Inferring molecular networks is a central challenge in computational biology. However, it has remained unclear whether causal, rather than merely correlational, relationships can be effectively inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge that focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results constitute the most comprehensive assessment of causal network inference in a mammalian setting carried out to date and suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess the causal validity of inferred molecular networks.
The fidelity of yeast RNA polymerase II (Pol II) was assessed in vivo with an assay in which errors in transcription of can1-100, a nonsense allele of CAN1, result in enhanced sensitivity to the toxic arginine analog canavanine. The Pol II accessory factor TFIIS has been proposed to play a role in transcript editing by stimulating the intrinsic nuclease activity of the RNA polymerase. However, deletion of DST1, the gene encoding the yeast homolog of TFIIS, had only a small effect on transcriptional fidelity, as determined by this assay. In contrast, strains containing a deletion of RPB9, which encodes a small core subunit of Pol II, were found to engage in error-prone transcription. rpb9⌬ strains also had increased steadystate levels of can1-100 mRNA, consistent with transcriptional errors that decrease the normal sensitivity of the can1-100 transcript to nonsense-mediated decay, a pathway that degrades mRNAs with premature stop codons. Sequences of cDNAs from rpb9⌬ strains confirmed a significantly increased occurrence of transcriptional substitutions and insertions. These results suggest that Rpb9 plays an important role in maintaining transcriptional fidelity, whereas TFIIS may serve a different primary purpose.any DNA polymerases possess 3Ј-5Ј exonuclease activity with an important role in the fidelity of DNA synthesis. This ''proofreading'' exonuclease excises nucleotides that have been added to the nascent DNA chain but do not correctly match the template base. Mutations that disable or eliminate the nuclease activities of DNA polymerases have demonstrated the importance of exonucleolytic proofreading to DNA replication fidelity. In cells harboring a nuclease-deficient DNA polymerase, the frequency of both base substitution and frameshift mutations can be substantially increased (ref. 1 and references therein).Interest in the possibility of an analogous proofreading mechanism during transcription was raised when multisubunit RNA polymerases were shown to have a nuclease activity that cleaves single nucleotides and short oligonucleotides from the 3Ј ends of nascent RNAs (reviewed in ref.2). The nuclease activity is intrinsic to RNA polymerases but in some cases can be stimulated by accessory proteins. The best characterized of such stimulatory proteins are GreA and GreB for Escherichia coli RNA polymerase and TFIIS (or SII) for eukaryotic RNA polymerase (Pol) II, the nuclear enzyme that synthesizes mRNAs. Both the Gre proteins and TFIIS have been proposed to participate directly in the nuclease mechanism by providing two carboxylic acid side chains that might help coordinate an essential Mg 2ϩ ion and position the nucleophilic water molecule (3-6). Consistent with a possible physiological role in transcriptional proofreading, both TFIIS and GreA are able to promote removal of misincorporated nucleotides in vitro (7-9).Less is known about the RNA polymerase residues that contribute to either the intrinsic or the factor-stimulated nuclease activities. Several lines of investigation have identified an importan...
Neuroinflammation occurs in acute and chronic CNS injury, including stroke, traumatic brain injury and neurodegenerative diseases. Microglia are specialized resident myeloid cells that mediate CNS innate immune responses. Disease relevant stimuli such as reactive oxygen species (ROS) can influence microglia activation. Previously we observed that p53, a ROS responsive transcription factor, modulates microglia behaviors in vitro and in vivo, promoting pro-inflammatory functions and suppressing down-regulation of the inflammatory response and tissue repair. Here we describe a novel mechanism by which p53 modulates the functional differentiation of microglia both in vitro and in vivo. Adult microglia from p53deficient mice have increased expression of the anti-inflammatory transcription factor c-Maf. To determine how p53 negatively regulates c-Maf, we examined the impact of p53 on known c-Maf regulators. MiR-155 is a microRNA (miRNA) that targets c-Maf. We observed that cytokine induced expression of miR-155 was suppressed in p53 deficient microglia. Furthermore, Twist2, a transcriptional activator of c-Maf, is increased in p53 deficient microglia. We identified recognition sites in the 3′ untranslated region of Twist2 mRNA that are predicted to interact with two p53 dependent miRNAs: miR-34a and miR-145. Here we demonstrate that miR-34a and -145 are regulated by p53 and negatively regulate Twist2 and c-Maf expression in microglia and the RAW macrophage cell line. Taken together, these findings support the hypothesis that p53 activation induced by local ROS or accumulated DNA damage, influences microglia functions and that one specific molecular target of p53 in microglia is c-Maf.
Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia, have pro-inflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete pro-inflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a pro-inflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions.
SummarySignaling networks downstream of receptor tyrosine kinases are among the most extensively studied biological networks, but new approaches are needed to elucidate causal relationships between network components and understand how such relationships are influenced by biological context and disease. Here, we investigate the context specificity of signaling networks within a causal conceptual framework using reverse-phase protein array time-course assays and network analysis approaches. We focus on a well-defined set of signaling proteins profiled under inhibition with five kinase inhibitors in 32 contexts: four breast cancer cell lines (MCF7, UACC812, BT20, and BT549) under eight stimulus conditions. The data, spanning multiple pathways and comprising ∼70,000 phosphoprotein and ∼260,000 protein measurements, provide a wealth of testable, context-specific hypotheses, several of which we experimentally validate. Furthermore, the data provide a unique resource for computational methods development, permitting empirical assessment of causal network learning in a complex, mammalian setting.
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