Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes.

A family of interferon (IFN) regulatory factors (IRFs) have been shown to play a role in transcription of IFN genes as well as IFN-stimulated genes. We report the identification of a member of the IRF family which we have named IRF-3. The IRF-3 gene is present in a single copy in human genomic DNA. It is expressed constitutively in a variety of tissues and no increase in the relative steady-state levels of IRF-3 mRNA was observed in virus-infected or IFN-treated cells. The IRF-3 gene encodes a 50-kDa protein that binds specifically to the IFN-stimulated response element (ISRE) but not to the IRF-1 binding site PRD-I. Overexpression of IRF-3 stimulates expression of the IFN-stimulated gene 15 (ISG15) promoter, an ISRE-containing promoter. The murine IFNA4 promoter, which can be induced by IRF-1 or viral infection, is not induced by IRF-3. Expression of IRF-3 as a Gal4 fusion protein does not activate expression of a chloramphenicol acetyltransferase reporter gene containing repeats of the Gal4 binding sites, indicating that this protein does not contain the transcription transactivation domain. The high amino acid homology between IRF-3 and ISG factor 3 gamma polypeptide (ISGF3 gamma) and their similar binding properties indicate that, like ISGF3 gamma, IRF-3 may activate transcription by complex formation with other transcriptional factors, possibly members of the Stat family. Identification of this ISRE-binding protein may help us to understand the specificity in the various Stat pathways.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

Inhibition of human immunodeficiency virus type 1 and vaccinia virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells.

ICSBP is a member of the interferon (IFN) regulatory factor (IRF) family that regulates expression of type I interferon (IFN) and IFN-regulated genes. To study the role of the IRF family in viral infection, a cDNA for the DNA-binding domain (DBD) of ICSBP was stably transfected into U937 human monocytic cells. Clones that expressed DBD exhibited a dominant negative phenotype and did not elicit antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing DBD were refractory to infection by vaccinia virus (VV) and human immunodeficiency virus type 1 (HIV-1). The inhibition of VV infection was attributed to defective virion assembly, and that of HIV-1 to low CD4 expression and inhibition of viral transcription in DBD clones. HIV-1 and VV were found to have sequences in their regulatory regions similar to the IFN-stimulated response element (ISRE) to which IRF family proteins bind. Accordingly, these viral sequences and a cellular ISRE bound a shared factor(s) expressed in U937 cells. These observations suggest a novel host-virus relationship in which the productive infection of some viruses is regulated by the IRF-dependent transcription pathway through the ISRE.

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-β enhanceosome in vitro

The transcriptional activity of an in vitro assembled human interferon-β gene enhanceosome is highly synergistic. This synergy requires five distinct transcriptional activator proteins (ATF2/c-JUN, interferon regulatory factor 1, and p50/p65 of NF-κB), the high mobility group protein HMG I(Y), and the correct alignment of protein-binding sites on the face of the DNA double helix. Here, we investigate the mechanisms of enhanceosome-dependent transcriptional synergy during preinitiation complex assembly in vitro. We show that the stereospecific assembly of the enhanceosome is critical for the efficient recruitment of TFIIB into a template-committed TFIID-TFIIA-USA (upstream stimulatory activity complex) and for the subsequent recruitment of the RNA polymerase II holoenzyme complex. In addition, we provide evidence that recruitment of the holoenzyme by the enhanceosome is due, at least in part, to interactions between the enhanceosome and the transcriptional coactivator CREB, cAMP responsive element binding protein (CBP). These studies reveal a unique role of enhanceosomes in the cooperative assembly of the transcription machinery on the human interferon-β promoter.

Ectopic expression of prion protein (PrP) in T lymphocytes or hepatocytes of PrP knockout mice is insufficient to sustain prion replication

The cellular form of the Prion protein (PrPC) is necessary for prion replication in mice. To determine whether it is also sufficient, we expressed PrP under the control of various cell- or tissue-specific regulatory elements in PrP knockout mice. The interferon regulatory factor-1 promoter/Eμ enhancer led to high PrP levels in the spleen and low PrP levels in the brain. Following i.p. scrapie inoculation, high prion titers were found in the spleen but not in the brain at 2 weeks and 6 months, showing that the lymphoreticular system by itself is competent to replicate prions. PrP expression directed by the Lck promoter resulted in high PrP levels on T lymphocytes only but, surprisingly, did not allow prion replication in the thymus, spleen, or brain following i.p. inoculation. A third transgenic line, which expressed PrP in the liver under the control of the albumin promoter/enhancer—albeit at low levels—also failed to replicate prions. These results show that expression of PrP alone is not sufficient to sustain prion replication and suggest that additional components are needed.

The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies intranuclear targeting of the AML1/CBFα2 transcription factor

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.

Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.

Proteolytic Processing and Ca2+-binding Activity of Dense-Core Vesicle Polypeptides in Tetrahymena

Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles.

An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

Muscle LIM Proteins Are Associated with Muscle Sarcomeres and Require dMEF2 for Their Expression during Drosophila Myogenesis

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and α-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.

An intrinsic genetic program for autonomous differentiation of muscle cells in the ascidian embryo.

The B-line presumptive muscle cells of ascidian embryos have extensive potential for self-differentiation dependent on determinants prelocalized in the myoplasm of fertilized eggs. Ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells. Experiments with egg fragments suggested that maternal mRNAs are one of the components of muscle determinants. Expression of larval muscle actin genes begins as early as the 32-cell stage, prior to the developmental fate restriction of the cells. The timing of initiation of the actin gene expression proceeds the expression of an ascidian homologue of vertebrate MyoD by a few hours. Mutations in the proximal E-box of the 5' flanking region of the actin genes did not alter the promoter activity for muscle-specific expression of reporter gene. These results, together with results of deletion constructs of fusion genes, suggest that muscle determinants regulate directly, or indirectly via regulatory factors other than MyoD, the transcription of muscle-specific structural genes leading to the terminal differentiation.

Chromatin structure and gene expression.

It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes. This is most obvious in the neighborhood of promoters and enhancers, where hypersensitivity to nucleases marks sites that no longer carry canonical nucleosomes, and to which transcription factors bind. To study the relationship between transcription factor binding and the generation of these hypersensitive regions, we mutated individual cis-acting regulatory elements within the enhancer that lies between the chicken beta- and epsilon-globin genes. Constructions carrying the mutant enhancer were introduced by stable transformation into an avian erythroid cell line. We observed that weakening the enhancer resulted in creation of two classes of site: those still completely accessible to nuclease attack and those that were completely blocked. This all-or-none behavior suggests a mechanism by which chromatin structure can act to sharpen the response of developmental systems to changing concentrations of regulatory factors. Another problem raised by chromatin structure concerns the establishment of boundaries between active and inactive chromatin domains. We have identified a DNA element at the 5' end of the chicken beta-globin locus, near such a boundary, that has the properties of an insulator; in test constructions, it blocks the action of an enhancer on a promoter when it is placed between them. We describe the properties and partial dissection of this sequence. A third problem is posed by the continued presence of nucleosomes on transcribed genes, which might prevent the passage of RNA polymerase. We show, however, that a prokaryotic polymerase can transcribe through a histone octamer on a simple chromatin template. The analysis of this process reveals that an octamer is capable of transferring from a position in front of the polymerase to one behind, without ever losing its attachment to the DNA.