Kara Lukin

Opposing effects of SWI/SNF and Mi-2/NuRD chromatin remodeling complexes on epigenetic reprogramming by EBF and Pax5

Transcriptionally silent genes are maintained in inaccessible chromatin. Accessibility of these genes requires their modification by chromatin remodeling complexes (CRCs), which are recruited to promoters by sequence-specific DNA-binding proteins. Early B-cell factor (EBF), which is crucial for B-cell lineage specification, reprograms mb-1 (Ig-α) promoters by increasing chromatin accessibility and initiating the loss of DNA methylation. In turn, this facilitates promoter activation by Pax5. Here, we investigated the roles of ATP-dependent CRCs in these mechanisms. Fusion of EBF and Pax5 with the ligand-binding domain of ERα allowed for 4-hydroxytamoxifen-dependent, synergistic activation of mb-1 transcription in plasmacytoma cells. Knock-down of the SWI/SNF ATPases Brg1 and Brm inhibited transcriptional activation by EBF:ER and Pax5:ER. In contrast, knock-down of the Mi-2/NuRD complex subunit Mi-2β greatly enhanced chromatin accessibility and mb-1 transcription in response to the activators. The reduction of Mi-2β also propagated DNA demethylation in response to EBF:ER and Pax5:ER, resulting in fully unmethylated mb-1 promoters. In EBF- or EBF/Pax5-deficient fetal liver cells, both EBF and Pax5 were required for efficient demethylation of mb-1 promoters. Together, our data suggest that Mi-2/NuRD is important for the maintenance of hypermethylated chromatin in B cells. We conclude that SWI/SNF and Mi-2/NuRD function in opposition to enable or limit the reprogramming of genes by EBF and Pax5 during B-cell development.

From hematopoietic progenitors to B cells: mechanisms of lineage restriction and commitment

The generation of B lymphocytes from hematopoietic progenitors requires lineage-specific transcription factors that progressively direct cell fate choices. Differentiation of hematopoietic stem cells to lymphoid progenitors requires Ikaros-dependent lineage priming and graded levels of PU.1, which are controlled by Ikaros and Gfi1. E2A drives expression of EBF1, which initiates B lineage specification. EBF1, in addition to Pax5, is necessary for commitment to the B cell lineage. As a model of gene activation in early B lymphopoiesis, mb-1 genes are activated sequentially by factors (e.g. EBF1) that initiate chromatin modifications before transcription. This review highlights the requisite interplay between transcription factors and epigenetic mechanisms in the context of B cell development.

Early B cell Factor: regulator of B lineage specification and commitment

B lymphocytes are generated from hematopoietic stem cells in a series of steps controlled by transcription factors. One of the most important regulators of this process is early B cell factor (EBF). Multiple lines of evidence indicate that expression of EBF is a principle determinant of the B cell fate. In the absence of EBF, progenitor cells fail to express classical markers of B cells, including immunoglobulins. EBF drives B cell differentiation by activating the Pax5 gene and other genes required for the pre-B and B cell receptors. New evidence suggests that expression of EBF in common lymphoid progenitors directs B cell fate decisions. Specification and commitment of cells to the B cell lineage are further established by Pax5, which increases expression of EBF. Recently, it was demonstrated that both EBF and Pax5 contribute to the commitment of cells to the B lineage. Together, these studies confirm that EBF is a keystone in the regulatory network that coordinates B cell lineage specification and commitment.

Compound haploinsufficiencies of Ebf1 and Runx1 genes impede B cell lineage progression

Early B cell factor (EBF)1 is essential for B lineage specification. Previously, we demonstrated the synergistic activation of Cd79a (mb-1) genes by EBF1 and its functional partner, RUNX1. Here, we identified consequences of Ebf1 haploinsufficiency together with haploinsufficiency of Runx1 genes in mice. Although numbers of “committed” pro-B cells were maintained in Ebf1+/−Runx1+/− (ERhet) mice, activation of B cell-specific gene transcription was depressed in these cells. Expression of genes encoding Aiolos, κ0 sterile transcripts, CD2 and CD25 were reduced and delayed in ERhet pro-B cells, whereas surface expression of BP-1 was increased on late pro-B cells in ERhet mice. Late pre-B and immature and mature B cells were decreased in the bone marrow of Ebf1+/− (Ehet) mice and were nearly absent in ERhet mice. Although we did not observe significant effects of haploinsuficiencies on IgH or Igκ rearrangements, a relative lack of Igλ rearrangements was detected in Ehet and ERhet pre-B cells. Together, these observations suggest that B cell lineage progression is impaired at multiple stages in the bone marrow of Ehet and ERhet mice. Furthermore, enforced expression of EBF1 and RUNX1 in terminally differentiated plasmacytoma cells activated multiple early B cell-specific genes synergistically. Collectively, these studies illuminate the effects of reduced Ebf1 dosage and the compounding effects of reduced Runx1 dosage. Our data confirm and extend the importance of EBF1 in regulating target genes and Ig gene rearrangements necessary for B cell lineage specification, developmental progression, and homeostasis.

A dose-dependent role for EBF1 in repressing non-B-cell-specific genes

In the absence of early B‐cell factor 1 (EBF1), B‐cell development is arrested at an uncommitted progenitor stage that exhibits increased lineage potentials. Previously, we investigated the roles of EBF1 and its DNA‐binding partner Runx1 by evaluating B lymphopoiesis in single (EBF1het and Runx1het) and compound haploinsufficent (Ebf1+/−Runx1+/−, ERhet) mice. Here, we demonstrate that decreased Ebf1 gene dosage results in the inappropriate expression of NK‐cell lineage‐specific genes in B‐cell progenitors. Moreover, prolonged expression of Ly6a/Sca‐1 suggested the maintenance of a relatively undifferentiated phenotype. These effects were exacerbated by reduced expression of Runx1 and occurred despite expression of Pax5. Repression of inappropriately expressed genes was restored in most pre‐B and all immature B cells of ERhet mice. Enforced EBF1 expression repressed promiscuous transcription in pro‐B cells of ERhet mice and in Ebf1−/−Pax5−/− fetal liver cells. Together, our studies suggest that normal levels of EBF1 are critical for maintaining B‐cell identity by directing repression of non‐B‐cell‐specific genes.

