Hes6 acts in a positive feedback loop with the neurogenins to promote neuronal differentiation.

During the development of the vertebrate nervous system, neurogenesis is promoted by proneural bHLH proteins such as the neurogenins, which act as potent transcriptional activators of neuronal differentiation genes. The pattern by which these proteins promote neuronal differentiation is thought to be governed by inhibitors, including a class of transcriptional repressors called the WRPW-bHLH proteins, which are similar to Drosophila proteins encoded by hairy and genes in the enhancer of split complex (E-(SPL)-C). Here, we describe the isolation and characterization of Hes6, which encodes a novel WRPW-bHLH protein expressed during neurogenesis in mouse and Xenopus embryos. We show that Hes6 expression follows that of neurogenins but precedes that of the neuronal differentiation genes. We provide several lines of evidence to show that Hes6 expression occurs in developing neurons and is induced by the proneural bHLH proteins but not by the Notch pathway. When ectopically expressed in Xenopus embryos, Hes6 promotes neurogenesis. The properties of Hes6 distinguish it from other members of the WRPW-bHLH family in vertebrates, and suggest that it acts in a positive-feedback loop with the proneural bHLH proteins to promote neuronal differentiation.

Activation of Xenopus genes required for lateral inhibition and neuronal differentiation during primary neurogenesis.

XNGN-1, a member of the neurogenin family of basic helix-loop-helix proteins, plays a critical role in promoting neuronal differentiation in Xenopus embryos. When ectopically expressed, XNGN-1 induces the expression of a set of genes required for neuronal differentiation such as XMyT1 and NeuroD. At the same time, however, XNGN-1 induces the expression of genes that antagonize neuronal differentiation by a process called lateral inhibition. Here, we present evidence that XNGN-1 activates the expression of genes required for differentiation and lateral inhibition by recruiting transcriptional coactivators p300/CBP (CREB-binding protein) or PCAF (p3OO/CBP-associated protein), both of which contain histone acetyltransferase (HAT) activity. Significantly, transcriptional activation of the genes in the lateral inhibitory pathway is less dependent on the HAT activity than is the activation of the genes that mediate differentiation. We propose that this difference enables the genes in the lateral inhibition pathway to be induced prior to the genes that promote differentiation, thus enabling lateral inhibition to establish a negative feedback loop and restrict the number of cells undergoing neuronal differentiation.

A two-step mechanism generates the spacing pattern of the ciliated cells in the skin of Xenopus embryos.

The skin of Xenopus embryos contains a population of specialized ciliated cells that are distributed in an evenly spaced pattern. Here we describe two successive steps that govern the differentiation and the generation of the spacing pattern of these ciliated cells. The first step occurs in the inner or sensorial layer of the non-neural ectoderm where a subset of cells are chosen to differentiate into ciliated-cell precursors. This choice is under the control of lateral inhibition mediated by a Suppressor of Hairless-dependent Notch signaling pathway, in which X-Delta-1 is the putative ligand driving the selection process, and a new Enhancer-of-Split-related gene is an epidermal target of Notch signaling. Because nascent ciliated-cell precursors prevent neighboring cells from taking on the same fate, a scattered pattern of these precursors is generated within the deep layer of the non-neural ectoderm. Ciliated-cell precursors then intercalate into the outer layer of cells in the epidermis. We show that the intercalation event acts as a second step to regulate the spacing of the mature ciliated cells. We propose that the differentiation of the ciliated cells is not only regulated by Notch-mediated lateral inhibition, but is also an example where differentiation is coupled to the movement of cells from one cell layer to another.

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.

Th2-specific DNase I-hypersensitive sites in the murine IL-13 and IL-4 intergenic region.

