Molecular cloning and functional characterization of a new Cap'n' collar family transcription factor Nrf3. (1/1416)

The NF-E2-binding sites or Maf recognition elements (MARE) are essential cis-acting elements in the regulatory regions of erythroid-specific genes recognized by the erythroid transcription factor NF-E2, composed of p45 and MafK. Recently, two p45-related factors Nrf1 and Nrf2 were isolated, and they are now collectively grouped as the Cap'n' collar (CNC) family. CNC factors bind to MARE through heterodimer formation with small Maf proteins. We report here the identification and characterization of a novel CNC factor, Nrf3, encoding a predicted 73-kDa protein with a basic region-leucine zipper domain highly homologous to those of other CNC proteins. In vitro and in vivo analyses showed that Nrf3 can heterodimerize with MafK and that this complex binds to the MARE in the chicken beta-globin enhancer and can activate transcription. Nrf3 mRNA is highly expressed in human placenta and B cell and monocyte lineage. Chromosomal localization of human Nrf3 is 7p14-15, which lies near the hoxA gene locus. As the genetic loci of p45, nrf1, and nrf2 have been mapped close to those of hoxC, hoxB, and hoxD, respectively, the present study strongly argues for the idea that a single ancestral gene for the CNC family members may have been localized near the ancestral Hox cluster and have diverged to give rise to four closely related CNC factors through chromosome duplication.  (+info)

Silencing of the Epstein-Barr virus latent membrane protein 1 gene by the Max-Mad1-mSin3A modulator of chromatin structure. (2/1416)

The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells.  (+info)

Transcriptional repression of human hepatitis B virus genes by a bZIP family member, E4BP4. (3/1416)

Box alpha is an essential element of both the upstream regulatory sequence of the core promoter and the second enhancer, which positively regulate the transcription of human hepatitis B virus (HBV) genes. In this paper, we describe the cloning and characterization of a box alpha binding protein, E4BP4. E4BP4 is a bZIP type of transcription factor. Overexpression of E4BP4 represses the stimulating activity of box alpha in the upstream regulatory sequence of the core promoter and the second enhancer in differentiated human hepatoma cell lines. E4BP4 can also suppress the transcription of HBV genes and the production of HBV virions in a transient-transfection system that mimics the viral infection in vivo. Expression of an E4BP4 antisense transcript can, instead, elevate the transcription of the core promoter. A low abundance of E4BP4 protein and mRNA in differentiated human hepatoma cell lines is detected, and E4BP4 is not a major component of box alpha binding proteins in untransfected differentiated human hepatoma cell lines. C/EBPalpha and C/EBPbeta, in contrast, are major components of the box alpha binding activity present in nuclear extracts. E4BP4 has a stronger binding affinity towards box alpha than the endogenous box alpha binding activity present in nuclear extracts. Structure and function analysis of E4BP4 reveals that DNA binding activity is sufficient to confer the negative regulatory function of E4BP4. These results indicate that binding site occlusion is the mechanism whereby E4BP4 suppresses transcription in HBV.  (+info)

Two distinct interleukin-3-mediated signal pathways, Ras-NFIL3 (E4BP4) and Bcl-xL, regulate the survival of murine pro-B lymphocytes. (4/1416)

Hematopoietic cells require cytokine-initiated signals for survival as well as proliferation. The pathways that transduce these signals, ensuring timely regulation of cell fate genes, remain largely undefined. The NFIL3 (E4BP4) transcription factor, Bcl-xL, and constitutively active mutants of components in Ras signal transduction pathways have been identified as key regulation proteins affecting murine interleukin-3 (IL-3)-dependent cell survival. Here we show that expression of NFIL3 is regulated by oncogenic Ras mutants through both the Raf-mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. NFIL3 inhibits apoptosis without affecting Bcl-xL expression. By contrast, Bcl-xL levels are regulated through the membrane proximal portion in the cytoplasmic domain of the receptor (betac chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor. Activation of either pathway alone is insufficient to ensure cell survival, indicating that multiple independent signal transduction pathways mediate the survival of developing B-lymphoid cells.  (+info)

Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) encodes a homologue of the Epstein-Barr virus bZip protein EB1. (5/1416)

