Helix-loop-helix proteins in lymphocyte lineage determination.

The cells of the lymphoid system develop from multipotent hematopoietic stem cells through a series of intermediate progenitors with progressively restricted developmental options. Commitment to a given lymphoid lineage appears to be controlled by numerous transcriptional regulatory proteins that activate lineage-specific gene expression programs and extinguish expression of lineage-inappropriate genes. In this review I discuss the function of transcription factors belonging to the helix-loop-helix protein family in the control of lymphoid cell fate decisions. A model of lymphocyte lineage determination based on the antagonistic activity of transcriptional activating and repressing helix-loop-helix proteins is presented.

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.

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.

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.

Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12.

The transcription factors encoded by the E2A and early B cell factor (EBF) genes are required for the proper development of B lymphocytes. However, the absence of B lineage cells in E2A- and EBF-deficient mice has made it difficult to determine the function or relationship between these proteins. We report the identification of a novel model system in which the role of E2A and EBF in the regulation of multiple B lineage traits can be studied. We found that the conversion of 70Z/3 pre-B lymphocytes to cells with a macrophage-like phenotype is associated with the loss of E2A and EBF. Moreover, we show that ectopic expression of the E2A protein E12 in this macrophage line results in the induction of many B lineage genes, including EBF, IL7Ralpha, lambda5, and Rag-1, and the ability to induce kappa light chain in response to mitogen. Activation of EBF may be one of the critical functions of E12 in regulating the B lineage phenotype since expression of EBF alone leads to the activation of a subset of E12-inducible traits. Our data demonstrate that, in the context of this macrophage line, E12 induces expression of EBF and together these transcription factors coordinately regulate numerous B lineage-associated genes.

In vitro tracking of IL-7 responsiveness and gene expression during commitment of bipotent B-cell/macrophage progenitors.

BACKGROUND: The development of B lymphocytes from multipotent hematopoietic stem cells occurs through a series of intermediate cell types with increasingly restricted developmental potential. Despite intensive investigation, the underlying basis for commitment to a given lineage or the restriction in developmental potential of multipotent cells is unknown. To gain insight into this process we have developed an in vitro system that tracks a bipotent progenitor, which has the capacity to give rise to both B lymphocytes and macrophages, as it makes the transition to a B-lineage-committed precursor. The development of mature B lymphocytes from bipotent progenitors is dependent on interleukin 7 (IL-7), a pre-B-cell growth factor, in addition to other stromal-cell-derived factors such as IL-11 and mast cell growth factor (MGF). IL-7 acts on pre-B lymphocytes, but the stage of differentiation at which B-lineage cells become responsive to this factor, and its potential role in lineage commitment have not been investigated thoroughly. Here, we examine the requirements for IL-7 during the development of B lymphocytes from bipotent progenitors. Furthermore, we define onset of B-lineage-associated gene expression during the development of committed B-lineage cells under defined culture conditions. RESULTS: We demonstrate that, under our experimental conditions, bipotent progenitors commit to differentiation through either the B or macrophage lineages within the first 3 days of culture. Cells that require IL-7 for survival first develop on day 3 of culture; however, commitment to the B lineage occurs at the same frequency in the presence or absence of this factor. After day 3 of culture, IL-7 is required both for the proliferation and survival of committed B-lineage progenitors and for the expression of several B-cell-associated genes, such as lambda5, VpreB, mb-1 and Rag1. CONCLUSIONS: Our results demonstrate that the growth factor combination of IL-11 and MGF provides sufficient support for bipotent progenitors to commit to either the B or the macrophage lineage. Single-cell cloning assays revealed that IL-7 does not influence the decision to commit to the B lineage, despite the observation that the bipotent cells potentially respond to IL-7, as indicated by an increase in cell number, prior to the commitment event. Furthermore, the addition of IL-7 to cells developing along the B-cell pathway promotes the expression of mRNA transcripts which encode several B-cell-specific genes.

Adaptor-mediated recruitment of RNA polymerase II to a signal-dependent activator.

The second messenger cAMP stimulates the expression of a number of target genes via the protein kinase A-mediated phosphorylation of CREB at Ser-133 (Gonzalez, G. A., and Montminy, M. R. (1989) Cell 59, 675-680). Ser-133 phosphorylation enhances CREB activity by promoting interaction with a 265-kDa CREB binding protein referred to as CBP (Arias, J., Alberts, A., Brindle, P., Claret, F., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994) Nature 370, 226-228; Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Nature 365, 855-859). The mechanism by which CBP in turn mediates induction of cAMP-responsive genes is unknown but is thought to involve recruitment of basal transcription factors to the promoter. Here we demonstrate that CBP associates specifically with RNA polymerase II in HeLa nuclear extracts. This association in turn permits RNA polymerase II to be recruited to CREB in a phospho-(Ser-133)-dependent manner. As anti-CBP antiserum, which inhibits recruitment of CBP and RNA polymerase II to phospho-(Ser-133) CREB, attenuates transcriptional induction by protein kinase A in vitro, our results demonstrate that the CBP-RNA polymerase II complex is critical for expression of cAMP-responsive genes.

In-vitro models of B-lineage commitment.

