Essential Transcriptional Activator Research Articles

Pancreatic agenesis and altered m6A methylation in the pancreas of PDX1-mutant cynomolgus macaques.

As an essential transcriptional activator, PDX1 plays a crucial role in pancreatic development and β-cell function. Mutations in the PDX1 gene may lead to type 4 maturity-onset diabetes of the young (MODY4) and neonatal diabetes mellitus. However, the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models. In this study, three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology, all of which succumbed shortly postpartum, exhibiting pancreatic agenesis. Notably, one tri-allelic PDX1-mutant cynomolgus macaque (designated as M4) developed a pancreas, whereas the two mono-allelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation. RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions, indicating developmental delay and PDX1 haploinsufficiency. A marked change in m6A methylation was identified in the M4 pancreas, confirmed through cultured PDX1-mutant islet organoids. Notably, overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids. This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.

Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

Upregulation of IQGAP2 by EBV transactivator Rta and its influence on EBV life cycle.

Epstein-Barr virus (EBV) is a human oncogenic γ-herpesvirus that establishes persistent infection in more than 90% of the world's population. EBV has two life cycles, latency and lytic replication. Reactivation of EBV from latency to the lytic cycle is initiated and controlled by two viral immediate-early transcription factors, Zta and Rta, encoded by BZLF1 and BRLF1, respectively. In this study, we found that IQGAP2 expression was elevated in EBV-infected B cells and identified Rta as a viral gene responsible for the IQGAP2 upregulation in both B cells and nasopharyngeal carcinoma cell lines. Mechanistically, we showed that Rta increases IQGAP2 expression through direct binding to the Rta-responsive element in the IQGAP2 promoter. We also demonstrated the direct interaction between Rta and IQGAP2 as well as their colocalization in the nucleus. Functionally, we showed that the induced IQGAP2 is required for the Rta-mediated Rta promoter activation in the EBV lytic cycle progression and may influence lymphoblastoid cell line clumping morphology through regulating E-cadherin expression. IMPORTANCE Elevated levels of antibodies against EBV lytic proteins and increased EBV DNA copy numbers in the sera have been reported in patients suffering from Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, indicating that EBV lytic cycle progression may play an important role in the pathogenesis of EBV-associated diseases and highlighting the need for a more complete mechanistic understanding of the EBV lytic cycle. Rta acts as an essential transcriptional activator to induce lytic gene expression and thus trigger EBV reactivation. In this study, scaffolding protein IQGAP2 was found to be upregulated prominently following EBV infection via the direct binding of Rta to the RRE in the IQGAP2 promoter but not in response to other biological stimuli. Importantly, IQGAP2 was demonstrated to interact with Rta and promote the EBV lytic cycle progression. Suppression of IQGAP2 was also found to decrease E-cadherin expression and affect the clumping morphology of lymphoblastoid cell lines.

M1BP is an essential transcriptional activator of oxidative metabolism during Drosophila development

Oxidative metabolism is the predominant energy source for aerobic muscle contraction in adult animals. How the cellular and molecular components that support aerobic muscle physiology are put in place during development through their transcriptional regulation is not well understood. Using the Drosophila flight muscle model, we show that the formation of mitochondria cristae harbouring the respiratory chain is concomitant with a large-scale transcriptional upregulation of genes linked with oxidative phosphorylation (OXPHOS) during specific stages of flight muscle development. We further demonstrate using high-resolution imaging, transcriptomic and biochemical analyses that Motif-1-binding protein (M1BP) transcriptionally regulates the expression of genes encoding critical components for OXPHOS complex assembly and integrity. In the absence of M1BP function, the quantity of assembled mitochondrial respiratory complexes is reduced and OXPHOS proteins aggregate in the mitochondrial matrix, triggering a strong protein quality control response. This results in isolation of the aggregate from the rest of the matrix by multiple layers of the inner mitochondrial membrane, representing a previously undocumented mitochondrial stress response mechanism. Together, this study provides mechanistic insight into the transcriptional regulation of oxidative metabolism during Drosophila development and identifies M1BP as a critical player in this process.

Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30

BackgroundTrichoderma reesei Rut-C30 is a hypercellulolytic mutant strain that degrades abundant sources of lignocellulosic plant biomass, yielding renewable biofuels. Although Zn2+ is an activator of enzymes in almost all organisms, its effects on cellulase activity in T. reesei have yet to be reported.ResultsAlthough high concentrations of Zn2+ severely suppressed the extension of T. reesei mycelia, the application of 1–4 mM Zn2+ enhanced cellulase and xylanase production in the high-yielding cellulase-producing Rut-C30 strain of T. reesei. Expression of the major cellulase, xylanase, and two essential transcription activator genes (xyr1 and ace3) increased in response to Zn2+ stimulation. Transcriptome analysis revealed that the mRNA levels of plc-e encoding phospholipase C, which is involved in the calcium signaling pathway, were enhanced by Zn2+ application. The disruption of plc-e abolished the cellulase-positive influence of Zn2+ in the early phase of induction, indicating that plc-e is involved in Zn2+-induced cellulase production. Furthermore, treatment with LaCl3 (a plasma membrane Ca2+ channel blocker) and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) indicated that calcium signaling is partially involved in this process. Moreover, we identified the zinc-responsive transcription factor zafA, the transcriptional levels of which declined in response to Zn2+ stress. Deletion of zafA indicates that this factor plays a prominent role in mediating the Zn2+-induced excessive production of cellulase.ConclusionsFor the first time, we have demonstrated that Zn2+ is toxic to T. reesei, although promotes a marked increase in cellulase production. This positive influence of Zn2+ is facilitated by the plc-e gene and zafA transcription factor. These findings provide insights into the role of Zn2+ in T. reesei and the mechanisms underlying signal transduction in cellulase synthesis.

Analyzing the impact of ATF3 in tumorigenesis and immune cell infiltration of ovarian tumor: a bioinformatics study.

ATF3 is an essential transcription activator in regulating cancer-related genetic expression. To identify the role of ATF3 in ovarian tumor, we investigated the correlation between ATF3 expression and the clinicopathological properties using multiple database. The cBioPortal and GEPIA database displayed the clinical information of ovarian patients harboring or without harboring ATF3 mutation. Furthermore, we assessed the relationship between survival and ATF3 expression level using Kaplan-Meier plotter, which reveals that the ovarian patients with higher expression of ATF3 suffered the worse overall survival and progression-free survival. The differentially expressed genes were analyzed using gene ontology, protein-protein interaction network, and gene set enrichment analysis to identify the hub gene and critical pathways, significantly affecting the tumorigenesis of ovarian tumor. Finally, we assessed the correlation between ATF3 and immune cell infiltration using Tumor Immunoassay Resource (TIMER) database. The results demonstrated that higher expression has a positive correlation with macrophage infiltration, expression for M1- and M2-type macrophages. Our study suggests that ATF3 can regulate the cell cycle and heme-related oxidative phosphorylation process, and it may be a critical factor to regulate the macrophage cell to be infiltrated into ovarian cancer. ATF3 can be used as a biomarker for diagnosis and therapy of ovarian tumor.

Chronic circadian shift leads to adipose tissue inflammation and fibrosis

The circadian clock exerts temporal coordination of metabolic pathways. Clock disruption is intimately linked with the development of obesity and insulin resistance, and our previous studies found that the essential clock transcription activator, Brain and Muscle Arnt-like 1 (Bmal1), is a key regulator of adipogenesis. However, the metabolic consequences of chronic shiftwork on adipose tissues have not been clearly defined. Here, using an environmental lighting-induced clock disruption that mimics rotating shiftwork schedule, we show that chronic clock dysregulation for 6 months in mice resulted in striking adipocyte hypertrophy with adipose tissue inflammation and fibrosis. Both visceral and subcutaneous depots display enlarged adipocyte with prominent crown-like structures indicative of macrophage infiltration together with evidence of extracellular matrix remodeling. Global transcriptomic analyses of these fat depots revealed that shiftwork resulted in up-regulations of inflammatory, adipogenic and angiogenic pathways with disruption of normal time-of-the-day-dependent regulation. These changes in adipose tissues are associated with impaired insulin signaling in mice subjected to shiftwork, together with suppression of the mTOR signaling pathway. Taken together, our study identified the significant adipose depot dysfunctions induced by chronic shiftwork regimen that may underlie the link between circadian misalignment and insulin resistance.

The clock regulator Bmal1 protects against muscular dystrophy

The muscle-intrinsic clock machinery is required for the maintenance of muscle growth, remodeling and function. Our previous studies demonstrated that the essential transcription activator of the molecular clock feed-back loop, Brain and Muscle Arnt-Like 1(Bmal1), plays a critical role in myogenic progenitor behavior to promote and regenerative myogenesis. Using genetic approaches targeting Bmal1 in the DMDmdx (mdx) dystrophic mouse model, here we report that the loss of Bmal1 function significantly accelerated dystrophic disease progression. In contrast to the mild dystrophic changes in mdx mice, the genetic loss-of-function of Bmal1 aggravated muscle damage in this dystrophic disease background, as indicated by persistently elevated creatine kinase levels, increased injury area and reduced muscle grip strength. Mechanistic studies revealed that markedly impaired myogenic progenitor proliferation and myogenic response underlie the defective new myofiber formation in the chronic dystrophic milieu. Taken together, our study identified the function of pro-myogenic clock gene Bmal1 in protecting against dystrophic damage, suggesting the potential for augmenting Bmal1 function to ameliorate dystrophic or degenerative muscle diseases.

Abstract 4403: Predicting therapeutic sensitivities from molecular interaction network rewiring of RUNX1-RUNX1T1 fusion

Abstract Cancer driver events are thought to converge on a subset of molecular systems that regulate the hallmark behaviors of cancer cells. However, the complexity of biological systems makes it difficult to evaluate the impact of individual mutations at the level of such cellular processes and behaviors. In order to overcome this, we propose to study the effects of cancer mutations by modeling them as perturbations to molecular network architectures, allowing network-based prediction of drug sensitivities. We first built the infrastructure to study protein-protein interaction network rewiring by variants by mapping different classes of variants onto edges of the PPI network using protein structure information. Mutations that cause a larger disruption to the underlying network architecture can create more vulnerabilities that can be used as drug targets. Therefore, in this project we decided to focus on fusion events which can interfere with many interactions of their parent proteins. In order to assess the disruption that can be caused by fusion events, we collected 2,699 cancer fusion proteins and demonstrated the centrality of their parents within a high confidence human PPI network of 12,047 proteins. This indicates that the parent proteins of fusion genes tend to interact with more protein partners and are more likely to display network topological properties suggesting a role in information transduction in molecular interaction networks. As leukemias are often impacted by chromosomal aberrations that can result in fusion and tend to harbor fewer somatic mutations than solid tumors, they provide a good setting to develop a network-based model to study fusion events in the context of drug responses. Therefore, we chose to study the RUNX1-RUNX1T1 translocation, t(8;21)(q22;q22), in AML which is thought to be a pre-leukemic event that can result in tumors after receiving additional mutations. This translocation replaces the transactivation domain of RUNX1 with most of the RUNX1T1 protein which changes its function as an essential transcriptional activator of hematopoiesis. In order to study the transcriptional effects of RUNX1-RUNX1T1 fusion on the underlying interactome network architecture, U937T cells induced with RUNX1-RUNX1T1 fusion were grown in triplicate and RNA was harvested prior to induction and at certain times post-induction. Wild type U937T cells with no perturbation serve as negative control. RNA sequencing was performed, followed by alignment of the reads to the reference genome. A differential gene expression analysis showed that 1,582 genes are differentially expressed in fusion induced cells versus wild type at FDR<0.05, among which 47 of them encode proteins in RUNX1-RUNX1T1 subnetwork. Gene set enrichment analysis using these genes revealed that 30 gene sets are significantly enriched at nominal p-value<0.01. Citation Format: Kivilcim Ozturk, Hannah Carter. Predicting therapeutic sensitivities from molecular interaction network rewiring of RUNX1-RUNX1T1 fusion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4403.

