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Results for “Interferon Regulatory Factor-4”

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At least 19 recordsLinked to original sources
PubMed1999

ICSAT overexpression is not sufficient to cause adult T-cell leukemia or multiple myeloma.

ICSAT (Interferon Consensus Sequence binding protein for Activated T cells) is a lymphocyte-specific member of the interferon regulatory factor (IRF) family of transcription factors, originally identified through Southwestern screening of the ATL(Adult T-cell leukemia)-16T expression library. In this study, we created transgenic mice overexpressing ICSAT in lymphocytes. Although spontaneous tumorigenesis was not observed, IL-2 production with Concanavalin A stimulation was significantly increased in transgenic mice overexpressing ICSAT. ICSAT overexpression in lymphocytes seems insufficient for the leukemogenesis of ATL or multiple myeloma (MM), however, it may regulate T cell activation and its overexpression may lead to leukemogenesis via controlling IL-2 production.

Adult↗
PubMed1999

Interferon regulatory transcription factors are constitutively expressed and spatially regulated in the mouse lens.

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulation of cell growth and immunological responses. Nine IRFs have been described and they are expressed in a variety of cells, except for ICSBP and LSIRF/Pip, which are thought to be expressed exclusively in immune cells. Here, we show that IRF-1, IRF-2, ICSBP, and LSIRF/Pip are constitutively expressed in the mouse lens. These IRFs are present in both the cytoplasm and the nuclei of lens cells. However, the nuclear and cytoplasmic proteins exhibit distinct mobilities on SDS/PAGE. We further show that in the developing mouse lens, IRF-1 and IRF-2 are expressed at high levels in differentiated lens fiber cells with very low and barely detectable levels in undifferentiated lens epithelial cells. Although the level of ICSBP expression is very low in the normal mouse lens, in transgenic mice with constitutive expression of interferon gamma in the lens, its level is markedly elevated and ICSBP expression is detected exclusively in the nuclei of undifferentiated lens cells. Taken together, our data suggest that expression of IRF transcription factors is spatially regulated in the lens and that distinct IRFs may contribute to differential gene regulation in the epithelial and fiber compartments of the vertebrate lens.

Animals↗
PubMed1996

Cloning of human lymphocyte-specific interferon regulatory factor (hLSIRF/hIRF4) and mapping of the gene to 6p23-p25.

The interferon regulatory factor (IRF) genes encode a family of transcription factors involved in the transcriptional regulation of interferon and the interferon stimulated genes through recognition of the interferon stimulated response element. We previously reported the cloning of a murine lymphocyte-specific IRF (mLSIRF), which was rapidly induced following B- or T-cell receptor crosslinking. To study the role of LSIRF in human lymphocyte development, we have cloned the complete 5.3-kb cDNA for the human homolog (hLSIRF). hLSIRF is a protein of 450 amino acids with a predicted molecular weight of 51.6 kDa and possesses 92% identity at the amino acid level to mLSIRF, including near identity in the DNA-binding domain. In Northern blot analysis, a single transcript of approximately 5 kb was highly expressed in spleen and peripheral blood lymphocyte. hLSIRF mRNA was rapidly induced in peripheral T cells after crosslinking the T-cell receptor. Analysis of tumor cell lines showed that hLSIRF mRNA was basally expressed in most B- but not T-cell lines. Surprisingly hLSIRF mRNA was also found in the melanoma line G361 and is expressed in normal melanocytes as well. Sequence from a genomic clone for hLSIRF was compared to that from mouse and revealed an identical exon-intron structure and a conserved PU-1-binding motif in the promoter. By FISH analysis, hLSIRF was mapped to 6p23-p25.

Amino Acid Sequence↗
PubMed1999

The influence of T cell receptor and cytokine genes on sarcoidosis susceptibility in African Americans.