The development of mature B lymphocytes from multipotent progenitors follows a pathway of differentiation marked by a progressive restriction in lineage options. The requirements for progression through the B lineage developmental pathway have been investigated intensively and a number of critical components of the differentiation process have been identified. However, the genetic basis for lineage determination remains unresolved. Recently, a number of in-vitro assays have been established which support the development of committed B cell progenitors from multipotent cells. These assays have provided a novel system in which the process of B lineage commitment can be followed and manipulated. In this review we present a model of B-lineage progression from multipotent progenitors to committed B-cell progenitors and discuss potential mediators of the commitment process.

Murine B cell development: commitment and progression from multipotential progenitors to mature B lymphocytes.

B lymphocytes, the cellular source of antibody, are critical components of the immune response. They develop from multipotential stem cells, progressively acquiring the traits that allow them to function as mature B lymphocytes. This developmental program is dependent on appropriate interactions with the surrounding environment. These interactions, mediated by cell-cell and cell-matrix interactions, provide the growth and differentiation signals that promote progression along the developmental pathway. This chapter addresses the properties of developing B lineage cells and the nature of the environmental signals that support B lineage progression.

Stromal cell independent growth of bipotent B cell--macrophage precursors from murine fetal liver.

We have established stromal cell independent culture conditions under which single murine hematopoietic progenitor cells give rise to B lymphocytes and macrophages. IL-11, mast cell growth factor, and IL-7 are sufficient for the growth of B lineage cells from AA4.1+B220-Mac-1-Ly6A+ fetal liver cells isolated at day 12 of gestation. These conditions are also sufficient for commitment to myeloid differentiation, although CSF-1 is required for substantial growth of macrophages. The progenitors responding under these culture conditions: (i) require 8 days in culture before they become competent to respond to B cell mitogens in the presence of stromal cells and (ii) undergo a minimum of three cell divisions prior to Ig heavy chain allotype restriction. The findings suggest that the stromal cell dependency of immature hematopoietic cells may reflect a requirement for stromal cell derived growth factors. Further, this provides an in vitro system in which commitment and differentiation can be studied in single cells.

Characterization of murine CD10, an endopeptidase expressed on bone marrow adherent cells.

The CD10/neutral endopeptidase (NEP) gene was identified in murine genomic DNA using a human CD10 cDNA probe. It is transcribed most abundantly in kidneys resulting in RNA transcripts of 3.4, 6.0, and 6.2 kb. The activity of the murine CD10/NEP shows identical kinetic parameters, Km and Ki, to those observed for this enzyme in other species. Two mAbs raised against rabbit NEP detect a 100 kDa protein by Western blot analysis; the antigen immunoprecipitated from extracts of lung shows specific NEP activity. CD10/NEP, as analyzed by Western blot and enzymatic activity, is expressed at high levels in kidney and lung, and at lower levels in liver, brain, thymus, spleen, and bone marrow. Analysis of bone marrow subpopulations indicate that the majority of CD10/NEP is associated with cells adherent to plastic and with subpopulations that do not express the surface markers AA4.1, B220, Mac-1, and Gr-1. These results suggest that CD10 is primarily associated with the stromal elements in murine bone marrow. A bone marrow stromal line, BMS 2.2, also expresses high levels of CD10/NEP. This peptidase activity on the surface of stromal cells may influence lymphopoiesis or other hematopoietic processes through the hydrolysis of regulatory peptides in the microenvironment.

CD10 and CD44 genes of leukemic cells and malignant cell lines show no evidence of transformation-related alterations.

The expression of CD10/CALLA is associated primarily with childhood leukemia of pre-B lymphocyte phenotype. We have compared the hybridization pattern of the CALLA gene from leukemic and normal cells digested with several restriction enzymes. No alterations were noticed with Eco RI, Sac I, Pvu II, Eco RV, Hind III, and Msp I. Since CALLA is also found on other malignancies, we analyzed DNA samples prepared from cell lines derived from leukemia, lymphoma, glioblastoma, retinoblastoma, and neuroblastoma. Normal restriction patterns were observed for all the lines regardless of their CALLA phenotype. Having demonstrated previously that CALLA was structurally identical to neutral endopeptidase 3.4.24.11 (NEP), we have now established a correlation between surface expression of CALLA and NEP activity on leukemia samples and on several cell lines. Malignant cells tested expressed a functionally active enzyme and no gross alteration was present in the CALLA gene. The CD44 gene is expressed on most cells of hemopoietic origin and on greater than 95% of cases of acute lymphoblastic leukemia and acute myeloblastic leukemia studied. It is also expressed on normal astrocytes and on malignant cells of glioma/astrocytoma types. We now report that a similar pattern of hybridization was observed with Sac I, Pvu II, and Eco RI for leukemic samples, normal cells, and malignant cell lines. A polymorphism was recently detected for CD44 using Hind III; leukemic cells and malignant lines also showed this normal polymorphism. Thus no deletion or insertion could be detected in the CD44 gene of leukemic cells and malignant lines, suggesting that no gross DNA alterations were involved. The correlation between surface expression and enzymatic activity of CD10/CALLA and the expression of CD44 on a variety of malignant cells would suggest that the structure and function of these two gene products are probably not altered by the process of transformation.