Mridul Kalita

RelA Ser276 Phosphorylation-Coupled Lys310 Acetylation Controls Transcriptional Elongation of Inflammatory Cytokines in Respiratory Syncytial Virus Infection

ABSTRACT Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections (LRTIs) in humans. In experimental models of RSV LRTI, the actions of the nuclear factor κB (NF-κB) transcription factor mediate inflammation and pathology. We have shown that RSV replication induces a mitogen-and-stress-related kinase 1 (MSK-1) pathway that activates NF-κB RelA transcriptional activity by a process involving serine phosphorylation at serine (Ser) residue 276. In this study, we examined the mechanism by which phospho-Ser276 RelA mediates expression of the NF-κB-dependent gene network. RelA-deficient mouse embryonic fibroblasts (MEFs) complemented with the RelA Ser276Ala mutant are deficient in CXCL2/Groβ, KC, and interleukin-6 (IL-6) expression, but NFKBIA/IκBα is preserved. We show that RSV-induced RelA Ser276 phosphorylation is required for acetylation at Lys310, an event required for transcriptional activity and stable association of RelA with the activated positive transcriptional elongation factor (PTEF-b) complex proteins, bromodomain 4 (Brd4), and cyclin-dependent kinase 9 (CDK9). In contrast to gene loading pattern of PTEF-b proteins produced by tumor necrosis factor (TNF) stimulation, RSV induces their initial clearance followed by partial reaccumulation coincident with RelA recruitment. The RSV-induced binding patterns of the CDK9 substrate, phospho-Ser2 RNA polymerase (Pol) II, follows a similar pattern of clearance and downstream gene reaccumulation. The functional role of CDK9 was examined using CDK9 small interfering RNA (siRNA) and CDK inhibitors, where RSV-induced NF-κB-dependent gene expression was significantly inhibited. Finally, although RSV induces a transition from short transcripts to fully spliced mRNA in wild-type RelA (RelA WT)-expressing cells, this transition is not seen in cells expressing RelA Ser276Ala. We conclude that RelA Ser276 phosphorylation mediates RelA acetylation, Brd4/CDK9 association, and activation of downstream inflammatory genes by transcriptional elongation in RSV infection.

Sources of Cell-to-cell Variability in Canonical Nuclear Factor-κB (NF-κB) Signaling Pathway Inferred from Single Cell Dynamic Images

The canonical nuclear factor-κB (NF-κB) signaling pathway controls a gene network important in the cellular inflammatory response. Upon activation, NF-κB/RelA is released from cytoplasmic inhibitors, from where it translocates into the nucleus, subsequently activating negative feedback loops producing either monophasic or damped oscillatory nucleo-cytoplasmic dynamics. Although the population behavior of the NF-κB pathway has been extensively modeled, the sources of cell-to-cell variability are not well understood. We describe an integrated experimental-computational analysis of NF-κB/RelA translocation in a validated cell model exhibiting monophasic dynamics. Quantitative measures of cellular geometry and total cytoplasmic concentration and translocated RelA amounts were used as priors in Bayesian inference to estimate biophysically realistic parameter values based on dynamic live cell imaging studies of enhanced GFP-tagged RelA in stable transfectants. Bayesian inference was performed on multiple cells simultaneously, assuming identical reaction rate parameters, whereas cellular geometry and initial and total NF-κB concentration-related parameters were cell-specific. A subpopulation of cells exhibiting distinct kinetic profiles was identified that corresponded to differences in the IκBα translation rate. We conclude that cellular geometry, initial and total NF-κB concentration, IκBα translation, and IκBα degradation rates account for distinct cell-to-cell differences in canonical NF-κB translocation dynamics.

CDK9-Dependent Transcriptional Elongation in the Innate Interferon-Stimulated Gene Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells

ABSTRACT Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3−/− MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser2 carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-β) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease.

