TIF1beta functions as a coactivator for C/EBPbeta and is required for induced differentiation in the myelomonocytic cell line U937.

Representational difference analysis (RDA) cloning has identified transcriptional intermediary factor 1 beta (TIF1beta) as a gene inducibly expressed early during myeloid differentiation of the promyelocytic cell lines HL-60 and U937. To assess the role of TIF1beta, U937 cell lines were made that expressed antisense-hammerhead ribozymes targeted specifically against TIF1beta mRNA. These cells failed to differentiate into macrophages, as determined by several criteria: a nonadherent morphology, a failure to arrest cell cycle, lowered levels of macrophage-specific cell surface markers, resistance to Legionella pneumophila infection, a loss of the ability to phagocytose and chemotax, and decreased expression of chemokine mRNAs. One way TIF1beta acts in macrophage differentiation is to augment C/EBPbeta transcriptional activity. Furthermore, we show by EMSA supershifts and coimmunoprecipitation that C/EBPbeta and TIF1beta physically interact. Although TIF1beta is necessary for macrophage differentiation of U937 cells, it is not sufficient, based on the inability of ectopically expressed TIF1beta to induce or augment phorbol ester-induced macrophage differentiation. We conclude that TIF1beta plays an important role in the terminal differentiation program of macrophages, which involves the coactivation of C/EBPbeta and induction of C/EBPbeta-responsive myeloid genes.

Human NM23/nucleoside diphosphate kinase regulates gene expression through DNA binding to nuclease-hypersensitive transcriptional elements.

NM23-H2/NDP kinase B has been identified as a sequence-specific DNA-binding protein with affinity for a nuclease-hypersensitive element of the c-MYC gene promoter (Postel et al., 1993). The ability of Nm23-H2 to activate c-MYC transcription in vitro and in vivo via the same element demonstrates the biological significance of this interaction. Mutational analyses have identified Arg34, Asn69 and Lys135 as critical for DNA binding, but not required for the NDP kinase reaction. However, the catalytically important His118 residue is dispensible for sequence-specific DNA binding, suggesting that sequence-specific DNA recognition and phosphoryl transfer are independent properties. Nm23-H2 also has an activity that cleaves DNA site-specifically, involving a covalent protein-DNA complex. In a DNA sequence-dependent manner, Nm23-H2 recognizes additional target genes for activation, including myeloperoxidase, CD11b, and CCR5, all involved in myeloid-specific differentiation. Moreover, both NM23-H1 and Nm23-H2 bind to nuclease hypersensitive elements in the platelet-derived growth factor PDGF-A gene promoter sequence-specifically, correlating with either positive or negative transcriptional regulation. These data support a model in which NM23/NDP kinase modulates gene expression through DNA binding and subsequent structural transactions.

A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis.

NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.

The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4.

The molecular basis for the distinctive cytokine expression of CD4+ T helper 1 (Th1) and T helper 2 (Th2) subsets remains elusive. Here, we report that the proto-oncogene c-maf, a basic region/leucine zipper transcription factor, controls tissue-specific expression of IL-4. c-Maf is expressed in Th2 but not Th1 clones and is induced during normal precursor cell differentiation along a Th2 but not Th1 lineage. c-Maf binds to a c-Maf response element (MARE) in the proximal IL-4 promoter adjacent to a site footprinted by extracts from Th2 but not Th1 clones. Ectopic expression of c-Maf transactivates the IL-4 promoter in Th1 cells, B cells, and nonlymphoid cells, a function that maps to the MARE and Th2-specific footprint. Furthermore, c-Maf acts in synergy with the nuclear factor of activated T cells (NF-ATp) to initiate endogeneous IL-4 production by B cells. Manipulation of c-Maf may alter Th subset ratios in human disease.

Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation.

The transcription factors NFAT and AP-1 have been shown to be essential for inducible interleukin-2 (IL-2) expression in activated T cells. NFAT has been previously reported to bind to two sites in the IL-2 promoter: in association with AP-1 at the distal antigen response element at -280 and at -135. On the basis of DNase I footprinting with recombinant NFAT and AP-1 proteins, gel shift assays, and transfection experiments, we have identified three additional NFAT sites in the IL-2 promoter. Strikingly, all five NFAT sites are essential for the full induction of promoter activity in response to T-cell receptor stimulation. Four of the five NFAT sites are part of composite elements able to bind AP-1 in association with NFAT. These sites display a diverse range of cooperativity and interdependency on NFAT and AP-1 proteins for binding. One of the NFAT sites directly overlaps the CD28-responsive element. We present evidence that CD28 inducibility is conferred by the AP-1 component in NFAT-AP-1 composite elements. These findings provide further insight into the mechanisms involved in the regulation of the IL-2 promoter.

