The Molecular Signatures Database (MSigDB) is one of the most widely used and comprehensive databases of gene sets for performing gene set enrichment analysis. Since its creation, MSigDB has grown beyond its roots in metabolic disease and cancer to include >10,000 gene sets. These better represent a wider range of biological processes and diseases, but the utility of the database is reduced by increased redundancy across, and heterogeneity within, gene sets. To address this challenge, here we use a combination of automated approaches and expert curation to develop a collection of “hallmark” gene sets as part of MSigDB. Each hallmark in this collection consists of a “refined” gene set, derived from multiple “founder” sets, that conveys a specific biological state or process and displays coherent expression. The hallmarks effectively summarize most of the relevant information of the original founder sets and, by reducing both variation and redundancy, provide more refined and concise inputs for gene set enrichment analysis.

https://www.cell.com/cell-systems/pdf/S2405-4712(15)00218-5.pdf

The Molecular Signatures Database Hallmark Gene Set Collection

Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction.

https://science.sciencemag.org/content/sci/264/5164/1415.full.pdf

Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins

Interferons play key roles in mediating antiviral and antigrowth responses and in modulating immune response. The main signaling pathways are rapid and direct. They involve tyrosine phosphorylation and activation of signal transducers and activators of transcription factors by Janus tyrosine kinases at the cell membrane, followed by release of signal transducers and activators of transcription and their migration to the nucleus, where they induce the expression of the many gene products that determine the responses. Ancillary pathways are also activated by the interferons, but their effects on cell physiology are less clear. The Janus kinases and signal transducers and activators of transcription, and many of the interferon-induced proteins, play important alternative roles in cells, raising interesting questions as to how the responses to the interferons intersect with more general aspects of cellular physiology and how the specificity of cytokine responses is maintained.

http://www3.uah.es/farmamol/Public/AnnReviews/PDF/Biochemistry/Interferons.pdf

How cells respond to interferons

STATs (signal transducers and activators of transcription) are a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular polypeptides. Biochemical and molecular genetic explorations have defined a single tyrosine phosphorylation site and, in a dimeric partner molecule, an Src homology 2 (SH2) phosphotyrosine-binding domain, a DNA interaction domain, and a number of protein-protein interaction domains (with receptors, other transcription factors, the transcription machinery, and perhaps a tyrosine phosphatase). Mouse genetics experiments have defined crucial roles for each known mammalian STAT. The discovery of a STAT in Drosophila , and most recently in Dictyostelium discoideum , implies an ancient evolutionary origin for this dual-function set of proteins.

http://science.sciencemag.org/content/sci/277/5332/1630.full.pdf

STATs and Gene Regulation

Interferon-gamma (IFN-gamma) coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. This article reviews the current understanding of IFN-gamma ligand, receptor, signal transduction, and cellular effects with a focus on macrophage responses and to a lesser extent, responses from other cell types that influence macrophage function during infection. The current model for IFN-gamma signal transduction is discussed, as well as signal regulation and factors conferring signal specificity. Cellular effects of IFN-gamma are described, including up-regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafficking. In addition, integration of signaling and response with other cytokines and pathogen-associated molecular patterns, such as tumor necrosis factor-alpha, interleukin-4, type I IFNs, and lipopolysaccharide are discussed.

https://jlb.onlinelibrary.wiley.com/doi/epdf/10.1189/jlb.0603252

Interferon-γ: an overview of signals, mechanisms and functions

p53 protects mammals from neoplasia by inducing apoptosis, DNA repair and cell cycle arrest in response to a variety of stresses. p53-dependent arrest of cells in the G1 phase of the cell cycle is an important component of the cellular response to stress. Here we review recent evidence that implicates p53 in controlling entry into mitosis when cells enter G2 with damaged DNA or when they are arrested in S phase due to depletion of the substrates required for DNA synthesis. Part of the mechanism by which p53 blocks cells at the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Cdc2 is inhibited simultaneously by three transcriptional targets of p53, Gadd45, p21, and 14-3-3σ. Binding of Cdc2 to Cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. p53 also represses the cdc2 gene, to help ensure that cells do not escape the initial block. Genotoxic stress also activates p53-independent pathways that inhibit Cdc2 activity, activation of the protein kinases Chk1 and Chk2 by the protein kinases Atm and Atr. Chk1 and Chk2 inhibit Cdc2 by inactivating Cdc25, the phosphatase that normally activates Cdc2. Chk1, Chk2, Atm and Atr also contribute to the activation of p53 in response to genotoxic stress and therefore play multiple roles. p53 induces transcription of the reprimo, B99, and mcg10 genes, all of which contribute to the arrest of cells in G2, but the mechanisms of cell cycle arrest by these genes is not known. Repression of the topoisomerase II gene by p53 helps to block entry into mitosis and strengthens the G2 arrest. In summary, multiple overlapping p53-dependent and p53-independent pathways regulate the G2/M transition in response to genotoxic stress.

/pdf/regulation-of-the-g2-m-transition-by-p53-36tx3reo2h.pdf

Regulation of the G2/M transition by p53.

The JAK-STAT pathway at twenty.

Increased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.

https://www.pnas.org/content/pnas/92/18/8493.full.pdf

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts

Signal transducer and activator of transcription 3 (STAT3), one of seven STAT family members, is activated in response to interleukin-6 (IL-6) (Akira et al. 1994). Many cytokines use the common gp130 receptor to activate the phosphorylation of STAT3 on tyrosine residue 705, leading to the formation of dimers through reciprocal phosphotyrosine–SH2 domain interactions. Several growth factors also stimulate STAT3 activation. STAT3 dimers bind to specific γ-interferon activation sequence (GAS) elements (TTCNNNGAA) in the promoters of the induced genes (Seidel et al. 1995).

