James V. Falvo

Reversal of intrinsic DNA bends in the IFNβ gene enhancer by transcription factors and the architectural protein HMG I(Y)

In this paper, we investigate DNA bending induced by proteins required for virus induction of the human interferon-beta (IFN beta) gene. We show that NF-kappa B-DNA complexes that are functionally distinct in the context of the IFN beta enhancer are also conformationally distinct and that two sites in the enhancer contain in-phase bends that are counteracted or reversed by the binding of NF-kappa B, ATF-2/c-Jun, and HMG I(Y). Strikingly, this modulation of intrinsic enhancer architecture results in an orientation that favors predicted protein-protein interactions in a functional nucleoprotein complex, the enhanceosome. Furthermore, the subtle modulation of DNA structure by HMG I(Y) in this process distinguishes it from other architectural factors.

A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo.

ABSTRACT The tumor necrosis factor alpha (TNF-α) gene is rapidly activated by lipopolysaccharide (LPS). Here, we show that extracellular signal-regulated kinase (ERK) kinase activity but not calcineurin phosphatase activity is required for LPS-stimulated TNF-α gene expression. In LPS-stimulated macrophages, the ERK substrates Ets and Elk-1 bind to the TNF-α promoter in vivo. Strikingly, Ets and Elk-1 bind to two TNF-α nuclear factor of activated T cells (NFAT)-binding sites, which are required for calcineurin and NFAT-dependent TNF-α gene expression in lymphocytes. The transcription factors ATF-2, c-jun, Egr-1, and Sp1 are also inducibly recruited to the TNF-α promoter in vivo, and the binding sites for each of these activators are required for LPS-stimulated TNF-α gene expression. Furthermore, assembly of the LPS-stimulated TNF-α enhancer complex is dependent upon the coactivator proteins CREB binding protein and p300. The finding that a distinct set of transcription factors associates with a fixed set of binding sites on the TNF-α promoter in response to LPS stimulation lends new insights into the mechanisms by which complex patterns of gene regulation are achieved.

Stimulus-Specific Assembly of Enhancer Complexes on the Tumor Necrosis Factor Alpha Gene Promoter

ABSTRACT The human tumor necrosis factor alpha (TNF-α) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-α enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-α gene transcription and selectively associates with the TNF-α promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-α transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.

Assembly of a Functional Beta Interferon Enhanceosome Is Dependent on ATF-2–c-jun Heterodimer Orientation

ABSTRACT Heterodimeric transcription factors, including the basic region-leucine zipper (bZIP) protein ATF-2–c-jun, are well-characterized components of an enhanceosome that mediates virus induction of the human beta interferon (IFN-β) gene. Here we report that within the IFN-β enhanceosome the ATF-2–c-jun heterodimer binds in a specific orientation, which is required for assembly of a complex between ATF-2–c-jun and interferon regulatory factor 3 (IRF-3). We demonstrate that correct orientation of the ATF-2–c-jun binding site is required for virus induction of the IFN-β gene and for IRF-3-dependent activation of a composite ATF-2– c-jun–IRF site in the IFN-β promoter. We also show that in vitro the DNA-bound ATF-2–c-jun heterodimer adopts a fixed orientation upon the binding of IRF-3 at an adjacent site in the IFN-β enhancer and that the DNA-binding domain of IRF-3 is sufficient to mediate this effect. In addition, we show that the DNA-binding domain of ATF-2 is necessary and sufficient for selective protein-protein interactions with IRF-3. Strikingly, in vivo chromatin immunoprecipitation experiments with IFN-β reporter constructs reveal that recruitment of IRF-3 to the IFN-β promoter upon virus infection is dependent on the orientation of the ATF-2–c-jun heterodimer binding site. These observations demonstrate functional and physical cooperativity between the bZIP and IRF transcription factor families and illustrate the critical role of heterodimeric transcription factors in formation of the IFN-β enhanceosome.

Transcriptional Control of the TNF Gene

The cytokine TNF is a critical mediator of immune and inflammatory responses. The TNF gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving the recruitment of distinct sets of transcription factors to a compact and modular promoter region. In this review, we describe our current understanding of the mechanisms through which TNF transcription is specifically activated by a variety of extracellular stimuli in multiple cell types, including T cells, B cells, macrophages, mast cells, dendritic cells, and fibroblasts. We discuss the role of nuclear factor of activated T cells and other transcription factors and coactivators in enhanceosome formation, as well as the contradictory evidence for a role for nuclear factor kappaB as a classical activator of the TNF gene. We describe the impact of evolutionarily conserved cis-regulatory DNA motifs in the TNF locus upon TNF gene transcription, in contrast to the neutral effect of single nucleotide polymorphisms. We also assess the regulatory role of chromatin organization, epigenetic modifications, and long-range chromosomal interactions at the TNF locus.

Activation-dependent intrachromosomal interactions formed by the TNF gene promoter and two distal enhancers

Here we provide a mechanism for specific, efficient transcription of the TNF gene and, potentially, other genes residing within multigene loci. We identify and characterize highly conserved noncoding elements flanking the TNF gene, which undergo activation-dependent intrachromosomal interactions. These elements, hypersensitive site (HSS)−9 and HSS+3 (9 kb upstream and 3 kb downstream of the TNF gene, respectively), contain DNase I hypersensitive sites in naive, T helper 1, and T helper 2 primary T cells. Both HSS-9 and HSS+3 inducibly associate with acetylated histones, indicative of chromatin remodeling, bind the transcription factor nuclear factor of activated T cells (NFAT)p in vitro and in vivo, and function as enhancers of NFAT-dependent transactivation mediated by the TNF promoter. Using the chromosome conformation capture assay, we demonstrate that upon T cell activation intrachromosomal looping occurs in the TNF locus. HSS-9 and HSS+3 each associate with the TNF promoter and with each other, circularizing the TNF gene and bringing NFAT-containing nucleoprotein complexes into close proximity. TNF gene regulation thus reveals a mode of intrachromosomal interaction that combines a looped gene topology with interactions between enhancers and a gene promoter.

NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor–viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5–LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.

Post-induction, Stimulus-specific Regulation of Tumor Necrosis Factor mRNA Expression

The tumor necrosis factor (TNF) gene is activated by multiple extracellular signals in a stimulus- and cell type-specific fashion. Based on the presence of κB-like DNA motifs in the region upstream of the TNF gene, some have proposed a direct role for NF-κB in lipopolysaccharide (LPS)-induced TNF gene transcription in cells of the monocyte/macrophage lineage. However, we have previously demonstrated a general and critical role for a minimal TNF promoter region bearing only one of the κB-like motifs, κ3, which is bound by nuclear factor of activated T cell proteins in lymphocytes and fibroblasts in response to multiple stimuli and Ets proteins in LPS-stimulated macrophages. Here, in an effort to resolve these contrasting findings, we used a combination of site-directed mutagenesis of the TNF promoter, quantitative DNase I footprinting, and analysis of endogenous TNF mRNA production in response to multiple stimuli under conditions that inhibit NF-κB activation (using the proteasome inhibitor lactacystin and using cells lacking either functional NF-κB essential modulator, which is the IκB kinase regulatory subunit, or the Nemo gene itself). We find that TNF mRNA production in response to ionophore is NF-κB-independent, but inhibition of NF-κB activation attenuates virus- and LPS-induced TNF mRNA levels after initial induction. We conclude that induction of TNF gene transcription by virus or LPS does not depend upon NF-κB binding to the proximal promoter; rather, a stimulus-specific post-induction mechanism involving NF-κB, yet to be characterized, is involved in the maintenance of maximal TNF mRNA levels.

A dimer-specific function of the transcription factor NFATp.

The transcription factor NFATp integrates multiple signal transduction pathways through coordinate binding with basic-region leucine zipper (bZIP) proteins and other transcription factors. The NFATp monomer, even in the absence of its activation domains, recruits bZIP proteins to canonical NFAT–bZIP composite DNA elements. By contrast, the NFATp dimer and its bZIP partner bind noncooperatively to the NFAT–bZIP element of the tumor necrosis factor (TNF) gene promoter. This observation raises the possibility that the function of the activation domains of NFATp is dimer-specific. Here, we determine the consensus DNA binding site of the NFATp dimer, describe monomer- and dimer-specific NFATp–DNA contact patterns, and demonstrate that NFATp dimerization and dimer-specific activation subdomains are required for transcriptional activation from the TNF NFAT–bZIP element. We also show that these NFATp subdomains interact with the coactivator CBP (CREB-binding protein), which is required for NFATp-dependent TNF gene transcription. Thus, the context-specific function of the activation domains of NFAT can be potentiated by DNA-directed dimerization.

Arc of a Vicious Circle: Pathways Activated by Mycobacterium tuberculosis That Target the HIV-1 Long Terminal Repeat

In this review, we examine how a subset of signal transduction cascades initiated by Mycobacterium tuberculosis (Mtb) infection modulates transcription mediated by the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). We describe two distinct phases of signaling that target transcription factors known to bind the HIV-1 LTR, and thus drive viral transcription and replication, in cells of the Mtb-infected host. First, Mtb-derived molecules, including cell wall components and DNA, interact with a number of host pattern recognition receptors. Second, cytokines and chemokines secreted in response to Mtb infection initiate signal transduction cascades through their cognate receptors. Given the variation in cell wall components among distinct clinical Mtb strains, the initial pattern recognition receptor interaction leading to direct LTR activation and differential cytokine and chemokine production is likely to be an important aspect of Mtb strain-specific regulation of HIV-1 transcription and replication. Improved understanding of these molecular mechanisms in the context of bacterial and host genetics should provide key insights into the accelerated viral replication and disease progression characteristic of HIV/TB coinfection.