IL-4 and IL-13 are cytokines preferentially produced by Th2 cells, and their genes are located in close proximity on human chromosome 5 and mouse chromosome 11. To identify potential regulatory elements that confer Th2-specific expression of IL-4 and IL-13 genes, we constructed a physical map of the IL-13/IL-4 locus and conducted DNase I-hypersensitive (DH) site analysis using Th clones and in vitro-differentiated effector Th cells obtained from TCR transgenic mice. Three DH sites, HSS1, HSS2 and HSS3, were identified within the intergenic region between IL-13 and IL-4 genes. HSS3 was observed both in Th1 and Th2 cells as well as CD4+ naive T cells, while HSS1 and HSS2 were detected exclusively in Th2 cells. The correlation between differentiation into Th2 subtype and the appearance of HSS1 and HSS2 suggests that these regions may play a role in subtype-specific expression of the IL-13/IL-4 locus.

Molecular cloning and functional characterization of murine cDNA encoding transcription factor NFATc.

Transcription factors of the NFAT (nuclear factor of activated T cells) family play important roles in immune and inflammatory responses by regulating the expression of genes encoding cytokines and immunoregulatory proteins. Here we describe cloning and characterization of full-length cDNA encoding murine (m) NFATc which predicts that the protein has all the conserved structural motifs of NFAT family members, including the rel homology domain, the NFAT homology domain and the nuclear translocation signals. mNFATc complexed with AP-1 bound specifically to the murine IL-2 NFAT recognition sequence and activated transcription from the co-transfected IL-2 promoter in COS-7 cells. Northern blot analysis showed that the cDNA probe hybridized with a 4.5 kb transcript which is highly inducible in murine T cells. By Northern and in situ hybridization, mNFATc transcript was detected from the early stage of development. In the mouse embryo, mNFATc transcript was strongly expressed in thymus, lung and submandibular gland and weakly in skeletal muscle and heart suggesting that mNFATc may have a role both in embryogenesis and in mature T cells.

Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins.

We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter. In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter. EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3. In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components. Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4. NFAT/AP-1 complex formed only with P and P2. Cross-competition assays among the P-like elements revealed element-specific and common complexes. Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did not. It would thus appear that components of each P-like element-binding complexes are not identical and may coordinately contribute to transcriptional activity.

Calcineurin-dependent nuclear translocation of a murine transcription factor NFATx: molecular cloning and functional characterization.

Members of the nuclear factor of activated T cells (NFAT) are involved in the induction of a number of cytokine genes. We report here cDNA cloning and chromosomal localization of a murine homologue of human NFATx, designated as mNFATx1, and its splicing variants mNFATx2 and m delta NFATx. Northern blot analysis showed mNFATx1 to be predominantly expressed in the thymus. mNFATx1, but not m delta NFATx, produced in COS-7 cells, bound to all NFAT-binding sites of the interleukin (IL)-2 and IL-4 promoters tested. Immunofluorescence assay showed that both mNFATx1 and m delta NFATx introduced into COS-7 cells localized predominantly to the cytoplasm, but did translocate to the nucleus, either by cotransfection with an active form of calcineurin or wild-type calcineurin followed by stimulation with calcium ionophore. Translocation of mNFATx1 correlated well with activation of the murine IL-2 promoter; mNFATx1 translocated under conditions described above, in combination with phorbol 12-myristate 13-acetate, activated the transiently transfected murine IL-2 promoter. Thus, nuclear-translocated mNFATx1 is involved in activation of the IL-2 promoter. These results provide the first evidence for the requirement of calcineurin in the control of mNFATx imported from the cytoplasm to the nucleus and implies that mNFATx may possibly be a substrate of calcineurin in vivo.

Specific versus cooperative regulatory mechanisms of the cytokine genes that are clustered on the same chromosome.

The genes for IL-3, IL-4, IL-5, IL-9, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to be clustered on human chromosome 5q and on mouse chromosome 11. IL-2 and interferon gamma (IFN-gamma) genes are located on separate chromosomes. It is well known that upon stimulation by antigen presentation, TH1 and TH2 subsets of T helper cells start to transcribe distinct sets of cytokine genes. Thus mechanisms should exist that transmit extracellular signals into the nucleus, thereby coordinately turning on transcriptional machinery in cell type-specific manners. Several different mechanisms exist in which specific as well as coordinated expression of cytokines are regulated at the transcriptional level. These include (1) regulation by proximal cis-elements, to which specific transcription factors bind, (2) regulation by distal cis-elements, such as enhancers or locus controlling elements, especially those located several kilobases away from the target gene, and (3) enhancement of transcription by viral trans-activators in a pathologic state. In this article, we review the recent studies on the above issues, with particular emphasis on our own results that support the presence of different modes of control mechanisms. We also discuss the possible approaches to the thorough understanding of the coordinated and specific regulation of cytokines.

Cyclic AMP inhibits expression of the IL-2 gene through the nuclear factor of activated T cells (NF-AT) site, and transfection of NF-AT cDNAs abrogates the sensitivity of EL-4 cells to cyclic AMP.

cAMP inhibits PMA-induced IL-2 production at the transcriptional level in EL-4, a mouse lymphoma line. The region of the mouse IL-2 promoter covering positions from -321 to +46 relative to the transcription initiation site is required for activation by PMA and inhibition by cAMP. This region contains the nuclear factor of activated T cells (NF-AT), nuclear factor-kappa B (NF-kappa B), AP-1, and Oct binding sites, and the role of each element in responding to PMA and/or cAMP signals was characterized. The IL-2 promoter carrying mutations in each element reduced response to PMA while it retained sensitivity to cAMP, thereby suggesting that multiple elements contribute to positive and negative responses to PMA and cAMP, respectively. Using reporter plasmid carrying multiple copies of each element, we then found that the NF-AT construct was most effective in responding to PMA activation and to cAMP inhibition. Electrophoretic mobility shift assay revealed that, after exposure of cells to Bt2cAMP, NF-AT binding complex changed in amount or in mobility as a function of time. Furthermore, overexpression of the cytoplasmic component of NF-AT abrogated the inhibitory action of cAMP. These results indicate that the NF-AT site is a target of the inhibitory action of cAMP. In addition, binding of the NF-kappa B (p50/p65) heterodimer to the NF-kappa B site was inhibited by cAMP. Taken together, our data show that cAMP in EL-4 cells inhibits mouse IL-2 gene transcription through cis regulatory elements that include the NF-AT site as well as the NF-kappa B site.

Molecular cloning of a novel human cDNA encoding a zinc finger protein that binds to the interleukin-3 promoter.

The CT/GC-rich region (-76 to -47) is one transcriptional regulatory region of the interleukin-3 (IL-3) gene which confers basic transcriptional activity and responds to trans-activation by human T-cell leukemia virus type I-encoded Tax. We isolated three types of cDNAs encoding Cys2/His2-type zinc finger proteins that bind to this region. Two were identical to known transcription factors, EGR1 and EGR2, and the other clone, named DB1, encoded a novel protein of 516 amino acids with six zinc finger motifs. DB1 mRNA was present in human tissues, ubiquitously. Two constitutive transcripts of 4.0 and 4.8 kb in length were present in Jurkat cells. Electrophoretic mobility shift assay, with specific antibodies, showed that DB1 constitutively binds to this region whereas EGR1 binds in a T-cell activation-dependent manner. Overexpression of DB1 in Jurkat cells had no detectable effect on the transcription activity of the IL-3 promoter, in a transient-transfection assay. EGR1 and EGR2 increased IL-3 promoter activity when the transfected cells were stimulated with phorbol-12-myristate-13-acetate and A23187. When DB1 was cotransfected with a Tax expression vector, transcription activity of the IL-3 promoter induced by Tax was significantly increased, while EGR1 and EGR2 were without effect. These results suggest that EGR1 has a role in inducible transcription of the IL-3 gene, while DB1 sustains basal transcriptional activity and also cooperates with Tax to activate the IL-3 promoter.

cAMP activates the IL-5 promoter synergistically with phorbol ester through the signaling pathway involving protein kinase A in mouse thymoma line EL-4.

