Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3' ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3' extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

Bam32 is an adaptor protein recruited to the plasma membrane upon B cell receptor (BCR) crosslinking in a phosphoinositol 3-kinase (PI3K)-dependent manner; however, its physiologic function is unclear. To determine its physiologic function, we produced Bam32-deficient mice. Bam32(-/-) B cells develop normally but have impaired T-independent antibody responses in vivo and diminished responses to BCR crosslinking in vitro. Biochemical analysis revealed that Bam32 acts in a novel pathway leading from the BCR to MAPK/ERK Kinases (MEK1/2), MAPK/ERK Kinase Kinase-1 (MEKK1), extracellular signal-regulated kinase (ERK), and c-jun NH2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (p38). This pathway appears to be initiated by hematopoietic progenitor kinase-1 (HPK1), which interacts directly with Bam32, and differs from all previously characterized BCR signaling pathways in that it is required for normal BCR-mediated proliferation but not for B cell survival.

The genetic basis of cognition and behavioral adaptation to the environment remains poorly understood. Here we demonstrate that the histone methyltransferase complex GLP/G9a controls cognition and adaptive responses in a region-specific fashion in the adult brain. Using conditional mutagenesis in mice, we show that postnatal, neuron-specific deficiency of GLP/G9a leads to derepression of numerous nonneuronal and neuron progenitor genes in adult neurons. This transcriptional alteration is associated with complex behavioral abnormalities, including defects in learning, motivation, and environmental adaptation. The behavioral changes triggered by GLP/G9a deficiency are similar to key symptoms of the human 9q34 mental retardation syndrome that is associated with structural alterations of the GLP/EHMT1 gene. The likely causal role of GLP/G9a in mental retardation in mice and humans suggests a key role for the GLP/G9a-controlled histone H3K9 dimethylation in regulation of brain function through maintenance of the transcriptional homeostasis in adult neurons.

B cell life depends critically on the cytokine B cell-activating factor of the tumor necrosis factor family (BAFF). Lack of BAFF signaling leads to B cell death and immunodeficiency. Excessive BAFF signaling promotes lupus-like autoimmunity. Despite the great importance of BAFF to B cell biology, its signaling mechanism is not well characterized. We show that BAFF initiates signaling and transcriptional programs, which support B cell survival, metabolic fitness, and readiness for antigen-induced proliferation. We further identify a BAFF-specific protein kinase C beta-Akt signaling axis, which provides a connection between BAFF and generic growth factor-induced cellular responses.

Transcriptional activation depends on the interplay of chromatin modifiers to establish a permissive epigenetic landscape. While histone 3 lysine 9 (H3K9) methylation has long been associated with gene repression, there is limited evidence to support a role for H3K9 demethylases in gene activation.

The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4(+) cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. These phenotypic subtypes segregate with largely unique genetic lesions, suggesting the involvement of different pathogenetic mechanisms. In this report we show that the BLIMP1/PRDM1 gene is inactivated by multiple mechanisms, including homozygous deletions, truncating or missense mutations, and transcriptional repression by constitutively active BCL6, in ∼53% of ABC-DLBCL. In vivo, conditional deletion of Blimp1 in mouse B cells promotes the development of lymphoproliferative disorders recapitulating critical features of the human ABC-DLBCL. These results demonstrate that BLIMP1 is a bona fide tumor-suppressor gene whose loss contributes to lymphomagenesis by blocking plasma cell differentiation.

Adult-onset diseases can be associated with in utero events, but mechanisms for this remain unknown(1,2). The Polycomb histone methyltransferase Ezh2 stabilizes transcription by depositing repressive marks during development that persist into adulthood(3-9), but its function in postnatal organ homeostasis is unknown. We show that Ezh2 stabilizes cardiac gene expression and prevents cardiac pathology by repressing the homeodomain transcription factor gene Six1, which functions in cardiac progenitor cells but is stably silenced upon cardiac differentiation. Deletion of Ezh2 in cardiac progenitors caused postnatal myocardial pathology and destabilized cardiac gene expression with activation of Six1-dependent skeletal muscle genes. Six1 induced cardiomyocyte hypertrophy and skeletal muscle gene expression. Furthermore, genetically reducing Six1 levels rescued the pathology of Ezh2-deficient hearts. Thus, Ezh2-mediated repression of Six1 in differentiating cardiac progenitors is essential for stable gene expression and homeostasis in the postnatal heart. Our results suggest that epigenetic dysregulation in embryonic progenitor cells is a predisposing factor for adult disease and dysregulated stress responses.

Studies investigating the causes of autism spectrum disorder (ASD) point to genetic, as well as epigenetic, mechanisms of the disease. Identification of epigenetic processes that contribute to ASD development and progression is of major importance and may lead to the development of novel therapeutic strategies. Here, we identify the bromodomain and extraterminal domain-containing proteins (BETs) as epigenetic regulators of genes involved in ASD-like behaviors in mice. We found that the pharmacological suppression of BET proteins in the brain of young mice, by the novel, highly specific, brain-permeable inhibitor I-BET858 leads to selective suppression of neuronal gene expression followed by the development of an autism-like syndrome. Many of the I-BET858-affected genes have been linked to ASD in humans, thus suggesting the key role of the BET-controlled gene network in the disorder. Our studies suggest that environmental factors controlling BET proteins or their target genes may contribute to the epigenetic mechanism of ASD.

Effective antiviral immunity depends on the ability of infected cells or cells triggered with virus-derived nucleic acids to produce type I interferon (IFN), which activates transcription of numerous antiviral genes. However, disproportionately strong or chronic IFN expression is a common cause of inflammatory and autoimmune diseases. We describe an epigenetic mechanism that determines cell type-specific differences in IFN and IFN-stimulated gene (ISG) expression in response to exogenous signals. We identify di-methylation of histone H3 at lysine 9 (H3K9me2) as a suppressor of IFN and IFN-inducible antiviral gene expression. We show that levels of H3K9me2 at IFN and ISG correlate inversely with the scope and amplitude of IFN and ISG expression in fibroblasts and dendritic cells. Accordingly, genetic ablation or pharmacological inactivation of lysine methyltransferase G9a, which is essential for the generation of H3K9me2, resulted in phenotypic conversion of fibroblasts into highly potent IFN-producing cells and rendered these cells resistant to pathogenic RNA viruses. In summary, our studies implicate H3K9me2 and enzymes controlling its abundance as key regulators of innate antiviral immunity.

Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.

Regulatory T (T reg) cells are indispensable for preventing autoimmunity. Incumbent to this role is the ability of T reg cells to exert their suppressor function under inflammatory conditions. We found that T reg cell-mediated tolerance is critically dependent on the Dicer-controlled microRNA (miRNA) pathway. Depletion of miRNA within the T reg cell lineage resulted in fatal autoimmunity indistinguishable from that in T reg cell-deficient mice. In disease-free mice lacking Dicer in all T cells or harboring both Dicer-deficient and -sufficient T reg cells, Dicer-deficient T reg cells were suppressive, albeit to a lesser degree, whereas their homeostatic potential was diminished as compared with their Dicer-sufficient counterparts. However, in diseased mice, Dicer-deficient T reg cells completely lost suppressor capacity. Thus, miRNA preserve the T reg cell functional program under inflammatory conditions.

