The epigenetics of early lymphocyte development.

The mammalian genome is highly structured, both spatially and functionally. Chromosomes are organized into specific territories, which are further folded into euchromatic or heterochromatic compartments. The euchromatic compartment often contains domains decorated with activating epigenetic marks, whereas heterochromatic regions lack activating marks or bear repressive ones. During lymphocyte development, gene segments move between these compartments. Additionally, some genes undergoing changes in transcriptional activity also display elaborate alterations in chromatin folding. Lineage-specific transcription factors help mediate these reconfigurations. Herein, I describe how genetic loci encoding for key regulators switch nuclear neighborhoods and reorganize their 3D structures to drive cell fate.

Localized gene-specific induction of accessibility to V(D)J recombination induced by E2A and early B cell factor in nonlymphoid cells.

Accessibility of immunoglobulin (Ig) gene segments to V(D)J recombination is highly regulated and is normally only achieved in B cell precursors. We previously showed that ectopic expression of E2A or early B cell factor (EBF) with recombination activating gene (RAG) induces rearrangement of IgH and IgL genes in nonlymphoid cells. VkappaI genes throughout the locus were induced to rearrange after transfection with E2A, suggesting that the entire Vkappa locus was accessible. However, here we show that Ig loci are not opened globally but that recombination is localized. Gene families are interspersed in the D(H), Vkappa, and Vlambda loci, and we show that certain families and individual genes undergo high levels of recombination after ectopic expression of E2A or EBF, while other families within the same locus are not induced to rearrange. Furthermore, in some families, induction of germline transcription correlates with the level of induced recombination, while in others there is no correlation, suggesting that recombination is not simply initiated by induction of germline transcription. The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism. We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.

Early thymocyte development is regulated by modulation of E2A protein activity.

The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

Regulation of the helix-loop-helix proteins, E2A and Id3, by the Ras-ERK MAPK cascade.

Activation of mitogen-activated protein kinase (MAPK) pathways leads to cellular differentiation and/or proliferation in a wide variety of cell types, including developing thymocytes. The basic helix-loop-helix (bHLH) proteins E12 and E47 and an inhibitor HLH protein, Id3, play key roles in thymocyte differentiation. We show here that E2A DNA binding is lowered in primary immature thymocytes consequent to T cell receptor (TCR)-mediated ligation. Whereas expression of E2A mRNA and protein are unaltered, Id3 transcripts are rapidly induced upon signaling from the TCR. Activation of Id3 transcription is regulated in a dose-dependent manner by the extracellular signal-regulated kinase (ERK) MAPK module. These observations directly connect the ERK MAPK cascade and HLH proteins in a linear pathway.

Transcription factor regulation of B lineage commitment.

The past year has provided insight into the mechanisms by which multipotent progenitors commit to differentiation through the B lymphocyte lineage. Mice lacking the Pax5 gene develop pro-B lymphocytes but these cells are not uniquely committed to the B lineage as they can give rise to all hematopoietic cell types if cultured under appropriate conditions. Regulators of lymphocyte proliferation and survival have also been identified that may allow lymphocytes to respond to information provided by the external environment.

Induction of a diverse T cell receptor gamma/delta repertoire by the helix-loop-helix proteins E2A and HEB in nonlymphoid cells.

During specific stages of thymocyte development, the T cell receptor (TCR) locus is assembled from variable (V), diversity (D), and joining (J) gene segments. Proper TCR gamma and delta V(D)J rearrangement during thymocyte development requires the presence of the E2A proteins. Here we show that E2A and a closely related protein, HEB, in the presence of recombination activating gene (RAG)1 and RAG2, each have the ability to activate TCR gamma and delta rearrangement in human kidney cells. The coding joints are diverse, contain nucleotide deletions, and occasionally show the presence of P nucleotides. Interestingly, only a subset of V, D, and J segments are targeted by the E2A and HEB proteins. Thus, E2A and HEB permit localized accessibility of the TCR gamma and delta loci to the recombination machinery. These data indicate that a distinct but diverse TCR repertoire can be induced in nonlymphoid cells by the mere presence of the V(D)J recombinase and the transcriptional regulators, E2A and HEB.

