Edvige Perrotti

Transcriptional regulation of the ferritin heavy-chain gene: the activity of the CCAAT binding factor NF-Y is modulated in heme-treated Friend leukemia cells and during monocyte-to-macrophage differentiation.

The ferritin H-chain gene promoter regulation was analyzed in heme-treated Friend leukemia cells (FLCs) and during monocyte-to-macrophage differentiation. In the majority of cell lines studied, the regulation of ferritin expression was exerted mostly at the translational level. However, in differentiating erythroid cells, which must incorporate high levels of iron to sustain hemoglobin synthesis, and in macrophages, which are involved in iron storage, transcriptional regulation seemed to be a relevant mechanism. We show here that the minimum region of the ferritin H-gene promoter that is able to confer transcriptional regulation by heme in FLCs to a reporter gene is 77 nucleotides upstream of the TATA box. This cis element binds a protein complex referred to as HRF (heme-responsive factor), which is greatly enhanced both in heme-treated FLCs and during monocyte-to-macrophage differentiation. The CCAAT element present in reverse orientation in this promoter region of the ferritin H-chain gene is necessary for binding and for gene activity, since a single point mutation is able to abolish the binding of HRF and the transcriptional activity in transfected cells. By competition experiments and supershift assays, we identified the induced HRF as containing at least the ubiquitous transcription factor NF-Y. NF-Y is formed by three subunits, A, B, and C, all of which are necessary for DNA binding. Cotransfection with a transdominant negative mutant of the NF-YA subunit abolishes the transcriptional activation by heme, indicating that NF-Y plays an essential role in this activation. We have also observed a differential expression of the NF-YA subunit in heme-treated and control FLCs and during monocyte-to-macrophage differentiation.

IRF-1 Is Required for Full NF-κB Transcriptional Activity at the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Enhancer

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-κB transcriptional activity. We provide evidence that IRF-1 and NF-κB form a functional complex at the long terminal repeat (LTR) κB sites, which is abolished by specific mutations in the two adjacent κB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-κB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.

IFN Regulatory Factor-1 Negatively Regulates CD4 + CD25 + Regulatory T Cell Differentiation by Repressing Foxp3 Expression

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance, mechanisms and molecules involved in the generation of Treg cells remain poorly understood. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4+CD25+ Treg cell development and function by specifically repressing Foxp3 expression. IRF-1-deficient (IRF-1−/−) mice showed a selective and marked increase of highly activated and differentiated CD4+CD25+Foxp3+ Treg cells in thymus and in all peripheral lymphoid organs. Furthermore, IRF-1−/− CD4+CD25− T cells showed extremely high bent to differentiate into CD4+CD25+Foxp3+ Treg cells, whereas restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells impaired their differentiation into CD25+Foxp3+ cells. Functionally, both isolated and TGF-β-induced CD4+CD25+ Treg cells from IRF-1−/− mice exhibited more increased suppressive activity than wild-type Treg cells. Such phenotype and functional characteristics were explained at a mechanistic level by the finding that IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and negatively regulates its transcriptional activity. We conclude that IRF-1 is a key negative regulator of CD4+CD25+ Treg cells through direct repression of Foxp3 expression.

Intracellular HIV-1 Tat protein represses constitutive LMP2 transcription increasing proteasome activity by interfering with the binding of IRF-1 to STAT1

The Tat protein is the transcriptional activator of HIV-1 gene expression, which is not only essential for viral replication, but also important in the complex HIV-induced pathogenesis of AIDS, as both an intracellular and an extracellular released protein. Accordingly, Tat is able to profoundly affect cellular gene expression, regulating several cellular functions, also in non-infected cells. We showed recently that Tat induces modification of immunoproteasomes in that it up-regulates LMP7 (low-molecular-mass polypeptide 7) and MECL1 (multicatalytic endopeptidase complex-like 1) subunits and down-modulates the LMP2 subunit, resulting in a change in the generation and presentation of epitopes in the context of MHC class I. In particular, Tat increases presentation of subdominant and cryptic epitopes. In the present study, we investigated the molecular mechanism responsible for the Tat-induced LMP2 down-regulation and show that intracellular Tat represses transcription of the LMP2 gene by competing with STAT1 (signal transducer and activator of transcription 1) for binding to IRF-1 (interferon-regulatory factor-1) on the overlapping ICS-2 (interferon consensus sequence-2)-GAS (gamma-interferon-activated sequence) present in the LMP2 promoter. This element is constitutively occupied in vivo by the unphosphorylated STAT1-IRF-1 complex, which is responsible for the basal transcription of the gene. Sequestration of IRF-1 by intracellular Tat impairs the formation of the complex resulting in lower LMP2 gene transcription and LMP2 protein expression, which is associated with increased proteolytic activity. On the other hand, extracellular Tat induces the expression of LMP2. These effects of Tat provide another effective mechanism by which HIV-1 affects antigen presentation in the context of the MHC class I complex and may have important implications in the use of Tat for vaccination strategies.

