Tom Kadesch

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.

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

ABSTRACT 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-Jκ, 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-Jκ 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.

Helix-loo-helix proteins in the regulation of immunoglobulin gene transcription

Transcriptional control of the Ig heavy chain and kappa light chain is dominated by single enhancers located within the body of each gene. These enhancers bind distinct, yet overlapping, sets of cell-type-specific and ubiquitous nuclear proteins. This review focuses on one particular subclass of enhancer-binding protein, termed helix-loop-helix (HLH) proteins, describing their role in the establishment of cell-type-specific transcription and suggesting how they may be regulated during B-cell maturation.

Repression of immunoglobulin enhancers by the helix-loop-helix protein Id : implications for B-lymphoid-cell development

It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.

Dual roles for the Notch target gene Hes-1 in the differentiation of 3T3-L1 preadipocytes.

ABSTRACT The process of adipogenesis involves a complex program of gene expression that includes down-regulation of the gene encoding Hes-1, a target of the Notch signaling pathway. To determine if Notch signaling affects adipogenesis, we exposed 3T3-L1 preadipocytes to the Notch ligand Jagged1 and found that differentiation was significantly reduced. This effect could be mimicked by constitutive expression of Hes-1. The block was associated with a complete loss of C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ) induction and could be overcome by retroviral expression of either C/EBPα or PPARγ2. Surprisingly, small interfering RNA (siRNA)-mediated reduction of Hes-1 mRNA in 3T3-L1 cells also inhibited differentiation, suggesting an additional, obligatory role for Hes-1 in adipogenesis. This role may be related to our observation that both Notch signaling and Hes-1 down-regulate transcription of the gene encoding DLK/Pref-1, a protein known to inhibit differentiation of 3T3-L1 cells. The results presented in this study establish a new target downstream of the Notch-Hes-1 pathway and suggest a dual role for Hes-1 in adipocyte development.

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.

The Notch Intracellular Domain Can Function as a Coactivator for LEF-1

ABSTRACT 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 β-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.

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 Deltex2ΔE (MDTX2ΔE), 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 MDTX2ΔE 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.

Inhibition of myogenesis by Notch: evidence for multiple pathways.

Notch signaling is critical for skeletal muscle development and regeneration, permitting the expansion of progenitor cells by preventing premature differentiation. We have interrogated the pathways through which ligand‐mediated signaling inhibits myogenesis by identifying Notch target genes and assessing their impact on differentiation in vitro. Notch activation led to the robust induction of the transcriptional repressors Hey1 and HeyL in myoblasts, but only constitutive expression of Hey1 blocked myogenesis. siRNA‐mediated knockdown of Hey1 had no effect on Notchs ability to inhibit differentiation, suggesting the existence of additional, possibly redundant pathways. We identified 82 genes whose expression was activated when C2C12 myoblasts were cultured in the presence of the Notch ligand Dll4. One of these, MyoR, is a novel Notch‐responsive gene, whose protein product is known to repress myogenesis in vitro. siRNA‐mediated knockdown of MyoR alone, or in combination with Hey1, was also ineffective at rescuing differentiation in the presence of Dll4. Our data support a model in which Notch signaling inhibits myogenesis through multiple pathways, two of which are defined by the Notch target genes Hey1 and MyoR. J. Cell. Physiol. 218: 84–93, 2009.

Identification and characterization of two functional domains within the murine heavy-chain enhancer.

We have investigated the effect of polymerizing defined segments of the immunoglobulin heavy-chain enhancer on the activity of a single, linked transcription unit. Transient assays in lymphoid cells have led to the following observations. First, polymerizing the entire enhancer led to an increase in overall transcription. Second, polymerizing defined DNA segments revealed two distinct functional domains within the enhancer. Although each domain alone possessed only partial enhancer activity, greater than wild-type levels of activity could be obtained upon polymerization. One of these domains contains three regions thought to be involved in protein binding in vivo and in vitro (E motifs E1, E2, and E3). The other domain contains the fourth E motif (E4) and the conserved octanucleotide, ATTTGCAT. We have tested the functional importance of these motifs by determining the effect of mutating these elements singly or in combination in the context of the isolated domains. Although E2, E3, E4, and the octanucleotide are clearly important for enhancer function, mutation of the E1 motif did not appear to have an effect on enhancer activity in our assay. Transient assays in mouse L cells indicate that nonlymphoid cells are able to use a distinct subset of these motifs.