The pathogenesis of sarcoidosis, a multisystem granulomatous disorder, is mediated through immunoregulatory pathways. While sarcoidosis clusters in families, inherited risk factors remain undefined. In search of possible sarcoidosis susceptibility genes, we examined anonymous polymorphic genetic markers tightly linked to six different candidate gene regions on chromosomes 2q13, 5q31, 6p23-25, 7p14-15, 14q11 and 22q11. These candidate regions contain T cell receptor, interleukin (IL) and interferon regulatory factor (IRF) genes. Our study population consisted of 105 African-American sarcoidosis cases and 95 unrelated healthy controls. The allelic frequency distribution of two out of the six markers, IL-1 alpha marker (p = 0.010) on 2q13 and the F13A marker (p = 0.0006) on 6p23-25, was statistically significantly different in cases compared with controls. The two alleles most strongly associated with sarcoidosis were IL-1 alpha*137 (Odds Ratio (OR) = 2.60; 95% confidence interval (CI) = 1.36-4.98) and F13A*188 (OR = 2.42; 95% CI = 1.37-4.30). Individuals that had both of these alleles were at a six-fold increased risk for sarcoidosis (OR = 6.19; 95% CI = 2.54-15.10). Restricting the analysis to cases with at least one first or second-degree relative affected with sarcoidosis increased the OR to 15.38. IL-1 levels are elevated in sarcoidosis and the F13A marker is tightly linked to a gene that codes for a newly identified interferon regulatory factor protein (IRF-4), which is thought to play a role in T cell effector functions. Our results suggest genetic susceptibility to sarcoidosis may be conferred by more than one immune-related gene that act synergistically on disease risk.

Adult↗
PubMed1999

Interferon regulatory factors: the next generation.

Interferons are a large family of multifunctional secreted proteins involved in antiviral defense, cell growth regulation and immune activation. Viral infection induces transcription of multiple IFN genes, a response that is in part mediated by the interferon regulatory factors (IRFs). The initially characterized members IRF-1 and IRF-2 are now part of a growing family of transcriptional regulators that has expanded to nine members. The functions of the IRFs have also expanded to include distinct roles in biological processes such as pathogen response, cytokine signaling, cell growth regulation and hematopoietic development. The aim of this review is to provide an update on the novel discoveries in the area of IRF transcription factors and the important roles of the new generation of IRFs--particularly IRF-3, IRF-4 and IRF-7.

Amino Acid Sequence↗
PubMed1999

PU.1 and Spi-B are required for normal B cell receptor-mediated signal transduction.

PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.

Animals↗
PubMed1997

The growing family of interferon regulatory factors.

Interferons (IFN) exert their multiple biological effects through the induction of expression of over 30 genes encoding proteins with antiviral, antiproliferative and immunomodulatory functions. Among the many IFN-inducible proteins are the Interferon Regulatory Factors (IRFs), a family of transcription regulators, originally consisting of the well-characterized IRF-1 and IRF-2 proteins; the family has now expanded to over 10 members and is still growing. The present review provides a detailed description of recently characterized IRF family members. Studies analyzing IRF-expressing cell lines and IRF knockout mice reveal that each member of the IRF family exerts distinct roles in biological processes such as pathogen response, cytokine signalling, cell growth regulation and hematopoietic development. Understanding the molecular mechanisms by which the IRFs affect these important cellular events and IFN expression will contribute to a greater understanding of events leading to various viral, immune and malignant disease states and will suggest novel strategies for antiviral and immune modulatory therapy.

Amino Acid Sequence↗
PubMed1998

Quantitative hydroxyl radical footprinting reveals cooperative interactions between DNA-binding subdomains of PU.1 and IRF4.

Quantitative hydroxyl radical footprinting and fluorescence polarization measurements have been used to determine the dissociation constants (Kd) of complexes between the ets domain of the murine transcription factor PU.1 and three different DNA fragments. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Iglambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex. With the use of quantitative hydroxyl radical footprinting we obtained binding isotherms for individual protected nucleotides and contact sites on both strands of the DNA. Kd values of (1.53 +/- 0. 12) x 10(-)8 M were found for the lambdaB element, (3.60 +/- 0.65) x 10(-)8 M for the SV40 enhancer site, and (2.28 +/- 0.27) x 10(-)8 M for the sequence used in the crystal structure. In addition, the binding of a second protein, the DNA binding domain of IRF4, to the lambdaB site by itself and in the presence of PU.1 was analyzed. The IRF4 DBD shows three footprints on the TTCC strand and one footprint on the GGAA strand of the lambdaB element. The dissociation constant for the binary IRF4 DBD-lambdaB complex equals (5.59 +/- 0.60) x 10(-)7 M. The Kd value of the IRF4-lambdaB interaction is reduced by a factor of 5 in the presence of two different DNA-bound PU.1 protein constructs, PU.1 DBD and a PU.1 construct containing the PEST domain (PU.1-PEST). A similar decrease of the Kd value was observed for the binding of PU.1-PEST in the presence of DNA-bound IRF4 DBD demonstrating a cooperative interaction between the PU. 1-PEST and IRF4 DBD. On the basis of the hydroxyl radical footprints in the ternary PU.1/IRF4/lambdaB complex, a model for the interactions between the two proteins and the lambdaB site was developed. The DNA binding domains of both proteins bind the DNA in the major groove with potential protein-protein interactions near the intervening minor groove.

Animals↗
PubMed1997

PU.1 can participate in an active enhancer complex without its transcriptional activation domain.

The transcription factor PU.1 is necessary for the development of multiple hematopoietic lineages and contributes to the activity of the immunoglobulin kappa 3' enhancer. A variety of proteins bind to the 3' enhancer (PU.1, PIP, ATF1, CREM, c-Fos, c-Jun, and E2A), but the mechanism of 3'-enhancer activity and the proteins necessary for its activity are presently unclear. We show here that PU.1 participates with other transcription factors in forming a higher-order complex with 3'-enhancer DNA sequences. Each protein is necessary for formation of this complex. Individually, transcription factors that bind to the 3' enhancer do not appreciably stimulate transcription in a cell type in which the 3' enhancer is normally silent (NIH 3T3). However, mixture of multiple transcription factors (PU.1, PIP, c-Fos, and c-Jun) can greatly activate the enhancer. PU.1 is necessary for maximal enhancer activity, but mutants of PU.1 that lack the transcriptional activation domain are nearly as efficient at stimulating enhancer activity as the wild-type PU.1 protein. PU.1 apparently can activate transcription by playing an architectural role in interactions with other transcription factors.

DNA-Binding Proteins↗
PubMed1999

Mutation analysis of the Pip interaction domain reveals critical residues for protein-protein interactions.

The PU.1 interaction partner (Pip) is a member of the interferon regulatory factor family that regulates gene expression through heterodimerization with the ETS transcription factor PU.1. Binding of Pip alone to DNA is weak, and usually it is recruited by phosphorylated PU.1 to form a strong ternary complex with specific DNA sequences. An approach combining sequence homology analysis, secondary structure predictions, and a precise mutational strategy has been used to determine critical residues within the Pip heterodimerization domain that contribute to ternary complex formation. We have delimited the Pip interaction domain to residues 245-422 by using deletion analysis. Site-directed mutagenesis of conserved polar amino acids within two predicted alpha-helices contained in this region, and which are highly conserved in the IRF family, confirmed the importance of these residues for Pip-PU.1 interaction with DNA as well as for trans-activation activity. Our results suggest the existence of a functional epitope essential for heterodimerization between Pip and PU.1 and possibly, in general, between interferon regulatory factor family members and their partners.

Amino Acid Sequence↗
PubMed1999

Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers.

Gene expression in higher eukaryotes appears to be regulated by specific combinations of transcription factors binding to regulatory sequences. The Ets factor PU.1 and the IRF protein Pip (IRF-4) represent a pair of interacting transcription factors implicated in regulating B cell-specific gene expression. Pip is recruited to its binding site on DNA by phosphorylated PU.1. PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative alpha-helix in Pip. Thus, a coordinated set of protein-protein and protein-DNA contacts are essential for PU.1-Pip ternary complex assembly. To analyze the function of these factors in vivo, we engineered chimeric repressors containing the ets and IRF DNA-binding domains connected by a flexible POU domain linker. When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin lambda gene, thereby establishing the functional importance of PU.1-Pip complexes in B cell gene expression. Comparative analysis of the wild-type dimer with a series of mutant dimers distinguished a gene regulated by PU.1 and Pip from one regulated by PU.1 alone. This strategy should prove generally useful in analyzing the function of interacting transcription factors in vivo, and for identifying novel genes regulated by such complexes.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE).