The proximal promoter of the IL-4 gene is composed of multiple essential regulatory sites that bind at least two distinct factors.

Immune responses to pathogens often lead to the generation of polarized T helper subsets designated Th1 and Th2. Th1 cells, characterized by the production of IL-2 and IFN-gamma, stimulate cellular immune responses important for protection against intracellular pathogens. In contrast, Th2 cells, which produce IL-4, are potent stimulators of B cells and stimulate protection against extracellular pathogens. IL-4 has also emerged as a key cytokine in T cell differentiation since it has been shown to direct the development of naive T cells toward a Th2 phenotype. Recent studies have provided insights into the transcriptional regulation of IL-4, including the identification of multiple binding sites for a subunit of the IL-2 transcription factor NF-AT. In this study we describe the characterization of an essential region of the IL-4 promoter located immediately upstream of the TATA element. High-resolution mutagenesis of this 33-bp region revealed multiple sites indispensable for inducible IL-4 transcription. Included in this region are overlapping binding sites for the cyclosporin A-sensitive factor NF-ATp and a novel constitutively expressed factor designated PCC. An additional sequence adjacent to the TATA element is also shown to be critical for IL-4 transcription in Th2 cells.

Coordinate and cooperative roles for NF-AT and AP-1 in the regulation of the murine IL-4 gene.

The transcription factor NF-AT plays an essential role in the inducible transcription of several cytokine genes during T cell activation. The distal NF-AT site of the murine IL-2 promoter binds both NF-AT and AP-1 proteins, and thus represents a composite regulatory site that integrates Ca(2+)- and PKC-dependent signaling pathways in T cell activation. However, the individual contributions of the NF-AT and AP-1 components to promoter activity via this composite site have not been resolved, owing to the absence of a clearly defined AP-1 binding site, which, when mutated abolishes AP-1 binding. We describe here an apparently analogous NF-AT/AP-1 composite site in the murine IL-4 promoter, which can be mutated to selectively block the recruitment of each component. We show that the cooperative and coordinate involvement of both NF-AT and AP-1 is necessary for full activity of the NF-AT/AP-1 composite site, and, ultimately, the entire IL-4 promoter.

A common factor regulates both Th1- and Th2-specific cytokine gene expression.

Murine T helper cell clones are classified into two distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), on the basis of cytokine secretion patterns. Th1 clones produce interleukin-2 (IL-2), tumor necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), while Th2 clones produce IL-4, IL-5, IL-6 and IL-10. These subsets differentially promote delayed-type hypersensitivity or antibody responses, respectively. The nuclear factor NF-AT is induced in Th1 clones stimulated through the T cell receptor-CD3 complex, and is required for IL-2 gene induction. The NF-AT complex consists of two components: NF-ATp, which pre-exists in the cytosol and whose appearance in the nucleus is induced by an increase of intracellular calcium, and a nuclear AP-1 component whose induction is dependent upon activation of protein kinase C (PKC). Here we report that the induction of the Th2-specific IL-4 gene in an activated Th2 clone involves an NF-AT complex that consists only of NF-ATp, and not the AP-1 component. On the basis of binding experiments we show that this 'AP-1-less' NF-AT complex is specific for the IL-4 promoter and does not reflect the inability of activated Th2 cells to induce the AP-1 component. We propose that NF-ATp is a common regulatory factor for both Th1 and Th2 cytokine genes, and that the involvement of PKC-dependent factors, such as AP-1, may help determine Th1-/Th2-specific patterns of gene expression.

Transforming growth factor beta 1 repression of the HLA-DR alpha gene is mediated by conserved proximal promoter elements.

Transforming growth factor beta 1 (TGF-beta 1) is a pleiotropic cytokine that decreases the expression of class II MHC Ag in the melanoma cell line Hs294T(c). To investigate the mechanism of this repression, we have examined the effect of TGF-beta 1 on expression of the HLA-DR alpha gene. Both the constitutive level of HLA-DR protein and DR alpha mRNA were repressed by treatment with TGF-beta 1. The proximal 176 bp of the DR alpha promoter were sufficient to confer TGF-beta 1 repression on a reporter gene. Deletional and mutational analysis of the DR alpha promoter revealed that the conserved S and X1 promoter elements were important for basal expression of DR alpha and also mediated the down-regulation by TGF-beta 1. Mobility shift assays and in vivo footprinting showed no change in occupancy of the proximal DR alpha promoter after TGF-beta 1 treatment. These results identify the DNA elements that mediate repression of the HLA-DR alpha gene by TGF-beta 1 and suggest that TGF-beta 1 acts at these sites without causing a change in promoter occupancy.

SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae gene SPT15, which encodes the TATA-binding protein TFIID, have been shown to cause pleiotropic phenotypes and to lead to changes in transcription in vivo. Here, we report the cloning and analysis of one such mutation, spt15-21, which causes a single-amino-acid substitution in a conserved residue of TFIID. Surprisingly, the spt15-21 mutation does not affect the stability of TFIID, its ability to bind to DNA or to support basal transcription in vitro, or the ability of an upstream activator to function in vivo. To study further the spt15-21 defect, extragenic suppressors of this mutation were isolated and analyzed. All of the extragenic suppressors of spt15-21 are mutations in the previously identified SPT3 gene. Suppression of spt15-21 by these spt3 mutations is allele-specific, suggesting that TFIID and SPT3 interact and that spt15-21 impairs this interaction in some way. Consistent with these genetic data, coimmunoprecipitation experiments demonstrate that the TFIID and SPT3 proteins are physically associated in yeast extracts. Taken together, these results suggest that SPT3 is a TFIID-associated protein, required for TFIID to function at particular promoters in vivo.

Cross-linking of surface IgM activates NF-kappa B in B lymphocyte.

In B lymphocytes, cross-linking of surface IgM activates changes in both the cell cycle and differentiation. In normal B cells and B cell tumors, many stimuli induce the activation of NF-kappa B and its translocation from the cytoplasm to the nucleus. In this study we sought to determine if cross-linking of surface IgM led to the activation of NF-kappa B. Our results show that activation of B cells by cross-linking anti-IgM antibodies activated NF-kappa B in the murine B lymphoid cell lines 70Z/3 and M12, and in the dense fraction of splenic cells. The activation of NF-kappa B required optimal doses of anti-IgM antibodies and took 5 to 10 min to reach maximal levels. Cross-linking of IgM has also been shown to activate protein kinases including protein kinase C (PKC). To test whether PKC activation was required for NF-kappa B translocation, we treated 70Z/3 cells for 18 h with phorbol 12-myristate 13-acetate, a procedure which depletes these cells of functional PKC. This treatment did not abrogate the nuclear translocation of NF-kappa B following anti-IgM cross-linking. These results indicate that the nuclear translocation of NF-kappa B is rapidly induced by surface IgM cross-linking and that this activation appears to use a pathway which does not require PKC.

Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

Evidence that interleukin-1 and phorbol esters activate NF-kappa B by different pathways: role of protein kinase C.

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor that affects expression of many genes, including immunoglobulin kappa (kappa), the interleukin-2 receptor alpha chain, and two genes in HIV-1. NF-kappa B can be activated by a number of stimuli, including pharmacological stimulation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and treatment in vitro with either protein kinase C or protein kinase A. This has lead to the proposal that these kinases are key enzymes in the physiological activation of NF-kappa B as well. We have used a murine B cell line, 70Z/3, and T cell line, EL-4 6.1 C10, to study the activation of NF-kappa B by two physiological activators, interleukin-1 alpha (IL-1) and lipopolysaccharide (LPS). There are four reasons to propose that these agents activate pathways that do not include protein kinase C as a major component in these cell lines. First, the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) strongly inhibited PMA-induced activation of NF-kappa B in 70Z/3 cells but had no effect on NF-kappa B activated by IL-1 or LPS. Second, depletion of protein kinase C by prolonged growth of 70Z/3 in PMA abrogated the capacity of the cells to activate NF-kappa B in response to further PMA treatment. However, these same cells activated NF-kappa B normally after either IL-1 or LPS treatment. Third, IL-1 effectively activated NF-kappa B in EL-4 6.1 C10 cells, but PMA did not. Fourth, interferon-gamma is a potent activator of protein kinase C in 70Z/3 cells, but is completely inactive in the mobilization of NF-kappa B. These results suggest that the physiological inducers IL-1 and LPS activate NF-kappa B by pathways independent of protein kinase C in both 70Z/3 and EL-4 6.1 C10 cells.

Role of cAMP in interleukin-1-induced kappa light chain gene expression in murine B cell line.