Differentiation and activation of T cells require the activity of numerous histone lysine methyltransferases (HMT) that control the transcriptional T cell output. One of the most potent regulators of T cell differentiation is the HMT Ezh2. Ezh2 is a key enzymatic component of polycomb repressive complex 2 (PRC2), which silences gene expression by histone H3 di/tri-methylation at lysine 27. Surprisingly, in many cell types, including T cells, Ezh2 is localized in both the nucleus and the cytosol. Here we show the presence of a nuclear-like PRC2 complex in T cell cytosol and demonstrate a role of cytosolic PRC2 in T cell antigen receptor (TCR)-mediated signaling. We show that short-term suppression of PRC2 precludes TCR-driven T cell activation in vitro. We also demonstrate that pharmacological inhibition of PRC2 in vivo greatly attenuates the severe T cell-driven autoimmunity caused by regulatory T cell depletion. Our data reveal cytoplasmic PRC2 is one of the most potent regulators of T cell activation and point toward the therapeutic potential of PRC2 inhibitors for the treatment of T cell-driven autoimmune diseases.

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3' untranslated region (3'UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3'UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3'UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3'UTR targeting as to harness the activity of several Ago proteins.

Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.

OCA-B was originally identified as a nuclear transcriptional coactivator that is essential for antigen-driven immune responses. The later identification of a membrane bound, myristoylated form of OCA-B suggested additional, unique functions in B cell signaling pathways. This study has shown that OCA-B also functions in the pre-B1-to-pre-B2 cell transition and, most surprisingly, that it directly interacts with SYK, a tyrosine kinase critical for pre-BCR and BCR signaling. This unprecedented type of interaction-a transcriptional coactivator with a signaling kinase-occurs in the cytoplasm and directly regulates SYK stability. This study indicates that OCA-B is required for pre-BCR and BCR signaling at multiple stages of B cell development through its nontranscriptional regulation of SYK. Combined with the deregulation of OCA-B target genes, this may help explain the multitude of defects observed in B cell development and immune responses of Oca-b-/- mice.

MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear.

Viral infection leads to a robust cellular response whereby the infected cell produces hundreds of molecular regulators to combat infection. Currently, non-canonical components, e.g., long noncoding RNAs (lncRNAs) have been added to the repertoire of immune regulators involved in the antiviral program. Interestingly, studies utilizing next-generation sequencing technologies show that a subset of the >10,000 lncRNAs in the mammalian genome contain small open reading frames (smORFs) associated with active translation, i.e., many lncRNAs are not noncoding. Here, we use genome-wide high-throughput methods to identify potential micropeptides in smORF-containing lncRNAs involved in the immune response. Using influenza as a viral infection model, we performed RNA-seq and ribosome profiling to track expression and translation of putative lncRNAs that may encode for peptides and identify tens of potential candidates. Interestingly, many of these peptides are highly conserved at the protein level, strongly suggesting biological relevance and activity. By perusing publicly available data sets, four potential peptides of interest seem common to stress induction and/or are highly conserved; potential peptides from the MMP24-AS1, ZFAS1, RP11-622K12.1, and MIR22HG genes. Interestingly, using an antibody against the potential peptide encoded by MIR22HG RNA, we show that the peptide is stably expressed in the absence of infection, and upregulated in response to infection, corroborating the prediction of the ribosome profiling results. These data show the utility of perturbation approaches in identifying potentially relevant novel molecules encoded in the genome.

Normal brain function depends on the interaction between highly specialized neurons that operate within anatomically and functionally distinct brain regions. Neuronal specification is driven by transcriptional programs that are established during early neuronal development and remain in place in the adult brain. The fidelity of neuronal specification depends on the robustness of the transcriptional program that supports the neuron type-specific gene expression patterns. Here we show that polycomb repressive complex 2 (PRC2), which supports neuron specification during differentiation, contributes to the suppression of a transcriptional program that is detrimental to adult neuron function and survival. We show that PRC2 deficiency in striatal neurons leads to the de-repression of selected, predominantly bivalent PRC2 target genes that are dominated by self-regulating transcription factors normally suppressed in these neurons. The transcriptional changes in PRC2-deficient neurons lead to progressive and fatal neurodegeneration in mice. Our results point to a key role of PRC2 in protecting neurons against degeneration.

To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs.