation of globin gene expression

Abstract We have studied the effect of DNA methylation on human γ-globin gene expression, using a novel in vitro DNA methylation technique. With this method we have methylated specific segments of the γ-globin gene and its 5′-flanking region, which were cloned for this purpose in the bacteriophage vector M13mp8. The in vitro methylated DNA was introduced into mouse L-cells by cotransfer using the herpes simplex virus thymidine kinase gene as a selective marker. Transformed cell lines were isolated to examine the inheritance of the segmental methylation pattern and its effect on the expression of the γ-globin gene. Cells transformed with DNA methylated throughout the M13 vector and γ-globin DNA sequences do not express the γ-globin gene. Methyl-C residues in either the M13 DNA sequences or the γ-globin structural gene have, however, no inhibitory effect on globin transcription. In contrast, methylation in the 5′ region of the γ-globin gene (from nucleotides −760 to +100) prevents transcription. These data indicate that DNA methylation in the 5′ region of a gene might play a direct role in the regulation of gene expression.

The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells

Regulatory T cells (Treg cells) are required for peripheral tolerance. Evidence indicates that Treg cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of Treg cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector Treg cells. Thus, our study defines a differentiation pathway that leads to the acquisition of Treg cell effector functions and requires both IRF4 and Blimp-1.