Kruppel-like factor 4 regulates developmental angiogenesis through disruption of the RBP-J-NICD-MAML complex in intron 3 of Dll4.

Angiogenesis is a multistep process that requires highly regulated endothelial cell (EC) behavior. The transcription factor Krüppel-like factor 4 (KLF4) is a critical regulator of several basic EC functions; we have recently shown that KLF4 disturbs pathological (tumor) angiogenesis by mediating the expression of members of VEGF and Notch signaling pathways. Notch signaling is central to orchestration of sprouting angiogenesis but little is known about the upstream regulation of Notch itself. To determine the role of KLF4 in normal (developmental) angiogenesis, we used a mouse retinal angiogenesis model. We found that endothelial-specific overexpression of KLF4 in transgenic mice (EC-K4 Tg) leads to increased vessel density, branching and number of tip cell filopodia as assessed on postnatal day 6 (P6). The hypertrophic vasculature seen with sustained KLF4 overexpression is not stable and undergoes prominent remodeling during P7-P12 resulting in a normal appearing retinal vasculature in adult EC-K4 Tg mice. We find that KLF4 inhibits Delta-like 4 (DLL4) expression in the angiogenic front during retinal vascular development. Furthermore, in an oxygen-induced retinopathy model, overexpression of KLF4 results in decreased vaso-obliteration and neovascular tuft formation that is similar to genetic or pharmacologic DLL4 inhibition. Mechanistically, we show that KLF4 disables the activity of the essential Notch transcriptional activator RBP-J by interfering with binding of co-activators NICD and MAML at intron 3 of the Notch ligand DLL4. In summary, our experimental results demonstrate a regulatory role of KLF4 in developmental angiogenesis through regulation of DLL4 transcription.

Investigation of the variable In(Lu) phenotype caused by KLF1 variants.

KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population. In(Lu) samples were sourced through screening and through the RBC reference laboratory. Blood donor samples (8036) were screened to identify weakened/absent Lub antigen. Samples were genotyped by massively parallel sequencing, while surface carbohydrates and blood group molecules were assessed by flow cytometry. Hemoglobin (Hb) types were analyzed by high-performance liquid chromatography. Four of 8036 donors were identified to be In(Lu), and two previously identified In(Lu) samples were provided from the RBC reference laboratory. Five different KLF1 variants were identified; two were novel: c.954G>C/p.Trp318Cys and c.421C>T/p.Arg141*. BCAM and CD44 were reduced in all samples, consistent with previous reports. As a group, In(Lu) RBCs had reduced CD35 (KN), ICAM4 (LW), and CD147 (OK), and demonstrated increased binding of lectins ECA and SNAI. One In(Lu) sample had elevated HbF and another elevated HbA2. Different KLF1 variants may potentially produce variable phenotypes. A framework for investigating KLF1 variants and their phenotypic impact has been provided. In the future, given available international databases, further testing algorithms (as advocated here) will allow for correlation of phenotype with genotype and therefore accurately document this variability between KLF1 variants.

Concerted Molecular Clock Control of Myogenic Precursor Properties Promotes Skeletal Muscle Growth and Remodeling

Circadian clock imparts temporal regulation on diverse physiological processes. We and others recently demonstrated that the clock circuit plays an important role in muscle growth and repair for maintenance of muscle mass, raising the possibility of targeting this machinery for muscle‐wasting disease therapies. Currently, the mechanisms underlying clock regulation of muscle growth processes are largely unknown. Here we show that the antagonistic actions of the essential clock transcriptional activator, Brain and Muscle Ant‐like 1 (Bmal1), and the clock repressor protein, Rev‐erbα, coordinately modulate myogenic precursor cell (MPC) differentiation and proliferation properties to facilitate muscle growth and remodeling. We were the first to report a novel function of Bmal1 in promoting MPC myogenic differentiation required for skeletal muscle regenerative repair. Current studies reveal that the clock transcription repressor Rev‐erbα suppresses myocyte development and its genetic loss of function promotes regenerative myogenesis, through its negative transcription control of target genes in the Wnt signaling pathway. Furthermore, we show that Bmal1 promotes, whereas Reverbα inhibits MPC proliferative behavior in vitro and upon muscle regeneration, mediated by their respective transcriptional regulations of cell cycle regulators. Notably, our work uncovers that mechanical stretch and exercise‐associated signals induce clock gene expression, suggesting their roles in entraining the cell‐autonomous muscle clock oscillators to physiological stimuli. Collectively, our studies reveal concerted functions of the muscle‐resident clock machinery in orchestrating MPC properties to contribute to muscle growth and consequently mass maintenance in accordance with myogenic cues. Elucidation of this temporal control in skeletal muscle tissue remodeling may offer novel therapeutic strategies to combat muscle‐wasting associated with aging, cancer or chronic diseases.Support or Funding InformationThis project is supported by grants American Heart Association 17GRNT33370012, Muscular Dystrophy Association 381294, and NIH DK 1R01DK112794 to KM.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcriptome analysis of Δmig1Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris.

Two catabolite repressor genes (MIG1 and MIG2) were previously identified in Pichia pastoris, and the derepression of alcohol oxidase (AOX) expression was realized in Δmig1 or Δmig1Δmig2 mutants grown in glycerol, but not in glucose. In this study, genome-wide RNA-seq analysis of Δmig1Δmig2 and the wild-type strain grown in glycerol revealed that the expression of numerous genes was greatly altered. Nearly 7% (357 genes) of approximately 5276 genes annotated in P. pastoris were significantly upregulated, with at least a two-fold differential expression in Δmig1Δmig2; the genes were mainly related to cell metabolism. Approximately 23% (1197 genes) were significantly downregulated; these were mainly correlated with the physiological characteristics of the cell. The methanol catabolism and peroxisome biogenesis pathways were remarkably enhanced, and the genes AOX1 and AOX2 were upregulated higher than 30-fold, which was consistent with the experimental results of AOX expression. The Mig proteins had a slight effect on autophagy when cells were grown in glycerol. The expression analysis of transcription factors showed that deletion of MIG1 and MIG2 significantly upregulated the binding of an essential transcription activator, Mit1p, with the AOX1 promoter, which suggested that Mig proteins might regulate the AOX1 promoter through the regulation of Mit1p. This work provides a reference for the further exploration of the methanol induction and catabolite repression mechanisms of AOX expression in methylotrophic yeasts.

SETD1B compensates loss of IRF8 expression to activate iNOS expression in myeloid-derived suppressor cells through an epigenetic mechanism

Abstract Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSCs). MDSCs use NO to nitrate the T cell receptor and STAT1 to inhibit T cell activation and the anti-tumor immune response. The molecular mechanism for iNOS expression regulation in tumor-induced MDSCs is unknown. We show that IRF8 deficiency results in diminished iNOS expression in mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo, indicating that IRF8 is an essential transcriptional activator for iNOS expression in myeloid cells under physiological conditions. Though IRF8 is silenced in tumor-induced MDSCs, iNOS expression is dramatically elevated in tumor-induced MDSCs suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathological conditions. Tumor-induced MDSCs exhibit diminished STAT1 and NF-κB Rel protein level, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs show increased SETD1B expression compared to their equivalent in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, a product of SETD1B enzymatic activity, is enriched at the nos2 promoter in tumor-induced MDSCs. Inhibition/silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. Our data determine that IRF8 is an essential transcriptional activator of iNOS in myeloid cells under physiological conditions. Tumor cells use the SETD1B-H3K4me3 epigenetic axis to compensate for loss of IRF8 expression to activate iNOS expression in MDSCs under pathological conditions.

Role of MIZ-1 in AMELX gene expression

Amelogenin (AMELX) is the main component of the developing tooth enamel matrix and is essential for enamel thickness and structure. However, little is known about its transcriptional regulation. Using gene expression analysis, we found that MIZ-1, a potent transcriptional activator of CDKN1A, is expressed during odontoblastic differentiation of hDPSCs (human dental pulp stem cells), and is essential for odontoblast differentiation and mineralization. We also investigated how MIZ-1 regulates gene expression of AMELX. Oligonucleotide-pull down assays showed that MIZ-1 binds to an MRE (MIZ-1 binding element) of the AMELX proximal promoter region (bp, −170 to −25). Combined, our ChIP, transient transcription assays, and promoter mutagenesis revealed that MIZ-1 directly binds to the MRE of the Amelx promoter recruits p300 and induces Amelx gene transcription. Finally, we show that the zinc finger protein MIZ-1 is an essential transcriptional activator of Amelx, which is critical in odontogenesis and matrix mineralization in the developing tooth.

An Essential Viral Transcription Activator Modulates Chromatin Dynamics.

Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes.

Abstract LB-265: IRF8 is an essential transcriptional activator of iNOS although MDSCs up-regulate iNOS expression via an IRF8-independent mechanism

Abstract Nitric Oxide Synthase (NOS) is a family of enzymes that catalyzes L-arginine metabolism to generate nitric oxide (NO). There are three main isoforms of NOS, named neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS or NOS2). These three distinct isoforms of NOS have different cellular localization, regulation, and catalytic properties. iNOS is the most extensively studied one among these three NOS isoforms. iNOS was originally identified in myeloid cells, and it is known that its expression is often induced after myeloid cell activation by endotoxins or cytokines to generate NO which then acts as a defense effector to suppress invading microorganisms or neoplastic cells. It is known that IFNγ signaling pathway and NF-κB synergistically regulate iNOS expression, and that IFN regulatory factor 8 (IRF8) enhances interaction between IFNγ signaling and NF-κB in regulation of iNOS expression. However, the molecular mechanism underlying iNOS expression regulation in myeloid cell lineage is still not fully understood. We report here that IRF8 is an essential transcription activator of iNOS in myeloid cells since IRF8-deficient myeloid cells lose iNOS expression. Furthermore, a key phenotype of IRF8 KO mice is the mass accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs), suggesting that IRF8 is a key suppressor of MDSC differentiation under physiological conditions. On the other hand, accumulation of MDSCs is a hallmark of cancers and iNOS expression is dramatically elevated in tumor-induced MDSCs, despite that tumor-induced MDSCs exhibit diminished IRF8 expression. Furthermore, the IFNγ signaling pathway is impaired and NF-κB subunits are undetectable in tumor-induced MDSCs. Our data therefore indicate that iNOS expression is activated by an IRF8-independent mechanism that is also IFNγ and NF-κB-independent in tumor-induced MDSCs under pathological conditions. At the functional level, we determined that iNOS-KO mice exhibit lower tumor incidence as compared to WT mice. However, the sizes of established tumors are not different between iNOS-KO mice and WT mice. Taken together, our data determine that IRF8 is an essential transcriptional activator of iNOS expression in myeloid cells under physiological conditions, although iNOS expression is activated by an IFNγ-, NF-κB-, and IRF8-independent mechanism in tumor-induced MDSCs under pathological conditions. iNOS acts as a suppressor of tumor cell colonization but not tumor growth. Citation Format: Mohammed M. Ibrahim, Amy V. Paschall, Priscilla S. Redd, Kebin Liu. IRF8 is an essential transcriptional activator of iNOS although MDSCs up-regulate iNOS expression via an IRF8-independent mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-265.