Systems biology approaches to understanding Epithelial Mesenchymal Transition (EMT) in mucosal remodeling and signaling in asthma

A pathological hallmark of asthma is chronic injury and repair, producing dysfunction of the epithelial barrier function. In this setting, increased oxidative stress, growth factor- and cytokine stimulation, together with extracellular matrix contact produces transcriptional reprogramming of the epithelial cell. This process results in epithelial-mesenchymal transition (EMT), a cellular state associated with loss of epithelial polarity, expression of mesenchymal markers, enhanced mobility and extracellular matrix remodeling. As a result, the cellular biology of the EMT state produces characteristic changes seen in severe, refractory asthma: myofibroblast expansion, epithelial trans-differentiation and subepithelial fibrosis. EMT also induces profound changes in epithelial responsiveness that affects innate immune signaling that may have impact on the adaptive immune response and effectiveness of glucocorticoid therapy in severe asthma. We discuss how this complex phenotype is beginning to be understood using systems biology-level approaches through perturbations coupled with high throughput profiling and computational modeling. Understanding the distinct changes induced by EMT at the systems level may provide translational strategies to reverse the altered signaling and physiology of refractory asthma.

Analysis of the TGFβ-induced program in primary airway epithelial cells shows essential role of NF-κB/RelA signaling network in type II epithelial mesenchymal transition

BackgroundThe airway epithelial cell plays a central role in coordinating the pulmonary response to injury and inflammation. Here, transforming growth factor-β (TGFβ) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-Seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it.ResultsGeneralized linear modeling was performed on a two-factor RNA-Seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-κB/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-κB/RelA pathway. This Type II EMT program was compared to Type III EMT in TGFβ stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGFβ-induced the NF-κB/RelA pathway by observing a 2-fold change in NF-κB/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGFβ-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression.ConclusionsThese data indicate that NF-κB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

Systems Approaches to Modeling Chronic Mucosal Inflammation

The respiratory mucosa is a major coordinator of the inflammatory response in chronic airway diseases, including asthma and chronic obstructive pulmonary disease (COPD). Signals produced by the chronic inflammatory process induce epithelial mesenchymal transition (EMT) that dramatically alters the epithelial cell phenotype. The effects of EMT on epigenetic reprogramming and the activation of transcriptional networks are known, its effects on the innate inflammatory response are underexplored. We used a multiplex gene expression profiling platform to investigate the perturbations of the innate pathways induced by TGFβ in a primary airway epithelial cell model of EMT. EMT had dramatic effects on the induction of the innate pathway and the coupling interval of the canonical and noncanonical NF-κB pathways. Simulation experiments demonstrate that rapid, coordinated cap-independent translation of TRAF-1 and NF-κB2 is required to reduce the noncanonical pathway coupling interval. Experiments using amantadine confirmed the prediction that TRAF-1 and NF-κB2/p100 production is mediated by an IRES-dependent mechanism. These data indicate that the epigenetic changes produced by EMT induce dynamic state changes of the innate signaling pathway. Further applications of systems approaches will provide understanding of this complex phenotype through deterministic modeling and multidimensional (genomic and proteomic) profiling.

Inducible Tumor Necrosis Factor (TNF) receptor-associated factor-1 expression couples the canonical to the non-canonical NF-κB pathway in TNF stimulation

Background: The NF-κB transcription factor mediates the inflammatory response through canonical and non-canonical pathways. Results: TRAF-1 binds and stabilizes NIK by disrupting its association with TRAF2·cIAP2 complex. Conclusion: These integrated computational-experimental studies identify TRAF1·NIK as a feed-forward complex coupling canonical and non-canonical pathways. Significance: These findings provide insight into how cells decode inflammatory signals into distinct genomic responses. The NF-κB transcription factor mediates the inflammatory response through distinct (canonical and non-canonical) signaling pathways. The mechanisms controlling utilization of either of these pathways are largely unknown. Here we observe that TNF stimulation induces delayed NF-κB2/p100 processing and investigate the coupling mechanism. TNF stimulation induces TNF-associated factor-1 (TRAF-1) that directly binds NF-κB-inducing kinase (NIK) and stabilizes it from degradation by disrupting its interaction with TRAF2·cIAP2 ubiquitin ligase complex. We show that TRAF1 depletion prevents TNF-induced NIK stabilization and reduces p52 production. To further examine the interactions of TRAF1 and NIK with NF-κB2/p100 processing, we mathematically modeled TRAF1·NIK as a coupling signaling complex and validated computational inference by siRNA knockdown to show non-canonical pathway activation is dependent not only on TRAF1 induction but also NIK stabilization by forming TRAF1·NIK complex. Thus, these integrated computational-experimental studies of TNF-induced TRAF1 expression identified TRAF1·NIK as a central complex linking canonical and non-canonical pathways by disrupting the TRAF2-cIAP2 ubiquitin ligase complex. This feed-forward kinase pathway is essential for the activation of non-canonical pathway.

