Structural Basis and Functional Consequences of Alternative ATF2-Jun Heterodimer Orientations at the Interferon-Beta Enhancer

The selective activation of genes is essential for diverse biological processes such as growth, development, and responses to environmental cues. Unlike lower organisms that often use individual proteins to control gene activation, transcription regulation in higher organisms generally requires cooperation among multiple proteins. Cooperation can be achieved via interactions between DNA-binding proteins that bind to adjoining DNA sequences. Such interactions can stabilize DNA binding by these proteins. Many eukaryotic transcription factors form heterodimers that can bind to DNA in two opposite orientations. Because of the asymmetry of such heterodimers, cooperative DNA binding has been predicted, and in some cases observed, to require a specific orientation of heterodimer binding. Interferon regulatory factor 3 (IRF3) and a heterodimer containing activating transcription factor 2 and c-Jun (ATF2-Jun) bind cooperatively to the human interferon-beta enhancer, and opposite orientations of ATF2-Jun binding have been observed using different experimental approaches. High mobility group protein I (HMGI) binds to sequences overlapping the ATF2-Jun-IRF3 site within the interferon-beta enhancer and facilitates DNA-binding and synergistic transcriptional activation by components of the enhancer complex, yet its effects on ATF2-Jun-IRF3 complex formation have not been investigated. This thesis presents the identification of the structural determinants of ATF2-Jun heterodimer orientation at the interferon-beta enhancer in vitro as well as functional characterization in cells. Using gel-based fluorescence resonance energy transfer analysis, I found that ATF2-Jun binds to the interferon-beta enhancer in both orientations alone and in association with IRF3 and HMGI. Two symmetry-related sets of amino acid residues in ATF2 and Jun facilitated the opposite orientations of heterodimer interactions with IRF3 at the interferon-beta enhancer. Expression of ATF2 and Jun variants that bound the interferon-beta enhancer in opposite orientations together with IRF3 produced distinct levels of interferon-beta transcription in Sendai-virus infected Hela cells. Expression of these proteins resulted in different relative levels of transcription of different genes regulated by ATF2 and Jun. Collectively, this work illustrates a novel mode of cooperative DNA-binding by transcription factors and suggests that alternative nucleoprotein arrangements can influence transcriptional activity through distinct mechanisms at different genes.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89855/1/vburns_1.pd

Structure-based Targeting of Transcriptional Regulatory Complexes Implicated in Human Disease: A Dissertation

Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders. Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease. Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers

Dynamics of KSHV gene expression during de novo infection and the role of LANA in immune modulation

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human herpes virus that has been linked to the development of multiple malignancies including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Like all members of the herpesvirus family, KSHV establishes a lifelong persistence in the infected host. However, immediately upon infection, the virus has to overcome many challenges in the hostile cellular environment before it can establish a long-term infection. The remarkable success of the virus in establishing lifelong persistence in the infected host indicates that the virus is well equipped to manipulate the host environment very efficiently, even before it has a chance to fully express its genome. In order to understand how KSHV manipulates the host environment immediately upon infection, we must first understand which molecules are packaged in the virions and which viral genes are expressed during the initial time points following entry of the virus into the host cells. In chapter I, we probe these questions by performing a comprehensive analysis of the viral transcriptome in the purified KSHV virions. We also examine the dynamics of viral transcriptome at very early time-points following de novo infection of multiple cell lines permissive to long-term infection of KSHV. For this comprehensive study of viral transcriptome analysis, we used a high throughput approach of next-generation RNA sequencing. The results of this study identified many viral transcripts that are packaged into the virions and the transcripts that are actively transcribed during the initial infection period. Overall, the results of this study lay a foundation for future research targeting the viral genes whose expression may be critical for establishing a successful viral infection. One critical aspect of KSHV infection is its ability to persist throughout the life of the host. This can be attributed to its ability to efficiently hide from the host's immune surveillance. In chapter II, we identify a novel mechanism that helps the virus hide from the radar of host immunity. Our data sheds light on how one of the viral proteins, Latency- associated nuclear antigen (LANA), reduces the expression of major histocompatibility class II (MHC II) molecules, which are the molecules that are critical for reporting viral antigens to the immune cells of the host. We demonstrated that LANA binds with the proteins of regulatory factor X (RFX) complex, which are essential components of MHC II transcription machinery. The association of LANA with RFX complex reduces binding of the transcription factor called class II transactivator (CIITA) to the highly conserved promoters of MHC II genes. Binding of the CIITA to MHC II promoters is absolutely necessary for the expression of MHC II genes; by reducing the association of CIITA with the promoters of MHC II genes, LANA inhibits the expression of MHC II genes. KSHV infection is known to induce multiple pro-angiogenic cytokines that aid in extensive angiogenesis associated with Kaposi's sarcoma tumors. In chapter III, we demonstrate that LANA induces expression of a secreted angiogenic cytokine epidermal growth factor-like multiple 7 (EGFL7). Our data shows that LANA induces expression of EGFL7 in B cells, suggesting that it may act in a paracrine fashion during KSHV infection. The results of this research provide insight into an additional mechanism used by KSHV to promote angiogenesis. Targeting EGFL7 in combination with other important angiogenic cytokines induced during KSHV infection may increase the effectiveness of currently available anti-angiogenic therapy for Kaposi's sarcoma