Highly Cooperative Recruitment of Ets-1 and Release of Autoinhibition by Pax5

Pax5 (paired box binding factor 5) is a critical regulator of transcription and lineage commitment in B lymphocytes. In B cells, mb-1 (Ig-alpha/immunoglobulin-associated alpha) promoter transcription is activated by Pax5 through its recruitment of E74-like transforming sequence (Ets) family proteins to a composite site, the P5-EBS (Pax5-Ets binding site). Previously, X-ray crystallographic analysis revealed a network of contacts between the DNA-binding domains of Pax5 and Ets-1 while bound to the P5-EBS. Here, we report that Pax5 assembles these ternary complexes via highly cooperative interactions that overcome the autoinhibition of Ets-1. Using recombinant proteins, we calculated K(d(app)) values for the binding of Pax5, Ets-1, and GA-binding proteins, separately or together, to the P5-EBS. By itself, Pax5 binds the P5-EBS with high affinity (K(d) approximately equal 2 nM). Ets-1(331-440) bound the P5-EBS by itself with low affinity (K(d)=136 nM). However, autoinhibited Ets-1(280-440) alone does not bind detectably to the suboptimal sequences of the P5-EBS. Recruitment of Ets-1(331-440) or Ets-1(280-440) resulted in highly efficient ternary complex assembly with Pax5. Pax5 counteracts autoinhibition and increases binding of Ets-1 of the mb-1 promoter by >1000-fold. Mutation of Pax5 Gln22 to alanine (Q22A) enhances promoter binding by Pax5; however, Q22A greatly reduces recruitment of Ets-1(331-440) and Ets-1(280-440) by Pax5 (8.9- or >300-fold, respectively). Thus, Gln22 of Pax5 is essential for overcoming Ets-1 autoinhibition. Pax5 wild type and Q22A each recruited GA-binding protein alpha/beta1 to the mb-1 promoter with similar affinities, but recruitment was less efficient than that of Ets-1 (reduced by approximately 8-fold). Our results suggest a mechanism that allows Pax5 to overcome autoinhibition of Ets-1 DNA binding. In summary, these data illustrate requirements for partnerships between Ets proteins and Pax5.

Maturational stage‐dependent thymocyte responses to TCR engagement

Thymocyte positive and negative selection are dependent on avidity‐driven TCR‐mediated recognition events in the thymus. High‐avidity recognition events result in negative selection, while low‐avidity recognition events result in positive selection. However, it has not been established how thymocytes maturation stages affect their responses to TCR signals of different avidities. We gained insight into this question when we reduced thymocyte selection to an in vitro system, in which full maturation of developmentally synchronized immature double‐positive thymocytes was induced on a cloned line of thymic epithelial cells. Our analysis of the kinetics of thymocyte development supports a multi‐phasic model of thymic selection. In it, thymocyte maturation stages as well as interaction avidity control the outcome TCR stimulation. Positive selection is initiated during a primary recognition event that proceeds independently of the TCR avidity. During a secondary recognition event the final fate of thymocyte, full maturation versus negative selection, is determined by TCR avidity.

The transcription factors GATA2 and microphthalmia-associated transcription factor regulate Hdc gene expression in mast cells and are required for IgE/mast cell–mediated anaphylaxis

Background Histamine is a critical mediator of IgE/mast cell–mediated anaphylaxis. Histamine is synthesized by decarboxylating the amino acid histidine, a reaction catalyzed by the histidine decarboxylase (Hdc) gene–encoded enzyme HDC. However, regulation of the Hdc gene in mast cells is poorly understood. Objective We sought to investigate the in vivo regulation of IgE/mast cell–mediated anaphylaxis by the transcription factors GATA2 and microphthalmia‐associated transcription factor (MITF) and the mechanisms by which GATA2 and MITF regulate Hdc gene expression in mouse and human mast cells. Methods Mice deficient in the transcription factors Gata2, aryl hydrocarbon receptor (Ahr), aryl hydrocarbon receptor repressor (Ahrr), or basic helix‐loop‐helix family member E40 (Bhlhe40) were assessed for anaphylactic reactions. Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers. Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene. The short hairpin RNA knockdown approach was used to determine the role of MITF in regulating mouse and human HDC gene expression. Results Connective tissue mast cell–specific Gata2‐deficient mice did not have IgE/mast cell‐mediated anaphylaxis. GATA2 induced the expression of Mitf, Ahr, Ahrr, and Bhlhe40 in mast cells. MITF, but not AHR, AHRR, or BHLHE40, was required for anaphylaxis. MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity. MITF overexpression largely restored Hdc gene expression in the Gata2‐deficient mast cells. In the human mast cell line LAD2, MITF was required for the HDC gene expression and histamine synthesis. Conclusion The transcription factors GATA2 and MITF regulate Hdc gene expression in mast cells and are required for IgE/mast cell–mediated anaphylaxis. Graphical abstract Figure. No Caption available.

Plasma Cells for Hire: Prior Experience Required

Activation of IgG⁺ memory B cells accounts for much of the antibodies in secondary immune responses. Here, Kometani et al. (2013) demonstrate that reduced amounts of Bach2 in antigen-experienced memory B cells control the robust production of IgG1⁺ plasma cells.