HIV-1, interferon and the interferon regulatory factor system: An interplay between induction, antiviral responses and viral evasion

Thirty years after the first isolation of the etiological agent of AIDS, the virus HIV-1 is still a major threat worldwide with millions of individuals currently infected. Although current combination therapies allow viral replication to be controlled, HIV-1 is not eradicated and persists in drug- and immune system-insensitive reservoirs and a cure is still lacking. Pathogens such as HIV-1 that cause chronic infections are able to adapt to the host in a manner that ensures long term residence and survival, via the evolution of numerous mechanisms that evade various aspects of the innate and adaptive immune response. One such mechanism is targeted to members of the interferon (IFN) regulatory factor (IRF) family of proteins. These transcription factors regulate a variety of biological processes including interferon induction, immune cell activation and downstream pattern recognition receptors (PRRs). HIV-1 renders IRFs harmless and hijacks them to its own advantage in order to facilitate its replication and evasion of immune responses. Type I interferon (IFN), the canonical antiviral innate response, can be induced in both acute and chronic HIV-1 infection in vivo, but in the majority of individuals this initial response is not protective and can contribute to disease progression. Type I IFN expression is largely inhibited in T cells and macrophages in order to successfully establish productive infection, whereas sustained IFN production by plasmacytoid dendritic cells is considered an important source of chronic immune activation, a hallmark to AIDS progression.

Role of Ets-1 in transcriptional regulation of transferrin receptor and erythroid differentiation.

High expression of transferrin receptor (TfR) on the membrane of erythroid cells accounts for the high level of iron required to sustain heme synthesis. Several studies indicate that during erythroid differentiation TfR expression is highly dependent on transcriptional regulation. In this study we characterized the minimal region able to confer transcriptional regulation during erythroid differentiation in Friend leukemia cells (FLC). This region of 120 bp, upstream the transcription start site, contains an overlapping consensus recognition sequence for AP1/CREB/ATF transcription factors and for proteins of the Ets family and a GC rich region. Here, we report that both the Ets and the Ap1/CRE like sites are essential for promoter activity during erythroid differentiation. We showed that Ets-1 binds to the EBS-TfR and its binding activity decreases in FLC induced to differentiate and during normal erythroid differentiation. Consistent with this, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. We conclude that Ets-1 binding activity is modulated during erythroid maturation and that a deregulated expression of this transcription factor interferes with terminal erythroid differentiation.

Critical Role of IRF-8 in Negative Regulation of TLR3 Expression by Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase-2 Activity in Human Myeloid Dendritic Cells

Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1– and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8–induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase–dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation.

Human Papillomavirus Type 16 E5 Protein Induces Expression of Beta Interferon through Interferon Regulatory Factor 1 in Human Keratinocytes

ABSTRACT Crucial steps in high-risk human papillomavirus (HR-HPV)-related carcinogenesis are the integration of HR-HPV into the host genome and loss of viral episomes. The mechanisms that promote cervical neoplastic progression are, however, not clearly understood. During HR-HPV infection, the HPV E5 protein is expressed in precancerous stages but not after viral integration. Given that it has been reported that loss of HPV16 episomes and cervical tumor progression are associated with increased expression of antiviral genes that are inducible by type I interferon (IFN), we asked whether E5, expressed in early phases of cervical carcinogenesis, affects IFN-β signaling. We show that the HPV type 16 (HPV16) E5 protein expression per se stimulates IFN-β expression. This stimulation is specifically mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs) as double-stranded RNA-dependent protein kinase R (PKR) and caspase 8. Our data show a new and unexpected role for HR-HPV E5 protein and indicate that HPV16 E5 may contribute to the mechanisms responsible for cervical carcinogenesis in part via stimulation of IFN-β and an IFN signature, with IRF-1 playing a pivotal role. HPV16 E5 and IRF-1 may thus serve as potential therapeutic targets in HPV-associated premalignant lesions.

IκB kinase ε targets interferon regulatory factor 1 in activated T lymphocytes.

ABSTRACT IκB kinase ε (IKK-ε) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present study, we investigated IKK-ε activation following T cell receptor (TCR)/CD28 stimulation of primary CD4+ T cells and its role in the stimulation of a type I IFN response. IKK-ε was activated following TCR/CD28 stimulation of primary CD4+ T cells; however, in T cells treated with poly(I·C), TCR/CD28 costimulation blocked induction of IFN-β transcription. We demonstrated that IKK-ε phosphorylated the transcription factor IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4+ T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-κB RelA subunit and interfered with PCAF-mediated acetylation of NF-κB RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-ε activation, which in turn contributes to suppression of IFN-β production.

Type I IFN – A blunt spear in fighting HIV-1 infection

For more than 50 years, Type I Interferon (IFN) has been recognized as critical in controlling viral infections. IFN is produced downstream germ-line encoded pattern recognition receptors (PRRs) upon engagement by pathogen-associated molecular patterns (PAMPs). As a result, hundreds of different interferon-stimulated genes (ISGs) are rapidly induced, acting in both autocrine and paracrine manner to build a barrier against viral replication and spread. ISGs encode proteins with direct antiviral and immunomodulatory activities affecting both innate and adaptive immune responses. During infection with viruses, as HIV-1, that can establish a persistent infection, IFN although produced, is not able to block the initial infection and a chronic IFN-mediated immune activation/inflammation becomes a pathogenic mechanism of disease progression. This review will briefly summarize when and how IFN is produced during HIV-1 infection and the way this innate immune response is manipulated by the virus to its own advantage to drive chronic immune activation and progression to AIDS.