Constitutive activation of STAT3 is observed in many types of tumors. Thus, STAT3 is an oncogene, promoting cell proliferation and survival (Haura et al. 2005; Hodge et al. 2005). STAT3 is persistently phosphorylated in many human cancer cell lines and primary tumors, including hepatocellular carcinomas, breast cancers, prostate cancers, and head and neck cancers, and also in several hematological malignancies. Furthermore, STAT3 is necessary for v-src-induced transformation, and a constitutively active mutant of STAT3 can transform fibroblasts in cooperation with other transcription factors (Joo et al. 2004). Genes encoding proteins that regulate cell survival, including Bcl-2, Bcl-xL, mcl-1, and Fas, are direct targets of STAT3, as are genes encoding the cell cycle regulatory proteins cyclin D1, cyclin E1, and p21. In addition, other transcription factors, including c-Myc, c-Jun, and c-Fos, are themselves STAT3 targets (Hirano et al. 2000). STAT3 also functions as a transcriptional repressor of p53 expression: Blocking STAT3 in cancer cells up-regulates the expression of p53, leading to p53-mediated apoptosis (Niu et al. 2005). Major mechanisms of STAT3 activation in tumor cells are autocrine production of IL-6 and paracrine activation by IL-6 from stroma and infiltrating inflammatory cells. Indeed, circulating IL-6 levels are usually high in cancer patients (Giannitrapani et al. 2002). STAT3 activation provides an important link between inflammation and cancer. For example, Tebbutt et al. (2002) generated gp130757F/F mice, which carry a Y757F point mutation that disrupts the binding of the negative regulators SOCS3 and SHP2 to gp130. As a result, these mice show hyperactivation of STAT3, resulting in chronic gastric inflammation and distal stomach tumors.

There are several reports that STAT3 and NF-κB interact with each other (Battle and Frank 2002). For example, Hagihara et al. (2005) demonstrated that STAT3 forms a complex with the p65 subunit of NFκB following stimulation of cells with IL-1 plus IL-6, and that the bound STAT3 interacts with nonconsensus sequences near the κB element of the SAA promoter. Moreover, they showed that a complex that includes STAT3, p65, and p300 is essential for the synergistic induction of the SAA gene by IL-1 plus IL-6. Yu et al. (2002) reported a physical and functional interaction between STAT3 and p65 that inhibits transcriptional activation of the iNOS gene. Yoshida et al. (2004) showed that STAT3 and p65 physically interact in vivo and that p65 homodimers can cooperate with unphosphorylated STAT3 (U-STAT3) when bound to a specific type of κB motif. Reciprocally, this interaction appears to inhibit the function of GAS-binding sites for STAT3. In contrast, the p50 subunit of NFκB can cooperate with phosphorylated STAT3 (P-STAT3) bound to GAS sites (Yoshida et al. 2004).

Previous work from this laboratory has shown that STAT1 can drive gene expression even in the absence of tyrosine phosphorylation. For example, Chatterjee-Kishore et al. (2000) showed that unphosphorylated STAT1 binds to IRF1, forming a complex that activates a half GAS-half ICS element in the LMP2 promoter. In the case of STAT3, our recent work (Yang et al. 2005) showed that its high-level expression drives the transcription of many genes in the complete absence of tyrosine phosphorylation, a function quite distinct from the role of P-STAT3 in driving inducible gene expression. This activity of STAT3 is likely to have important physiological functions, since the STAT3 gene has a GAS element that drives a major increase in the concentration of STAT3 protein in response to signal-dependent tyrosine phosphorylation of STAT3 (Kojima et al. 1996; Narimatsu et al. 2001). We now address the mechanism through which U-STAT3 regulates gene expression, showing that, for many genes, it does so through its ability to interact with NFκB. To understand how U-STAT3 functions, we expressed the Y705F mutant of STAT3, which cannot be phosphorylated on residue 705, at a high level in untransformed human mammary epithelial hTERT-HME1 cells and used coimmunoprecipitation and chromatin immunoprecipitation (ChIP) assays to identify cofactors and DNA elements to which Y705F-STAT3 binds. Our data reveal that Y705F-STAT3 forms a complex with unphosphorylated NFκB (U-NFκB), binding to the κB elements of promoters, such as that of the RANTES (CCL5) gene, to induce their transcription.

/pdf/unphosphorylated-stat3-accumulates-in-response-to-il-6-and-3u6jkmeugh.pdf

Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFκB

The seven members of the signal transducer and activator of transcription (STAT) family of transcription factors are activated in response to many different cytokines and growth factors by phosphorylation of specific tyrosine residues. The STAT1 and STAT3 genes are specific targets of activated STATs 1 and 3, respectively, resulting in large increases in the levels of these unphosphorylated STATs (U-STATs) in response to the interferons (STAT1) or ligands that active gp130, such as IL-6 (STAT3). U-STATs drive gene expression by novel mechanisms distinct from those used by phosphorylated STAT (P-STAT) dimers. In this review, we discuss the roles of U-STATs in transcription and regulation of gene expression.

/pdf/roles-of-unphosphorylated-stats-in-signaling-3gblwtzgxo.pdf

George R. Stark

Papers

Regulation of the G2/M transition by p53.

The JAK-STAT pathway at twenty.

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts

Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFκB

Roles of unphosphorylated STATs in signaling.