Id3 inhibits B lymphocyte progenitor growth and survival in response to TGF-beta.

E proteins function in many developmental processes and are essential for the formation of lymphocyte progenitors. However, it is not known whether E proteins regulate lymphocyte survival, proliferation or differentiation or how their activity is regulated during lymphocyte development. We show here a role for Id3, an inhibitor of E protein activity, in the induction of apoptosis and growth arrest. Id3 is induced in response to transforming growth factor beta (TGF-beta), a pleiotropic cytokine that inhibits the growth and survival of normal and transformed lymphocyte progenitors. In the absence of Id3, the response of lymphocyte progenitors to TGF-beta is perturbed, which indicates that Id3 is a mediator of this response. Our data show a key role for E proteins in lymphocyte survival and link the activity of E proteins, and their antagonists, to members of the TGF-beta family of cytokines.

The function of E- and Id proteins in lymphocyte development.

Helix-loop-helix proteins are essential factors for lymphocyte development and function. In particular, E-proteins are crucial for commitment of lymphoid progenitors to the B- and T-cell lineages. E-proteins are negatively regulated by the Id class of helix-loop-helix proteins. The Id proteins function as dominant-negative inhibitors of E-proteins by inhibiting their ability to bind DNA. Here, we review the role of E-proteins and their Id protein antagonists in lymphocyte proliferation and developmental progression. In addition, we discuss how E-protein activity and Id gene expression are regulated by T-cell receptor (TCR) and pre-TCR-mediated signalling.

The regulation and function of the Id proteins in lymphocyte development.

Helix-loop-helix (HLH) proteins are essential factors for lymphocyte development and function. One class of HLH proteins, the E-proteins, regulate many aspects of lymphocyte maturation, survival, proliferation, and differentiation. E-proteins are negatively regulated by another class of HLH proteins known as the Id proteins. The Id proteins function as dominant negative inhibitors of E-proteins by inhibiting their ability to bind DNA. Here we discuss the function and regulation of the Id proteins in lymphocyte development.

E2A proteins: essential regulators at multiple stages of B-cell development.

The development of mature B lymphocytes from multipotent progenitor cells requires the co-ordinated activities of a number of transcriptional regulatory proteins. The transcription factors encoded by the E2A gene are required for the development of committed B-lineage cells and regulate the expression of essential B-lineage genes at multiple stages of differentiation and activation. In this review we discuss the evidence that the E2A gene products function in the regulation of 1) transcription factors required for B-lineage determination, 2) essential proteins involved in pro-B and pre-B-cell development, 3) accessibilty and recombination of the IgH and IgL chain loci, and 4) isotype switching in activated, mature B lymphocytes.

E2A and EBF act in synergy with the V(D)J recombinase to generate a diverse immunoglobulin repertoire in nonlymphoid cells.

Immunoglobulin (Ig) and T cell receptor (TCR) genes are assembled during lymphocyte maturation through site-specific V(D)J recombination events. Here we show that E2A proteins act in concert with RAG1 and RAG2 to activate Ig VK1J but not Iglambda VlambdaIII-Jlambda1 rearrangement in an embryonic kidney cell line. In contrast, EBF, but not E2A, promotes VlambdaIII-Jlambda1 recombination. Either E2A or EBF activate IgH DH4J recombination but not V(D)J rearrangement. The Ig coding joints are diverse, contain nucleotide deletions, and lack N nucleotide additions. IgK VJ recombination requires the presence of the E2A transactivation domains. These observations indicate that in nonlymphoid cells a diverse Ig repertoire can be generated by the mere expression of the V(D)J recombinase and a transcriptional regulator.

Neuronal death and perinatal lethality in voltage-gated sodium channel alpha(II)-deficient mice.