Analysis of the recently completed genomic sequence of Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) revealed that ORF 50 encodes a protein with homology to the Epstein-Barr virus (EBV) transcription factor R. In this report, we show that ORF K8, contiguous to ORF 50, is interrupted by two introns and that the spliced RNA is translated into a bZip protein that has homology to the EBV transcription factor EB1. The newly characterized K8 protein forms homodimers but does not heterodimerize with other members of the bZip protein family.  (+info)

Multiple regulatory elements control the expression of the yeast ACR1 gene. (6/1416)

The ACR1 gene, encoding a succinate-fumarate transporter, is required by the yeast Saccharomyces cerevisiae for ethanol utilization. Accordingly, gene expression is induced by ethanol and repressed by glucose. Here, we investigated three carbon source response elements present in its promoter region. Specific deletions as well as functional analysis of the elements in a heterologous promoter confirmed their role in transcriptional regulation. Protein binding to carbon source response elements of the ICL1 promoter was competed by all three elements to various extents by the respective ACR1 sequences. In addition, two putative stress response promoter elements present in the ACR1 promoter were investigated in deletion analyses and shown to contribute to gene expression.  (+info)

A novel human DNA-binding protein with sequence similarity to a subfamily of redox proteins which is able to repress RNA-polymerase-III-driven transcription of the Alu-family retroposons in vitro. (7/1416)

In this study we identified a novel protein which may contribute to the transcriptional inactivity of Alu retroposons in vivo. A human cDNA clone encoding this protein (ACR1) was isolated from a human expression library using South-western screening with an Alu subfragment, implicated in the regulation of Alu in vitro transcription and interacting with a HeLa nuclear protein down-regulated in adenovirus-infected cells. Bacterially expressed ACR1 is demonstrated to inhibit RNA polymerase III (Pol III)-dependent Alu transcription in vitro but showed no repression of transcription of a tRNA gene or of a reporter gene under control of a Pol II promoter. ACR1 mRNA is also found to be down-regulated in adenovirus-infected HeLa cells, consistent with a possible repressor function of the protein in vivo. ACR1 is mainly (but not exclusively) located in cytoplasm and appears to be a member of a weakly characterized redox protein family having a central, highly conserved sequence motif, PGAFTPXCXXXXLP. One member of the family identified earlier as peroxisomal membrane protein (PMP)20 is known to interact in a sequence-specific manner with a yeast homolog of mammalian cyclosporin-A-binding protein cyclophilin, and mammalian cyclophilin A (an abundant ubiquitously expressed protein) is known to interact with human transcriptional repressor YY1, which is a major sequence-specific Alu-binding protein in human cells. It appears, therefore, that transcriptional silencing of Alu in vivo is a result of complex interactions of many proteins which bind to its Pol III promoter.  (+info)

Ubiquitination and degradation of ATF2 are dimerization dependent. (8/1416)

Ubiquitination and proteasome-dependent degradation are key determinants of the half-lives of many transcription factors. Homo- or heterodimerization of basic region-leucine zipper (bZIP) transcription factors is required for their transcriptional activities. Here we show that activating transcription factor 2 (ATF2) heterodimerization with specific bZIP proteins is an important determinant of the ubiquitination and proteasome-dependent degradation of ATF2. Depletion of c-Jun as one of the ATF2 heterodimer partners from the targeting proteins decreased the efficiency of ATF2 ubiquitination in vitro, whereas the addition of exogenously purified c-Jun restored it. Similarly, overexpression of c-Jun in 293T human embryo kidney cells increased ATF2 ubiquitination in vivo and reduced its half-life in a dose-dependent manner. Mutations of ATF2 that disrupt its dimerization inhibited ATF2 ubiquitination in vitro and in vivo. Conversely, removal of residues 150 to 248, as in a constitutively active ATF2 spliced form, enhanced ATF2 dimerization and transactivation, which coincided with increased ubiquitination and decreased stability. Our findings indicate the increased sensitivity of transcriptionally active dimers of ATF2 to ubiquitination and proteasome-dependent degradation. Based on these observations, we conclude that increased targeting of a transcriptionally active ATF2 form indicates the mechanism by which the magnitude and the duration of the cellular stress response are regulated.  (+info)

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