Expression of the IL-5 gene in T cells is induced in response to Ag stimulation; however, functional analysis of the IL-5 gene has been limited by lack of an appropriate transfection assay to facilitate measurement of the IL-5 promoter activity in response to T cell activation signals. Here, we describe a transient transfection system with which the IL-5 promoter activity can be assayed quantitatively. Using mouse thymoma line EL-4 cells, which produce several lymphokines including IL-2, IL-3, IL-4, IL-10, and GM-CSF in response to PMA, the effect of cAMP on IL-5 production was examined. These cells produce a low level of IL-5 when stimulated with PMA alone; however, N6, O2-dibutyryl cAMP (Bt2cAMP), in combination with PMA, augmented by more than tenfold the IL-5 production at the mRNA and the protein levels. Likewise, a transient transfection assay revealed that Bt2cAMP activated the IL-5 promoter more than tenfold, in a PMA-dependent manner, thereby indicating that two signals, PMA and cAMP, are required for optimal activation of the IL-5 promoter. Activation of the IL-5 promoter in response to Bt2cAMP and PMA depends on the region spanning from nucleotide position -1,200 to +33 relative to the transcription initiation site. Action of cAMP on the IL-5 promoter is mimicked by cotransfection of the expression plasmid containing cDNA encoding the catalytic subunit of protein kinase A, hence, cAMP probably exerts its action through the signaling pathway that involves protein kinase A. In contrast, Bt2cAMP almost completely inhibited the PMA-dependent activation of the endogenous IL-2 gene as well as the transfected IL-2 promoter. These results indicate that the IL-5 gene in EL-4 cells is positively regulated by cAMP in a manner opposite that for the IL-2 gene.

Reconstitution of the functional granulocyte macrophage colony stimulating factor promoter: evidence for distinct activation mechanisms that mediate the response to phorbol ester/calcium and human T cell leukemia virus type I Tax signals.

Functional elements in the promoter region of the mouse granulocyte-macrophage colony stimulating factor (GM-CSF) gene were assessed by constructing chimeric promoters linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and by employing a transient transfection assay of human T cell leukemia Jurkat cells. We previously reported that CLE2/GC-box (at positions -95 to -73, which is homologous to the NF-kappa B binding site) and CLE0 (at positions to -40) of the mouse GM-CSF promoter are essential for transcriptional activation in response to phorbol-12-myristate-13-acetate (PMA)/calcium ionophore (A23187). Here we show that CLE2/GC-box and the NF-kappa B binding motif are functionally interchangeable and that CLE2/GC-box and CLE0 as a unit activate the basic GM-CSF promoter in response to PMA/calcium signals. This unit is also capable of activating heterologous promoters in response to PMA/calcium signals. In addition, we show that Tax, the trans-activator encoded by human T cell leukemia virus type I (HTLV-I), activates the GM-CSF promoter via CLE2/GC-box without the involvement of CLE0. These results indicate that PMA/A23187-dependent and Tax-dependent activation of the GM-CSF gene proceeds through distinct mechanisms.

Activation of lymphokine genes in T cells: role of cis-acting DNA elements that respond to T cell activation signals.

Activation of T cells is initiated by the recognition of antigen on antigen presenting cells to exert the effector functions in immune and inflammatory responses. Two types of helper T cell (Th) clones (Th1 and Th2) are defined on the basis of different patterns of cytokine (lymphokine) secretion. They determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Although lymphokine genes are coordinately regulated upon antigen stimulation, they are regulated by the mechanisms common to all as well as those which are unique to each gene. For most lymphokine genes, a combination of phorbol esters (phorbol 12-myristate 13 acetate, PMA) and calcium ionophores (A23187) is required for their maximal induction. Yet phorbol ester alone or calcium ionophore alone produce several lymphokines. The production of the granulocyte-macrophage colony stimulating factor (GM-CSF) is completely dependent on the two signals. We have previously found a cis-acting region spanning the GM-CSF promoter region (positions -95 to +27) that confers inducibility to reporter genes in transient transfection assays. Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and conserved lymphokine element (CLE0). GM2 defines a binding site for protein(s) whose binding is inducible by PMA. One protein, NF-GM2 is similar to the transcription factor NF-kB. GC-box is a binding site for constitutively bound proteins. CLEO defines a binding site for protein(s) whose optimum binding is stimulated by PMA and A23187. Viral trans-activators such as Tax (human T cell leukemia virus-1, HTLV-1) and E2 (bovine papilloma virus, BPV) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor (TCR) mediated signaling. The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them. The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Sp1 to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation.