The notch intracellular domain can function as a coactivator for LEF-1.

Notch signaling commences with two ligand-mediated proteolysis events that release the Notch intracellular domain, NICD, from the plasma membrane. NICD then translocates into the nucleus and interacts with the DNA binding protein CSL to activate transcription. We found that NICD expression also potentiates activity of the transcription factor LEF-1. NICD stimulation of LEF-1 activity was context dependent and occurred on a subset of promoters distinct from those activated by beta-catenin. Importantly, the effect of NICD does not appear to be mediated through canonical components of the Wnt signaling pathway or downstream components of the Notch pathway. In vitro assays show a weak association between the C-terminal transactivation domain of NICD and the high-mobility group domain of LEF-1, suggesting that the two proteins interact in vivo. Our data therefore describe a new nuclear target of Notch signaling and a new coactivator for LEF-1.

Identification of a novel activation domain in the Notch-responsive transcription factor CSL.

CSL is the primary target of the Notch signaling pathway in mammalian cells. It is a DNA binding protein that generally represses transcription in the absence of Notch signaling and activates transcription upon formation of a ternary complex with NICD, the protease-generated intracellular domain of NOTCH: Previous mapping experiments identified the central third of CSL as both necessary and sufficient for DNA binding and activation by NOTCH: Here we show that CSL promotes transcription in 293T cells in the absence of added NICD and that this activity requires both the central domain plus the C-terminal third of the protein. Evidence is presented that argues against a contribution of endogenous NICD and instead supports the possibility that distinct coactivators may directly stimulate the activity of CSL in a cell type-specific manner. This conclusion supports a recent finding that Drosophila CSL (Suppressor of Hairless) can also mediate transcriptional activation in the absence of NOTCH:

Murine homologs of deltex define a novel gene family involved in vertebrate Notch signaling and neurogenesis.

Notch signaling plays an important role in cell-fate specification in multicellular organisms by regulating cell-cell communication. The Drosophila deltex gene encodes a modulator of the Notch pathway that has been shown to interact physically with the Ankyrin repeats of Notch. We isolated four distinct cDNAs corresponding to mouse homologs of deltex - mouse Deltex1 (MDTX1), mouse Deltex2 (MDTX2), mouse Deltex2DeltaE (MDTX2DeltaE), and mouse Deltex3 (MDTX3). Deduced amino acid sequences of these four cDNAs showed a high degree of similarity to Drosophila Deltex and its human homolog, DTX1 throughout their lengths, even though they possess distinct structural features. MDTX proteins formed homotypic and heterotypic multimers. We found that these genes were expressed in the central, peripheral nervous system and in the thymus, overlapping with those of mouse Notch1. In mammalian tissue culture cells, overexpression of any of the four mouse deltex homologs suppressed the transcriptional activity of E47, a basic helix-loop-helix (bHLH) protein, in a manner similar to suppression by an activated form of human Notch1 or human DTX1. In addition, overexpression of MDTX2 and MDTX2DeltaE in C2C12 cells under differentiation-inducing conditions suppressed the expression of myogenin, one of the myogenic transcriptional factors; this was also similar to a previously reported activity of constitutively activated Notch. Furthermore, misexpression of any of the MDTX genes in Xenopus embryos resulted in an expansion of the region expressing the neural cell adhesion molecule (N-CAM) gene, a marker for the neuroepithelium. Collectively, our results suggest that these mouse deltex homologs are involved in vertebrate Notch signaling and regulation of neurogenesis.

Hematopoietic expression of HOXB4 is regulated in normal and leukemic stem cells through transcriptional activation of the HOXB4 promoter by upstream stimulating factor (USF)-1 and USF-2.

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34(+) cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.

Isolation and characterization of the notch ligand delta4.