Control of Ebola hemorrhagic fever: vaccine development and our Ebola project in Sierra Leone.

Since December 2013, West Africa has experienced the worst Ebola virus outbreak in recorded history. Of the 28,639 cases reported to the World Health Organization as of March 2016, nearly half (14,124) occurred in Sierra Leone. With a case fatality rate of approximately 40%, this outbreak has claimed the lives of 11,316 individuals. No FDA-approved vaccines or drugs are available to prevent or treat Ebola virus infection. Experimental vaccines and therapies are being developed; however, their safety and efficacy are still being evaluated. Therefore, there is an urgent need to develop control measures to prevent or limit future Ebola virus outbreaks.Previously, we developed a replication-defective Ebola virus that lacks the coding region for the essential viral transcription activator VP30 (Ebola ΔVP30 virus). Here, we evaluated the vaccine efficacy of Ebola ΔVP30 virus in a non-human primate model and describe our collaborative Ebola project in Sierra Leone.

PGC-1α regulates the expression and activity of IRF-1.

Interferon regulatory transcription factor 1 (IRF-1) regulates downstream signals of tumor necrosis factor α (TNF-α). The activity of IRF-1 is mediated by Jak/Stat signaling pathway. In this study, we found that PPAR γ coactivator-1α (PGC-1α) is able to suppress the induction of IRF-1. Treatment with TNF-α in MC3T3 cells leads to a sustainable increase in the expression of IRF-1 and its target gene cyclooxygenase 2 (COX-2). In contrast, TNF-α treatment led to a sustainable reduction in expression of PGC-1α. Interestingly, we found that overexpression of PGC-1α attenuated the induction of IRF-1 and COX-2. However, silence of PGC-1α exacerbated the induction of IRF-1 and COX-2. Importantly, we found that the effect of PGC-1α on repressing IRF-1 expression and activity is facilitated by the reduction in phosphorylation of STAT1 at position 727 (S727P), an essential transcriptional activator of IRF-1. Finally, we found that calyculin A, a pharmacological inhibitor of protein phosphatase 2A (PP2A) and PP1 abolishes the repression of STAT1 phosphorylation mediated by PGC-1α, suggesting a new mechanism of PGC-1α in regulating STAT1/IRF-1 pathway.

The adipocyte clock controls brown adipogenesis through the TGF-β and BMP signaling pathways.

The molecular clock is intimately linked to metabolic regulation, and brown adipose tissue plays a key role in energy homeostasis. However, whether the cell-intrinsic clock machinery participates in brown adipocyte development is unknown. Here, we show that Bmal1 (also known as ARNTL), the essential clock transcription activator, inhibits brown adipogenesis to adversely affect brown fat formation and thermogenic capacity. Global ablation of Bmal1 in mice increases brown fat mass and cold tolerance, and adipocyte-selective inactivation of Bmal1 recapitulates these effects and demonstrates its cell-autonomous role in brown adipocyte formation. Further loss- and gain-of-function studies in mesenchymal precursors and committed brown progenitors reveal that Bmal1 inhibits brown adipocyte lineage commitment and terminal differentiation. Mechanistically, Bmal1 inhibits brown adipogenesis through direct transcriptional control of key components of the TGF-β pathway together with reciprocally altered BMP signaling; activation of TGF-β or blockade of BMP pathways suppresses enhanced differentiation in Bmal1-deficient brown adipocytes. Collectively, our study demonstrates a novel temporal regulatory mechanism in fine-tuning brown adipocyte lineage progression to affect brown fat formation and thermogenic regulation, which could be targeted therapeutically to combat obesity.