Analysis of the molecular mechanisms underlying the activity of the Ets-1/USF-1 transcription facotr complex on the HIV-1 LTR

To assure cell type specific gene transcription cells have developed strategies to control the transcription of a large number of genes with a limited number of transcription factors. This is achieved by combinatorial control in which a complex array of transcription factors regulates promoters and enhancers. Recognition of regulatory elements is governed by both protein-DNA and protein-protein interactions. Many transcription factors can engage in multiple protein-protein interactions that form larger complexes required for adequate gene expression. In this PhD thesis I will present results that illuminate the multifaceted interplay between the transcription factors Ets-1 and USF-1. The ETS proteins act synergistically with a variety of other transcription factors to regulate many cellular and viral promoters and enhancers. Transcription of human immunodeficiency virus 1 (HIV-1), integrated into the host cell genome, also depends on the concerted action of cellular and viral transcription factors recruited to the HIV-1 long terminal repeat (LTR). The cellular transcription factors Ets-1 and USF-1 have been shown to form a complex on adjacent DNA binding sites present in the distal enhancer of the HIV-1 provirus and to cooperate in DNA binding and transactivation. DNA binding of Ets-1 is governed by autoinhibition that is exerted by two distinct inhibitory modules situated N- and C-terminally to the ETS DNA binding domain. The objective of my thesis project was to unravel the molecular mechanisms that govern the cooperation between Ets-1 and USF-1. I could show that USF-1 interacts with the C-terminal autoinhibitory module of Ets-1 and that this interaction is required to relieve autoinhibition of Ets-1 DNA binding. Reciprocally DNA binding by USF-1 is also facilitated by interaction with Ets-1. Furthermore, I provide evidence that synergistic transactivation by Ets-1 and USF-1 is not only the consequence of increased DNA binding potential but of additional cooperative mechanisms that affect transactivation function itself. I could reveal a novel mechanism of transcription factor cooperativity by showing that the C-terminal autoinhibitory module of Ets-1 can directly activate transactivation capacity of USF-1. In addition, I show that the transcriptional cofactor CBP is implicated in the mediation of Ets-1/USF-1 cooperativity. CBP interacts physically with both transcription factors and is required for synergistic transactivation. I could map the domain in USF-1 necessary for interaction with CBP to a stretch of 22 amino acids. Deletion of this domain abolishes both transactivation capacity of USF-1 on the HIV-1 LTR reporter and cooperativity with Ets-1. Together, these data provide new insights into the molecular mechanisms underlying Ets-1/USF-1 cooperativity. They indicate that transcription factor interaction results in significant conformational changes that affect both DNA binding and transactivation function of the complex. The example of Ets-1 and USF-1 could serve as a model for the hypothesis that transcription factors do not act as individual entities but that their functionality is only revealed in the complex with other partner molecules, similar to other multiprotein machineries in the cell

Investigations of the DNA-binding activity and gene regulatory properties of IRF3, IRF5, and IRF7 homodimers