The murine lymphoid cell line 70Z/3 has been extensively used to study the intracellular mechanisms of interleukin-1 (IL-1) action. In these cells IL-1 is known to induce kappa gene expression but the signal transduction pathway has yet to be defined. IL-1-induced kappa expression is associated with stimulation of Na+/H+ exchange and activation of protein kinase C, but these events are not sufficient to trigger kappa expression. Thus, other signals must be present. Because cAMP is a well recognized second messenger, we sought to determine whether cAMP is the signal that triggers IL-1-induced kappa expression. To that end we first measured intracellular levels of cAMP following IL-1 treatment. The results showed that exposure of 70Z/3 cells to IL-1 alpha induced a rapid and a transient increase in cAMP, it peaked at 5 min and was back to base-line level at 20 min. Prostaglandin E2 (PGE2) also increased cAMP with similar kinetics to IL-1 alpha but the increased levels were far greater. IL-1 alpha-induced increase in cAMP proved not be a sufficient signal because an increase in intracellular cAMP by N6,O2'-dibutyryl cAMP (Bt2cAMP) or PGE2 failed to increase surface IgM or to increase kappa mRNA level. Although when used alone they had no effect, Bt2cAMP and PGE2 were found to amplify the IL-1 alpha-induced kappa expression. IL-1 alpha transiently activated NF-kappa B transcription factor. But this effect could not be simulated by Bt2cAMP or PGE2. This observation provides further evidence that cAMP is not a trigger of kappa expression. Although Bt2cAMP or PGE2 when used alone had no effect, they did consistently modify the level of NF-kappa B activity induced by IL-1 alpha. Results of this study show that cAMP is not sufficient to induce NF-kappa B or kappa expression. Therefore, the role of cAMP may not be trigger but rather to modulate the IL-1 alpha-induced kappa expression. Regulation of the response could occur at one or a number of points along the signal pathway. Such a regulatory role is supported by the observation that cAMP modulates the IL-1 alpha-induced NF-kappa B activity.

Slow response variant of the B lymphoma 70Z/3 defective in LPS activation of NF-kappa B.

The mouse B cell lymphoma 70Z/3 is membrane immunoglobulin M (mIgM) negative, but treatment of the cells with bacterial lipopolysaccharide (LPS) induces the expression of kappa (kappa) light chain synthesis, and the cells become mIgM+. In wild type cells, this reaction is maximal after 24 h; we have isolated a variant, 1B8, which becomes mIgM+ only after a more prolonged incubation with LPS. This delayed response results from a reduced rate of accumulation of (kappa) mRNA and protein. The transcription factor, NF-kappa B is present in the cytoplasm of both the wild type and the variant cells in its inactive form. The delay in kappa expression is correlated with the failure of NF-kappa B to be activated and translocated to the nucleus. Although NF-kappa B cannot be activated by LPS, it can be activated by treatment with phorbol ester (PMA). In contrast to the clear defect in NF-kappa B, LPS treatment of 1B8 cells causes the octamer-binding factor OTF-2 to increase normally. We conclude that the defect in 1B8 cells is in an early part of the LPS activation pathway, prior to the activation of NF-kappa B, but after the signal for OTF-2 induction. The phenotype of 1B8 demonstrates that an increase in OTF-2 alone is sufficient to cause a large increase in kappa transcription in 70Z/3 cells, but that without NF-kappa B, the response is slow to develop. In this view, NF-kappa B functions to facilitate kappa transcription and to speed its rate of increase, but is not required for the long-term response of 70Z/3 cells to LPS.

1.3E2, a variant of the B lymphoma 70Z/3, defective in activation of NF-kappa B and OTF-2.

The mouse B-cell cell lymphoma 70Z/3 is a convenient model system in which to study the regulation of immunoglobulin synthesis. Three transcriptional activators of kappa (kappa) light chain synthesis have been identified for these cells: bacterial lipopolysaccharide (LPS), interferon-gamma (IFN), and interleukin-1 (IL-1). The response of the kappa gene in 70Z/3 cells to LPS is mediated by increases in two transcription factors: NF-kappa B and OTF-2. In contrast, IFN has no effect on either of these factors in 70Z/3 cells. We have isolated by immunoselection an LPS- IFN+ variant of 70Z/3 called 1.3E2. We show here that LPS treatment of these cells causes no increase in nuclear localization of either NF-kappa B or OTF-2. Although they have normal levels of cytoplasmic NF-kappa B, it cannot be activated by LPS or by phorbol 12-myristate 13-acetate (PMA) treatment of the cells. These experiments expand the genetic dissection of the molecular pathways of activation of kappa transcription in 70Z/3 cells.

A gamma interferon-unresponsive variant of cell line 70Z/3, IFN-4, can be partially rescued by phorbol myristate acetate.

Lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) induce kappa transcription in 70Z/3 cells by different mechanisms; LPS treatment induces both NF-kappa B and OTF-2 transcription factors, but IFN-gamma treatment does not. We have dissected these two activation pathways by selecting and analyzing an LPS+ IFN- variant of 70Z/3.