I. TOOLS FOR CELL CIRCUIT RECONSTRUCTION: EXPERIMENTAL METHODS

We proposes to create an experimental toolkit for unbiased studies of cell circuits, addressing several unmet needs. First, we will develop methods to genomically profile not only nucleic acids (mRNA, DNA) and their interactions, but also proteins and their modifications. Second, we will improve methods to perturb individual circuit components, including proteins and non-coding RNAs, alone and in combinations, in any cell type. Third, an important goal is to measure complex molecular phenotypes – reflecting a circuit’s output and its internal state – under many perturbations. To do this, we will reduce the costs and input required for genome-scale profiling and develop meso-scale approaches for fast and cost-effective measurement of molecular signatures. Our toolbox will be useful for deriving a mechanistic circuit in any mammalian cell type.

Publications

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.

Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells.

Citation: Ram O, Goren A, Amit I, Shoresh N, Yosef N, Ernst J, Kellis M, Gymrek M, Issner R, Coyne M, Durham T, Zhang X, Donaghey J, Epstein CB, Regev A, Bernstein BE. Cell. 2011 Dec 23;147(7):1628-39.
Link to journal: http://dx.doi.org/10.1016/j.cell.2011.09.057

Abstract: Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.

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.

Comprehensive comparative analysis of strand-specific RNA sequencing methods.

Citation: Levin JZ, Yassour M, Adiconis X, Nusbaum C, Thompson DA, Friedman N, Gnirke A, Regev A. Nat Methods. 2010 Sep;7(9):709-15. Epub 2010 Aug 15.
Link to journalhttp://dx.doi.org/10.1038/nmeth.1491

Abstract: Strand-specific, massively parallel cDNA sequencing (RNA-seq) is a powerful tool for transcript discovery, genome annotation and expression profiling. There are multiple published methods for strand-specific RNA-seq, but no consensus exists as to how to choose between them. Here we developed a comprehensive computational pipeline to compare library quality metrics from any RNA-seq method. Using the well-annotated Saccharomyces cerevisiae transcriptome as a benchmark, we compared seven library-construction protocols, including both published and our own methods. We found marked differences in strand specificity, library complexity, evenness and continuity of coverage, agreement with known annotations and accuracy for expression profiling. Weighing each method's performance and ease, we identified the dUTP second-strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the current availability of paired-end sequencing. Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in other organisms.

Comprehensive mapping of long-range interactions reveals folding principles of the human genome.

Citation: Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J. Science. 2009 Oct 9;326(5950):289-93.
Link to journal:  http://dx.doi.org/10.1126/science.1181369

Abstract: We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.