The innate immune response is an essential component of the mammalian immune system that responds rapidly to pathogens. This response to pathogens is initiated by the detection of pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs). PRR signaling activates antipathogen gene programs via transcription factors (TFs) such as the interferon regulatory factors (IRFs). IRF3, IRF5, and IRF7 (IRF3/5/7) are key signal-dependent TFs that have overlapping, yet distinct, roles in the mammalian response to pathogens. To examine the role that DNA-binding specificity plays in delineating IRF3/5/7-specific gene regulation, we used protein-binding microarrays (PBMs) to characterize the DNA binding of IRF3/5/7 homodimers. We identified both common and dimer-specific DNA binding sites, and show that DNA-binding differences can translate into dimer-specific gene regulation. Central to the antiviral response, IRF3/5/7 regulate type I interferon (IFN) genes. We show that IRF3 and IRF7 bind to many interferon-stimulated response element (ISRE)-type sites in the virus-response elements (VREs) of IFN promoters. However, strikingly, IRF5 does not bind the VREs, suggesting evolutionary selection against IRF5 homodimer binding. Mutational analysis identified a a critical specificity-determining residue that inhibits IRF5 binding to the ISRE-variants present in the IFN gene promoters. Integrating PBM and reporter gene data we find that both DNA-binding affinity and affinity-independent mechanisms determine the transcriptional activation ability of DNA-bound IRF dimers, suggesting that DNA-based allostery plays a role in IRF binding site function. To assay the sequence determinants of IRF-dependent transcriptional regulation, we propose using a modified massively parallel reporter assay (MPRA). The proposed MPRA leverages unique molecular identifiers to improve the accuracy of reporter gene quantitation. This work provides new insights into the role and limitations of DNA-binding affinity in delineating IRF3/5/7-specific gene expression and lays groundwork for further understanding the complexities of IRF-dependent transcriptional regulation of innate immune genes

DNA-BINDING SITE RECOGNITION BY bHLH AND MADS-DOMAIN TRANSCRIPTION FACTORS

Herewithin, two transcription factor (TF) regulatory complexes were investigated. A bHLH–MYB–WDR (BMW) DNA-binding complex from maize was the first complex to be studied. R, a maize bHLH involved in the activation of genes in the anthocyanin pathway, had been characterized to indirectly bind DNA despite the presence of a functional DNA-binding domain. Findings presented here reveal that this is only partially correct. Direct DNA-binding by R was found to be dependent upon two distinct dimerization domains that function as a switch. This switch-like mechanism allows R to be repurposed for the activation of promoters of differing cis-element structure. The second regulatory complex studied was of the Arabidopsis thaliana MIKC-MADS TF family. For many TFs, DNA-binding site recognition is relatively straightforward and very sequence specific, while others exhibit relaxed sequence specificity. MADS-domain TFs are one family of TFs with a wider range of cis-element sequences. Though consensus cis-element sequences have been determined for various MADS-domains, correctly predicting and identifying biologically functional cis-elements has been a challenge. In order to study the influence of nucleobase associations within the cis-element, a DNA-Protein Interaction (DPI)-ELISA method was modified and optimized to screen a panel of specific probes. Screening of the SEP3 homodimer against a panel of sequential, palindromic probes revealed that nucleobases in position -1:+1 of the CArG-box influence binding strength between the MADS-domain and DNA. Additionally, the specificity of AGL15 towards CT-W6-AG forms was discovered to be determined by the functional groups present in the minor groove at position -4:+4 using inosine:cytosine (I:C) base pairs. Finally, the FLC–SVP MADS-domain heterodimer, bound to a native cis-element, was modeled and binding simulated using molecular dynamics. In conjunction with simulations of AGL15 and SEP3 homodimers, a potential binding mechanism was identified for this unique heterodimer. DNA sequence recognition by the MADS-domain was found to occur asymmetrically. In the case of the FLC–SVP heterodimer, the direction of asymmetrical DNA-binding in heterodimers was found to be fixed. Furthermore, the molecular dynamics simulations provided insight towards understanding the results generated from previous DPI-ELISA experiments, which should provide an improved means for predicting biologically significant CArG-boxes around genes