Interferon regulatory factor (IRF) genes encode a family of DNA-binding proteins that are involved in the transcriptional regulation of type-I interferon and/or interferon-inducible genes. We report here the characterization of LSIRF, a new member of the IRF gene family cloned from mouse spleen by the polymerase chain reaction using degenerate primers. LSIRF was found to encode a 51 kDa protein that shares a high degree of amino acid sequence homology in the DNA-binding domain with other IRF family members. LSIRF expression was detectable only in lymphoid cells. In contrast to other IRF genes, LSIRF expression was not induced by interferons, but rather by antigen-receptor mediated stimuli such as plant lectins, CD3 or IgM crosslinking. In in vitro DNA binding studies, LSIRF was able to bind to the interferon-stimulated response element (ISRE) of the MHC class I promoter. The expression pattern and DNA binding activities suggest that LSIRF plays a role in ISRE-targeted signal transduction mechanisms specific to lymphoid cells.

Amino Acid Sequence↗
PubMed1995

Domains of the Epstein-Barr virus nuclear antigen 2 (EBNA2) involved in the transactivation of the latent membrane protein 1 and the EBNA Cp promoters.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) is one of the first EBV-encoded gene products expressed after infection of primary B lymphocytes. EBNA2 is essential for the growth-transforming potential of the virus and it functions as a transcriptional activator of a set of viral and cellular genes. Sequence-specific DNA-binding by EBNA2 has not been demonstrated but the molecule is targeted to specific DNA regions by a cellular protein, RBP-J kappa, which recognizes the GTGGGAA sequence present in the regulatory region of all EBNA2-responsive promoters defined so far. We have determined the contribution of a RBP-J kappa recognition sequence, an adjacent interferon-stimulated response element (ISRE) motif and a PU.1-binding site in the LMP1 regulatory sequence (LRS) to EBNA2-induced transactivation of the promoter by site-directed mutagenesis of LRS-carrying reporter plasmids. EBNA2 responsiveness was reduced by approximately twofold when either or both of the RBP-J kappa-binding and ISRE sequences were mutated. ISRE seemed to function as an EBNA2-independent positive element. On the other hand, mutation of the PU box resulted in a drastic reduction of EBNA2 responsiveness, irrespective of whether the RBP-J kappa site or the ISRE motif was present. A comparative study by deletion mutation identified regions of EBV B95-8 EBNA2 involved in the transactivation of the LMP1 and the EBNA Cp promoters. Two domains of EBNA2 defined by deletion of amino acids 247-337 and 437-476 were found to be important for the activation of both promoters, while two different domains corresponding to residues 4-18 and 118-198 were required solely for the LMP1 promoter. Thus, EBNA2 must activate the LMP1 and Cp promoters by different mechanisms. All deletions involved in transcriptional activation of the two promoters contained regions that are conserved in EBNA2 of B95-8 EBV (type 1), AG876 EBV (type 2) and herpesvirus papio origin.

Antigens, Viral↗
PubMed1995

PU box-binding transcription factors and a POU domain protein cooperate in the Epstein-Barr virus (EBV) nuclear antigen 2-induced transactivation of the EBV latent membrane protein 1 promoter.

Expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP1) is regulated by virus- and host cell-specific factors. The EBV nuclear antigen 2 (EBNA2) has been shown to transactivate a number of viral and cellular gene promoters including the promoter for the LMP1 gene. EBNA2 is targeted to at least some of these promoters by interacting with a cellular DNA binding protein, RBP-J kappa. In the present report we confirm and extend our previous observation that the LMP1 promoter can be activated by EBNA2 in the absence of the RBP-J kappa-binding sequence in the LMP1 promoter regulatory region (LRS). We show that two distinct LRS regions, -106 to +40 and -176 to -136, contribute to EBNA2 responsiveness. Site-directed mutagenesis analysis of the upstream -176/-136 EBNA2 responsive element revealed that two critical cis-acting elements are required for full promoter function. These same elements analysed by electrophoretic mobility shift assays define two binding sites recognized by nuclear factors derived from B cells. An octamer-like sequence (-147 to -139) contained overlapping binding sites for an unidentified transcriptional repressor on the one hand and a factor(s) belonging to the POU domain family but distinct from Oct-1 and Oct-2 on the other. An adjacent purine tract (-171 to -155) held a PU.1 binding site, which was also recognized by a related factor. The results suggest that the POU domain protein and either of two PU box-binding factors bind simultaneously to LRS, creating a ternary complex that might be in part responsible for mediating the transactivation of the LMP1 promoter by EBNA2. There were no qualitative differences between EBV-negative and EBV-positive cells with regard to transcription factor binding to the octamer-like sequence and the PU.1 recognition site, as revealed by electrophoretic mobility shift assays.

Antigens, Viral↗
PubMed1996

Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1.

Pip is a lymphoid-restricted IRF transcription factor that is recruited to composite elements within immunoglobulin light-chain gene enhancers through a specific interaction with the Ets factor PU.1. We have examined the transcriptional regulatory properties of Pip as well as the requirements for its interaction with PU.1 and DNA to form a ternary complex. We demonstrate that Pip is a dichotomous regulator; it specifically stimulates transcription in conjunction with PU.1, but represses alpha/beta-interferon-inducible transcription in the absence of PU.1. Thus, during B-cell activation and differentiation, Pip may function both as an activator to promote B cell-specific gene expression and as a repressor to inhibit the antiproliferative effects of alpha/beta-interferons. Mutational analysis of Pip reveals a carboxy-terminal segment that is important for autoinhibition of DNA binding and ternary complex formation. A domain of Pip containing this segment confers autoinhibition and PU.1-dependent binding activity to the DNA-binding domain of the related IRF family member, p48. On the basis of these and other data we propose a model for PU.1/Pip ternary complex formation.

Animals↗
PubMed1995

Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator.

The immunoglobulin light-chain gene enhancers E kappa 3', E lambda 2-4, and E lambda 3-1 contain a conserved cell type-specific composite element essential for their activities. This element binds a B cell-specific heterodimeric protein complex that consists of the Ets family member PU.1 and a second factor (NF-EM5), whose participation in the formation of the complex is dependent on the presence of DNA-bound PU.1. In this report we describe the cloning and characterization of Pip (PU.1 interaction partner), a lymphoid-specific protein that is most likely NF-EM5. As expected, the Pip protein binds the composite element only in the presence of PU.1; furthermore, the formation of this ternary complex is critically dependent on phosphorylation of PU.1 at serine-148. The Pip gene is expressed specifically in lymphoid tissues in both B- and T-cell lines. When coexpressed in NIH-3T3 cells, Pip and PU.1 function as mutually dependent transcription activators of the composite element. The amino-terminal DNA-binding domain of Pip exhibits a high degree of homology to the DNA-binding domains of members of the interferon regulatory factor (IRF) family, which includes IRF-1, IRF-2, ICSBP, and ISGF3 gamma.

Amino Acid Sequence↗
PubMed1997

Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function.

Lymphocyte-specific interferon regulatory factor (LSIRF) (now called IRF4) is a transcription factor expressed only in lymphocytes. Mice deficient in IRF4 showed normal distribution of B and T lymphocyes at 4 to 5 weeks of age but developed progressive generalized lymphadenopathy. IRF4-deficient mice exhibited a profound reduction in serum immunoglobulin concentrations and did not mount detectable antibody responses. T lymphocyte function was also impaired in vivo; these mice could not generate cytotoxic or antitumor responses. Thus, IRF4 is essential for the function and homeostasis of both mature B and mature T lymphocytes.

Animals↗
PubMed1996

A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes.

We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by "differential repression" is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.

Amino Acid Sequence↗