Comparative Genomics and Immunoinformatics Approach for the Identification of Vaccine Candidates for Enterohemorrhagic Escherichia coli O157:H7

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic-uremic syndrome worldwide. Thus far, there is no vaccine for humans against EHEC infections. In this study, a comparative genomics analysis was performed to identify EHEC-specific antigens useful as potential vaccines. The genes present in both EHEC EDL933 and Sakai strains but absent in nonpathogenic E. coli K-12 and HS strains were subjected to an in silico analysis to identify secreted or surface-expressed proteins. We obtained a total of 65 gene-encoding protein candidates, which were subjected to immunoinformatics analysis. Our criteria of selection aided in categorizing the candidates as high, medium, and low priority. Three members of each group were randomly selected and cloned into pVAX-1. Candidates were pooled accordingly to their priority group and tested for immunogenicity against EHEC O157:H7 using a murine model of gastrointestinal infection. The high-priority (HP) pool, containing genes encoding a Lom-like protein (pVAX-31), a putative pilin subunit (pVAX-12), and a fragment of the type III secretion structural protein EscC (pVAX-56.2), was able to induce the production of EHEC IgG and sIgA in sera and feces. HP candidate-immunized mice displayed elevated levels of Th2 cytokines and diminished cecum colonization after wild-type challenge. Individually tested HP vaccine candidates showed that pVAX-12 and pVAX-56.2 significantly induced Th2 cytokines and production of fecal EHEC sIgA, with pVAX-56.2 reducing EHEC cecum colonization. We describe here a bioinformatics approach able to identify novel vaccine candidates potentially useful for preventing EHEC O157:H7 infections.

Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1 chromatin target was further demonstrated using chromatin immunoprecipitation (ChIP). These data indicate that CDK9 is a dynamic multifunctional enzyme complex mediating not only transcriptional elongation, but also alternative RNA splicing and potentially translational control.

Mixed-effects model of epithelial–mesenchymal transition reveals rewiring of signaling networks

The type II epithelial-mesenchymal transition (EMT) produces airway fibrosis and remodeling, contributing to the severity of asthma and chronic obstructive pulmonary disease. While numerous studies have been done on the mechanisms of the transition itself, few studies have investigated the system effects of EMT on signaling networks. Here, we use mixed effects modeling to develop a computational model of phospho-protein signaling data that compares human small airway epithelial cells (hSAECs) with their EMT-transformed counterparts across a series of perturbations with 8 ligands and 5 inhibitors, revealing previously uncharacterized changes in signaling in the EMT state. Strong couplings between menadione, TNFα and TGFβ and their known phospho-substrates were revealed after mixed effects modeling. Interestingly, the overall phospho-protein response was attenuated in EMT, with loss of Mena and TNFα coupling to heat shock protein (HSP)-27. These differences persisted after correction for EMT-induced changes in phospho-protein substrate abundance. Construction of network topology maps showed significant changes between the two cellular states, including a linkage between glycogen synthase kinase (GSK)-3α and small body size/mothers against decapentaplegic (SMAD)2. The model also predicted a loss of p38 mitogen activated protein kinase (p38MAPK)-independent HSP27 signaling, which we experimentally validated. We further characterized the relationship between HSP27 and signal transducers and activators of transcription (STAT)3 signaling, and determined that loss of HSP27 following EMT is only partially responsible for the downregulation of STAT3. These rewired connections represent therapeutic targets that could potentially reverse EMT and restore a normal phenotype to the respiratory mucosa.