Mitochondria and peroxisomes : role within cellular antiviral defense

Mestrado em Biomedicina MolecularThe present paper presents a review and compilation of all the scientifically relevant bibliography to date, regarding the antiviral signalling pathways implicated in the cellular innate immune system in humans. Emphasizing the mitochondrial antiviral signalling adaptor (MAVS), this paper explores the special features of the signal transduction pathways and their components in two specific organelles: mitochondria and peroxisomes. These pathways, ultimately, result in the expression of interferon-stimulated genes (ISGs), which are primarily responsible for fighting against viral replication, viral particle assembly and virion release within the cell. In this paper, several proposals for further investigation are also presented, since there is still a lot to learn about the role of peroxisomes in the antiviral innate immune responses.O presente trabalho propõe-se a rever e compilar toda a bibliografia cientificamente relevante até à data, no que respeita as vias de sinalização antivirais implicadas na imunidade celular inata em células humanas. Com ênfase na proteína adaptadora MAVS, este trabalho explora as particularidades das vias de transdução de sinal e respetivos intervenientes em dois organelos celulares específicos: mitocôndrias e peroxissomas. Estas vias, em última instância, resultam na expressão de genes estimulados por interferões (ISGs), principais responsáveis pelo combate celular eficaz contra a replicação viral, montagem de partículas virais e libertação de viriões na célula infetada. Neste trabalho são ainda apresentadas propostas para investigações futuras, uma vez que ainda muito pouco se sabe sobre o papel dos peroxissomas nas respostas imunitárias inatas contra infeções virais

Genetic analysis of equine 2', 5'-oligoadenylate synthetase (OASI) and ribonculclease L (RNASEL) polymorphims and association to severe West Nile Virus disease

West Nile virus (WNV), a member of the Flaviviridae family of RNA viruses, was first introduced to the United States in 1999 with rapid transmission across a variety of hosts throughout the continental states. Genetic research to identify genes involved in resistance and susceptibility to WNV began in mice, where it was observed that natural populations were resistant or fatally susceptible. Further investigation led to the identification of the Flavivirus resistance gene as the oligoadenylate synthetase 1b gene in mice. A nonsense mutation was found within the coding region of this gene that associated absolutely with susceptibility to WNV. A two-stage association study was conducted to identify similar genetic associations to West Nile encephalitis in naturally susceptible and resistant populations of horses in the United States. Genomic sequence of a majority of the equine 2’,5’-oligoadenylate synthetase 1 (OAS1) gene was assembled by shotgun-sequencing CHORI BAC 100:I10 (3.95X). A contig map spanning the entire gene was constructed, including 8 kilobases of promoter sequence upstream of the first exon. Coding regions of equine OAS1 and ribonuclease L (RNASEL) genes, as well as the OAS1 promoter, were screened for mutations from a random sample of horses of multiple breeds. Numerous polymorphisms were identified for case-control analyses. Analysis using Fisher’s Exact test identified allelic and genotypic associations. Odds ratios were also determined to measure strength of the associations. Case-control analysis of haplotype frequencies identified significant differences in haplotype frequencies between populations and association to West Nile encephalitis. A conserved interferon-stimulated response element was mapped to within 518 basepairs upstream of the transcription start site of OAS1. Promoter polymorphisms were not found to affect induction by interferon-tau; however, additional analyses are necessary to further characterize the equine OAS1 promoter and the host factors involved in regulating expression. Statistical analyses of the genotype data from the case and control populations identified significant associations between polymorphisms of the OAS1 and RNASEL genes with severe West Nile encephalitis. The similarity between human and horse OAS immunity genes suggests that the horse may provide a genetic model to further investigate mammalian SNP-associated viral susceptibility

Proto-oncogene c-jun expression is induced by AML1-ETO in a JNK dependent manner:possible role in the pathogenesis of acute myeloid leukemia