Notch signaling plays a critical role in a variety of developmental programs. In vertebrates, the complexity of the process is underscored by the existence of multiple Notch receptors and multiple ligands, each of which displays a distinct expression profile. Furthermore, the ligands can be subdivided into two families, the Serrate/Jagged family and the Delta family. Here we present the isolation of a novel Notch ligand, Delta4. Expression analyses indicate that mouse Delta4 is highly expressed in the eye and lung during embryogenesis and in the heart, lung, liver, and kidney of the adult. Functionally, Delta4 is indistinguishable from Jagged1 in its abilities to inhibit myogenesis and to stimulate transcription through Notch1 and the DNA binding protein CSL.

Notch1 expression in early lymphopoiesis influences B versus T lineage determination.

Notch receptors regulate fate decisions in many cells. One outcome of Notch signaling is differentiation of bipotential precursors into one cell type versus another. To investigate consequences of Notch1 expression in hematolymphoid progenitors, mice were reconstituted with bone marrow (BM) transduced with retroviruses encoding a constitutively active form of Notch1. Although neither granulocyte or monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced BM contained immature CD4+ CD8+ T cells in the BM and exhibited a simultaneous block in early B cell lymphopoiesis. These results suggest that Notch1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions, possibly by influencing lineage commitment from a common lymphoid progenitor cell.

A histone deacetylase corepressor complex regulates the Notch signal transduction pathway.

The Delta-Notch signal transduction pathway has widespread roles in animal development in which it appears to control cell fate. CBF1/RBP-Jkappa, the mammalian homolog of Drosophila Suppressor of Hairless [Su(H)], switches from a transcriptional repressor to an activator upon Notch activation. The mechanism whereby Notch regulates this switch is not clear. In this report we show that prior to induction CBF1/RBP-Jkappa interacts with a corepressor complex containing SMRT (silencing mediator of retinoid and thyroid hormone receptors) and the histone deacetylase HDAC-1. This complex binds via the CBF1 repression domain, and mutants defective in repression fail to interact with the complex. Activation by Notch disrupts the formation of the repressor complex, thus establishing a molecular basis for the Notch switch. Finally, ESR-1, a Xenopus gene activated by Notch and X-Su(H), is induced in animal caps treated with TSA, an inhibitor of HDAC-1. The functional role for the SMRT/HDAC-1 complex in CBF1/RBP-Jkappa regulation reveals a novel genetic switch in which extracellular ligands control the status of critical nuclear cofactor complexes.

Transcriptional repression of the IL-2 gene in Th cells by ZEB.

Th1- and Th2-type cells mediate distinct effector functions via cytokine secretion in response to immunologic challenge. Precursor Th cells transcribe IFN-gamma, IL-2, and IL-4 upon activation. Repeated stimulation of Th precursor cells in the presence of IL-4 leads to terminally differentiated Th2 cells that have lost the ability to transcribe the IL-2 gene. We provide evidence that repression of IL-2 gene expression in Th2 cells and partial repression in Th1 cells are mediated by ZEB, a zinc finger, E box-binding transcription factor. This factor binds to a negative regulatory element, NRE-A, in the IL-2 promoter, thereby acting as a potent repressor of IL-2 transcription.

Human deltex is a conserved regulator of Notch signalling.

A fundamental cell-fate control mechanism regulating multicellular development is defined by the Notch-signalling pathway. Developmental and genetic studies of wild type and activated Notch-receptor expression in diverse organisms suggest that Notch plays a general role in development by governing the ability of undifferentiated precursor cells to respond to specific signals. Notch signalling has been conserved throughout evolution and controls the differentiation of a broad spectrum of cell types during development. Genetic studies in Drosophila have led to the identification of several components of the Notch pathway. Two of the positive regulators of the pathway are encoded by the suppressor of hairless [Su(H)] and deltex (dx) genes. Drosophila dx encodes a ubiquitous, novel cytoplasmic protein of unknown biochemical function. We have cloned a human deltex homologue and characterized it in parallel with its Drosophila counterpart in biochemical assays to assess deltex function. Both human and Drosophila deltex bind to Notch across species and carry putative SH3-binding domains. Using the yeast interaction trap system, we find that Drosophila and human deltex bind to the human SH3-domain containing protein Grb2 (ref. 10). Results from two different reporter assays allow us for the first time to associate deltex with Notch-dependent transcriptional events. We present evidence linking deltex to the modulation of basic helix-loop-helix (bHLH) transcription factor activity.