Neural activity is crucial for cell survival and fine patterning of neuronal connectivity during neurodevelopment. To investigate the role in vivo of sodium channels (NaCh) in these processes, we generated knockout mice deficient in brain NaChalpha(II). NaChalpha(II)(-/-) mice were morphologically and organogenically indistinguishable from their NaChalpha(+/-) littermates. Notwithstanding, NaChalpha(II)(-/-) mice died perinatally with severe hypoxia and massive neuronal apoptosis, notably in the brainstem. Sodium channel currents recorded from cultured neurons of NaChalpha(II)(-/-) mice were sharply attenuated. Death appears to arise from severe hypoxia consequent to the brainstem deficiency of NaChalpha(II). NaChalpha(II) expression is, therefore, redundant for embryonic development but essential for postnatal survival.

Thymocyte selection is regulated by the helix-loop-helix inhibitor protein, Id3.

E2A, HEB, E2-2, and daughterless are basic helix-loop-helix (bHLH) proteins that play key roles in multiple developmental pathways. The DNA binding activity of E2A, HEB, and E2-2 is regulated by a distinct class of inhibitor HLH proteins, the Id gene products. Here, we show that Id3 is required for major histocompatability (MHC) class I- and class II-restricted thymocyte positive selection. Additionally, H-Y TCR-mediated negative selection is severely perturbed in Id3 null mutant mice. Finally, we show that E2A and Id3 interact genetically to regulate thymocyte development. These observations identify the HLH inhibitory protein Id3 as an essential component required for proper thymocyte maturation.

Thymocyte maturation is regulated by the activity of the helix-loop-helix protein, E47.

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I- and class II-restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I-restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection.

E2A activity is induced during B-cell activation to promote immunoglobulin class switch recombination.

The basic helix-loop-helix protein, E2A, is required for proper early B lymphopoiesis. Specifically, in E2A-deficient mice, B-cell development is blocked at the progenitor stage prior to the onset of immunoglobulin (Ig) V(D)J recombination. Here, we demonstrate that E2A plays an additional role during peripheral B lymphopoiesis. Upon activation of primary mature B lymphocytes, both E2A protein levels and DNA-binding activity are induced. Furthermore, we show that mature B cells, expressing a dominant-negative E2A antagonist, proliferate normally in response to mitogenic signaling and appropriately express the early and late activation markers CD69, CD44, IgD and B220. However, in the absence of E2A activity, B lymphocytes are blocked in their ability to express secondary Ig isotypes. We demonstrate that the defect lies at the level of DNA rearrangements between the Ig switch regions. These data suggest that E2A is an essential target during B-cell activation and its induction is required to promote Ig class switch recombination.

Transcription factors in hematopoiesis.

The advent of gene targeting in the mouse has led to rapid advances in the identification of factors controlling gene expression that are essential for normal hematopoietic development. Recent work has also uncovered roles for some of these factors in leukemogenesis and in the global regulation of chromatin structure.

Oncogenic homeodomain transcription factor E2A-Pbx1 activates a novel WNT gene in pre-B acute lymphoblastoid leukemia.

A large fraction of pediatric pre-B acute lymphoblastoid leukemias (ALL) consistently contain a t(1;19) chromosomal translocation. The t(1;19) translocation results in the production of a chimeric transcription factor containing the N-terminal transactivation domain of E2A fused to the C-terminal DNA-binding homeodomain of Pbx1. Here, we show that the E2A-Pbx1 fusion protein activates the expression of a novel WNT gene, WNT-16. WNT-16 normally is expressed in peripheral lymphoid organs such as spleen, appendix, and lymph nodes, but not in bone marrow. In contrast, high levels of WNT-16 transcripts are present in bone marrow and cell lines derived from pre-B ALL patients carrying the E2A-Pbx1 hybrid gene. Inhibition of E2A-Pbx1 expression leads to a significant decrease in WNT-16 mRNA levels, suggesting that WNT-16 is a downstream target of E2A-Pbx1. Three putative WNT receptors, FZ-2, FZ-3, and FZ-5, are expressed in cells of the B lineage, including pre-B ALL cells aberrantly expressing WNT-16. We propose that a WNT-16-mediated autocrine growth mechanism contributes to the development of t(1;19) pre-B ALL.