Overexpression of proto-oncogene c-jun and constitutive activation of the Jun NH2-terminal kinase (JNK) signaling pathway have been implicated in the leukemic transformation process. However, c-jun expression has not been investigated in acute myeloid leukemia (AML) cells containing the most common chromosomal translocations. t(8;21) is one of the most common AML-associated translocation and results in the AML1-ETO fusion protein. Overexpression of AML1-ETO in NIH3T3 cells leads to increased phosphorylation of Ser63 in c-Jun, which is generally JNK dependent. The role of the JNK signaling pathway for the functional properties of AML1-ETO is, however, unknown. In the present study we found high expression levels of c-jun mRNA in t(8;21), t(15;17) or inv(16) positive patient cells by microarray analysis. Within t(8;21) positive patient samples, there was a correlation between AML1-ETO and c-jun mRNA expression levels. In myeloid U937 cells, c-jun mRNA and c-Jun protein expression levels increased upon induction of AML1-ETO. AML1-ETO transactivated the human c-jun promoter through the proximal AP-1 site via activating the JNK signaling pathway. JNK targets c-Jun and ATF-2, which also bind to the proximal AP-1 site in U937 cells, were also phosphorylated upon AML1-ETO induction. Furthermore, AML1-ETO induction increased the DNA binding capacity of c-Jun and ATF-2 to the proximal AP-1 site of the c-jun promoter, which might result in their enhanced transactivation capacities. Interference with JNK and c-Jun activation by using JIP-1 or a JNK inhibitor reduced the transactivation capacity of AML1-ETO on the c-jun promoter and the pro-apoptotic function of AML1-ETO in U937 cells. AML1-ETO seems to activate the JNK signaling pathway by inducing the expression of a cytoplasmic factor, possibly G-CSF, because supernatant of AML1-ETO expressing cells was sufficient to induce phosphorylation of JNK and c-Jun in wildtype U937 cells. This data demonstrates a novel mechanism of how AML1-ETO might exert positive effects on target gene expression and identifies the proto-oncogene c-jun as a common target gene in AML patient cells.Überexpression des Proto-Onkogens c-jun und konstitutive Aktivierung des Jun NH2-terminalen Kinase (JNK)-Signaltransduktionsweges sind wichtig für die leukämische Transformation in der Chronischen Myeloischen Leukämie. Die Expression von c-jun bei Akuter Myeloischer Leukämie (AML) mit den häufigsten reziproken Translokationen ist jedoch unbekannt. Bei einer der häufigsten AML Translokation t(8;21) wurde in Fibroblastenzellen gezeigt, daß das AML1-ETO-Fusionsgen die Phosphorylierung des Serin 63 in c-Jun erhöht. Die Rolle des JNK-Signalweges, der c-Jun am Serin 63 phosphorylieren kann, für die Funktion von AML1-ETO wurde bisher jedoch nicht untersucht. Weiterhin kann aktiviertes c-Jun durch eine positive Rückkoppelungsschleife über den c-jun Promotor zur Erhöhung der c-Jun Expression führen. In der vorliegenden Arbeit konnten wir zeigen, daß AML Patientenzellen mit den häufigen Translokationen: t(8;21), t(15;17) oder inv(16) mehr c-jun mRNA besitzen im Vergleich zu Knochenmarkszellen gesunder Probanden. Weiterhin fanden wir eine hohe Korrelation zwischen der AML1-ETO und der c-jun mRNA bei t(8;21) positiven Patientenzellen. Induktion von AML1-ETO in der myeloischen U937 Zellinie erhöhte sowohl c-jun mRNA als auch c-Jun Proteinexpression. Damit konnten wir zeigen, daß AML1-ETO die Erhöhung der c-jun Expression bewirkt. Wir untersuchten den molekularen Mechanismus in U937 Zellen mittels transienter Transfektionen und fanden, daß AML1-ETO den c-jun Promotor durch die proximale AP-1 Seite transaktiviert. Diese Transaktivierung erfolgte indirekt über Aktivierung des JNK-Signaltransduktionsweges durch AML1-ETO. AML1-ETO-Induktion führte auch zur Phosphorylierung der JNK-Zielproteine c-Jun und ATF-2. Diese konnten im Gelretardierungsassay an die proximale AP-1 Seite des c-jun Promotors binden und wurden durch AML1-ETO-Induktion in ihrer Bindungsfähigkeit verstärkt. Deshalb nehmen wir an, daß die Transaktivierungskapazität des c-jun Promotors durch AML1-ETO über die Aktivierung des JNK-Signalweges läuft