Notch inhibition of E47 supports the existence of a novel signaling pathway.

E47 is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, we found that activated Notch1 and Notch2 effectively inhibit E47 activity. Only the intact E47 protein was inhibited by Notch-fusion proteins containing isolated DNA binding and activation domains were unaffected-suggesting that Notch targets an atypical E47 cofactor. Although overexpression of the coactivator p300 partially reversed E47 inhibition, results of several assays indicated that p300/CBP is not a general target of Notch. Notch inhibition of E47 did not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in drosophila. Importantly, E47 was inhibited independently of CBF1/RPB-Jkappa by Deltex, a second Notch-interacting protein. We provide evidence that Notch and Deltex may act on E47 by inhibiting signaling through Ras because (i) full E47 activity was found to be dependent on Ras and (ii) both Notch and Deltex inhibited GAL4-Jun, a hybrid transcription factor whose activity is dependent on signaling from Ras to SAPK/JNK.

Decreased immunoglobulin deposition in tumors and increased immature B cells in p53-null mice.

Recent studies have hinted that there may be a relationship between p53 and the immune response. In preliminary experiments, we found significantly decreased levels of immunoglobulin deposition in 13 of 16 p53-null tumors compared with 2 of 17 tumors derived from p53 +/- mice. We further explored the effect of p53 on B-cell development and function. p53-null mice contained more splenic white pulp and more immature B cells in the bone marrow compared with p53 +/- mice. p53-null B cells were hyperresponsive to proliferative challenge but were not more resistant to signal-induced apoptosis. Several p53 DNA-binding sites were localized to the regulatory regions of immunoglobulin heavy and light chain genes, including the KII site, which serves as an enhancer for rearrangement of the mouse kappa chain J cluster genes. Levels of p53 protein and the kappa chain sterile transcript increased after exposure of pre-B cells to the DNA damaging agents etoposide and Adriamycin. Our observations suggest that p53 may be involved in B-cell maturation and may relay certain stress signals to affect B-cell function.

Selective utilization of basic helix-loop-helix-leucine zipper proteins at the immunoglobulin heavy-chain enhancer.

The microE3 E box within the immunoglobulin heavy-chain (IgH) enhancer binds several proteins of the basic helix-loop-helix-leucine zipper (bHLHzip) class, including TFE3, USF1, and Max. Both TFE3 and USF have been described as transcriptional activators, and so we investigated their possible roles in activating the IgH enhancer in vivo. Although TFE3 activated various enhancer-based reporters, both USF1 and Max effectively inhibited transcription. Inhibition by USF correlated with the lack of a strong activation domain and was the result of the protein neutralizing the microE3 site. The effects of dominant-negative derivatives of TFE3 and USF1 confirmed that TFE3, or a TFE3-like protein, is the primary cellular bHLHzip protein that activates the IgH enhancer. In addition to providing a physiological role for TFE3, our results call into question the traditional view of USF1 as an obligate transcriptional activator.

Phosphorylation of E47 as a potential determinant of B-cell-specific activity.

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.

HMG box-activating factors 1 and 2, two HMG box transcription factors that bind the human Ig heavy chain enhancer.

We present the isolation of two cDNAs that encode distinct, yet related, proteins that bind the HE2 region of the human Ig heavy chain (IgH) enhancer. Designated HMG box-activating factors (HAF) 1 and 2, the two proteins are new members of the HMG box family of DNA binding proteins. Both are potent transcription activators when expressed 1) as GAL4 fusions targeted to promoters containing GAL4 operators, or 2) as intact proteins targeted to minimal promoters containing binding sites derived from the IgH enhancer. HAF-1 and HAF-2 mRNAs are apparently expressed in both B cells and non-B cells. However, activity generated by the isolated HE2 region in B cells is dependent on both an intact HAF-1/HAF-2 binding site and at least one additional site that has been reported previously to bind a B cell-restricted protein. Our results suggest a collaborative role for either or both HAF-1 and HAF-2 in establishing the B cell activity of the human IgH enhancer.

E47 activates the Ig-heavy chain and TdT loci in non-B cells.

The E2A proteins, E12 and E47, are basic helix-loop-helix (bHLH) proteins essential for the B-cell lineage. Initially identified as immunoglobulin enhancer-binding proteins, they have also been shown to activate immunoglobulin enhancer-based reporters in transient transfection assays. Here, we examine the relationship between E2A DNA binding activity and activation of the endogenous, chromosomal immunoglobulin heavy chain (IgH) locus. Using sterile I(mu) transcription as an indicator of IgH enhancer activity, we see a direct correlation between E2A DNA binding activity and I(mu) transcription in stable BxT hybrids. We also observe a 1000-fold stimulation of endogenous I(mu) transcription in fibroblasts that express high levels of E47 and less stimulation in cells that express E12. By contrast, none of the other IgH enhancer-binding proteins tested (E2-2, Pu.1, Oct-2, OCA-B, TFE3 and USF) were able to activate I(mu) transcription. E47 overexpression also resulted in transcriptional activation of the endogenous gene encoding TdT, indicating that it, too, is a target of E2A proteins early in the B-cell lineage. Our results indicate that E2A proteins have the distinctive property of activating silent, chromatin-embedded B-cell-specific genes, underscoring their crucial role in B-cell development.

Cloning of a cDNA encoding a mouse transcriptional repressor displaying striking sequence conservation across vertebrates.

The nucleotide sequence encoding an approx. 120-kDa transcriptional repressor (MEB1) was determined from a cDNA which was cloned from a mouse brain library. An alignment of the deduced amino-acid sequences of the putative functional domains of MEB1 with those from the human, hamster and chicken homologues reveals a dramatic degree of conservation.

B-cell-specific DNA binding by an E47 homodimer.

B cells express a unique E-box-binding activity that contains basic helix-loop-helix (bHLH) proteins encoded by the E2A gene. E2A proteins play a central role in immunoglobulin gene transcription and are also required for the generation of the B-lymphocyte lineage. In muscle, E2A proteins bind DNA as heterodimers with muscle-specific bHLH partners, such as MyoD and myogenin, and these heterodimers are thought to be both necessary and sufficient for muscle determination in cultured cells. Our results indicate that in B cells, the bHLH partners for E2A proteins are not B-cell-restricted proteins, but are the E2A proteins themselves. UV cross-linking, gel purification, and the analysis of "forced heterodimers" indicate that BCF1 is primarily a homodimer of the E2A protein E47. Since E47 is widely expressed, our results argue for a difference in the inherent DNA-binding properties of the E47 protein in B cells and may help explain the restricted B-lineage defect observed in E2A-deficient mice.

Constitutively expressed Oct-2 prevents immunoglobulin gene silencing in myeloma x T cell hybrids.

Recent experiments involving disruption of the Oct-2 gene have shown that this largely B cell-restricted transcription factor is not required in the early stages of B cell development. However, B cells that lack Oct-2 may be blocked from differentiation past the surface immunoglobulin-positive stage. To identify a possible function for Oct-2 in the late stage immunoglobulin-secreting cell, we have used the method of somatic cell fusion. When the immunoglobulin-producing myeloma MPC11 is fused to a T lymphoma, Oct-2 production ceases, as does the expression of immunoglobulin, J chain, and several other B cell-specific gene products. In the present study, we show that by preventing the loss of Oct-2 in the hybrid cells, we can preserve expression of all other tested B cell-specific genes. These results establish a central role for Oct-2 in maintaining the genetic program of the immunoglobulin-secreting plasmacyte.