III. CELL CIRCUIT RECONSTRUCTION: innate immune responses to pathogens in dendritic cells -multi-layer control of signaling and transcription

We will generate a multi-layered, integrated circuit of an environmental response in a mammalian cell specifically in the immune dendritic cells that detect pathogen-derived molecules and induce appropriate immune responses.  We will profile mRNA levels, splice forms, protein levels, binding and modifications along a time course following stimulation of different TLRs, and will use these to build a provisional model of the TLR circuit.  We will use the model to choose targets for pertubation and to determine signatures of the circuit's output and its internal state.  Then we will perturb each candidate regulator and measure its effect on the different signatures under different TLR stimulations in hundreds of perturbations.  After generating these large meso-scale data sets, we can use the data output to build a validated and refined circuit model, which we will further refine by additional perturbations, profiles, and physical interaction experiments.


High-resolution view of the yeast meiotic program revealed by ribosome profiling.

Citation: Brar GA, Yassour M, Friedman N, Regev A, Ingolia NT, Weissman JS.Science. 2012 Feb 3;335(6068):552-7. Epub 2011 Dec 22.
Link to journal: http://dx.doi.org/10.1126/science.1215110

Abstract: Meiosis is a complex developmental process that generates haploid cells from diploid progenitors. We measured messenger RNA (mRNA) abundance and protein production through the yeast meiotic sporulation program and found strong, stage-specific expression for most genes, achieved through control of both mRNA levels and translational efficiency. Monitoring of protein production timing revealed uncharacterized recombination factors and extensive organellar remodeling. Meiotic translation is also shifted toward noncanonical sites, including short open reading frames (ORFs) on unannnotated transcripts and upstream regions of known transcripts (uORFs). Ribosome occupancy at near-cognate uORFs was associated with more efficient ORF translation; by contrast, some AUG uORFs, often exposed by regulated 5' leader extensions, acted competitively. This work reveals pervasive translational control in meiosis and helps to illuminate the molecular basis of the broad restructuring of meiotic cells.

Strategies to discover regulatory circuits of the mammalian immune system.

Citation: Amit I, Regev A, Hacohen N. Nat Rev Immunol. 2011 Nov 18;11(12):873-80. doi: 10.1038/nri3109.
Link to journal: http://dx.doi.org/10.1038/nri3109

Abstract: Recent advances in technologies for genome- and proteome-scale measurements and perturbations promise to accelerate discovery in every aspect of biology and medicine. Although such rapid technological progress provides a tremendous opportunity, it also demands that we learn how to use these tools effectively. One application with great potential to enhance our understanding of biological systems is the unbiased reconstruction of genetic and molecular networks. Cells of the immune system provide a particularly useful model for developing and applying such approaches. Here, we review approaches for the reconstruction of signalling and transcriptional networks, with a focus on applications in the mammalian innate immune system.

Systematic Discovery of TLR Signaling Components Delineates Viral-Sensing Circuits.

Citation: Chevrier N, Mertins P, Artyomov MN, Shalek AK, Iannacone M, Ciaccio MF, Gat-Viks I, Tonti E, DeGrace MM, Clauser KR, Garber M, Eisenhaure TM, Yosef N, Robinson J, Sutton A, Andersen MS, Root DE, von Andrian U, Jones RB, Park H, Carr SA, Regev A, Amit I, Hacohen N. Cell. 2011 Nov 11;147(4):853-67.
Link to journal: http://dx.doi.org/10.1016/j.cell.2011.10.022

Abstract: Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.

Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells.

Citation: Rabani M, Levin JZ, Fan L, Adiconis X, Raychowdhury R, Garber M, Gnirke A, Nusbaum C, Hacohen N, Friedman N, Amit I, Regev A. Nat Biotechnol. 2011 May;29(5):436-42. Epub 2011 Apr 24.
Link to journalhttp://dx.doi.org/10.1038/nbt.1861

Abstract: Cellular RNA levels are determined by the interplay of RNA production, processing and degradation. However, because most studies of RNA regulation do not distinguish the separate contributions of these processes, little is known about how they are temporally integrated. Here we combine metabolic labeling of RNA at high temporal resolution with advanced RNA quantification and computational modeling to estimate RNA transcription and degradation rates during the response of mouse dendritic cells to lipopolysaccharide. We find that changes in transcription rates determine the majority of temporal changes in RNA levels, but that changes in degradation rates are important for shaping sharp 'peaked' responses. We used sequencing of the newly transcribed RNA population to estimate temporally constant RNA processing and degradation rates genome wide. Degradation rates vary significantly between genes and contribute to the observed differences in the dynamic response. Certain transcripts, including those encoding cytokines and transcription factors, mature faster. Our study provides a quantitative approach to study the integrative process of RNA regulation.

A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals.

Citation: Garber M., Raychowdhury R., Thielke A., Guttman M., Robinson J., Goren A., Minie B., Chevrier N., Itzhaki Z., Weiner A., Friedrich D., Meldrim J., Ram O., Cheng C., Gnirke A., Fisher S., Friedman N., Wong B., Bernstein B., Nusbaum C., Hacohen N., Regev A., Amit I.. In Review. (2011).

Unbiased reconstruction of a mammalian transcriptional network mediating pathogen responses.

Citation: Amit I, Garber M, Chevrier N, Leite AP, Donner Y, Eisenhaure T, Guttman M, Grenier JK, Li W, Zuk O, Schubert LA, Birditt B, Shay T, Goren A, Zhang X, Smith Z,Deering R, McDonald RC, Cabili M, Bernstein BE, Rinn JL, Meissner A, Root DE, Hacohen N, Regev A. Science. 2009 Oct 9;326(5950):257-63. Epub 2009 Sep 3.
Link to journal: http://dx.doi.org/10.1126/science.1179050

Abstract: Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data but have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We applied this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells to pathogens. Our approach revealed the regulatory functions of 125 transcription factors, chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 fine-tuners that help to explain how pathogen-sensing pathways achieve specificity. This study establishes a broadly applicable, comprehensive, and unbiased approach to reveal the wiring and functions of a regulatory network controlling a major transcriptional response in primary mammalian cells.

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