The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

Yeast can anticipate the depletion of a preferred nutrient by preemptively activating genes for alternative nutrients; the degree of this preparation varies across natural strains and is subject to a fitness tradeoff.

Natural environments are filled with multiple, often competing, signals. In contrast, biological systems are often studied in "wellcontrolled" environments where only a single input is varied, potentially missing important interactions between signals. Catabolite repression of galactose by glucose is one of the best-studied eukaryotic signal integration systems. In this system, it is believed that galactose metabolic (GAL) genes are induced only when glucose levels drop below a threshold. In contrast, we show that GAL gene induction occurs at a constant external galactose:glucose ratio across a wide range of sugar concentrations. We systematically perturbed the components of the canonical galactose/glucose signaling pathways and found that these components do not account for ratio sensing. Instead we provide evidence that ratio sensing occurs upstream of the canonical signaling pathway and results from the competitive binding of the two sugars to hexose transporters. We show that a mutant that behaves as the classical model expects (i.e., cannot use galactose above a glucose threshold) has a fitness disadvantage compared with wild type. A number of common biological signaling motifs can give rise to ratio sensing, typically through negative interactions between opposing signaling molecules. We therefore suspect that this previously unidentified nutrient sensing paradigm may be common and overlooked in biology.nutrient signaling | signal integration | gene regulation | ratio sensing | yeast

In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality we used genomic replacement experiments. Sequences within non-coding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bi-directional fluorescent protein reporter construct was introduced into the yeast non-essential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide de-repression of nascent divergent non-coding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation together with the nucleosome remodeler SWI/SNF facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the non-coding RNAs.

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In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it is difficult to perform this technique on frozen samples because freezing cells before extracting nuclei can impair nuclear integrity and alter chromatin structure, especially in fragile cells such as neurons. Our aim was to develop a protocol for freezing neuronal cells that is compatible with ATAC-Seq; we focused on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells (iMNs) from a patient affected by spinal muscular atrophy. We found that while flash-frozen iMNs are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved cells. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved iMNs quantitatively agree.Since its establishment, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has revolutionized the study of epigenetics 1,2 . This technique detects open-chromatin regions and maps transcription factor binding events genome-wide by means of direct in vitro transposition of native chromatin. Specifically, hyperactive Tn5 transposase is used to interrogate chromatin accessibility by inserting high-throughput DNA sequencing adapters into open genomic regions, which allows for the preferential amplification of DNA fragments located at sites of active chromatin. Because the DNA sites directly bound by DNA-binding proteins are protected from transposition, this approach enables the inference of transcription factor occupancy at the level of individual functional regulatory regions. Furthermore, ATAC-Seq can be utilized to decode nucleosome occupancy and positioning, by exploiting the fact that the Tn5 transposase cuts DNA with a periodicity of about 150-200 bp, corresponding to the length of the DNA fragments wrapped around histones 3 . This periodicity is maintained up to six nucleosomes and provides information about the spatial organization of nucleosomes within accessible chromatin. ATAC-Seq signals thus allow for the delineation of fine-scale architectures of the regulatory framework by correlating occupancy patterns with other features, such as chromatin remodeling and global gene induction programs. Compared to other epigenetic methodologies, such as FAIRE-Seq and conventional DNase-Seq, ATAC-Seq requires a small number of cells. Therefore, it is suitable for work on precious samples, including differentiated cells derived from induced pluripotent stem cells (iPSCs), primary cell culture, and limited clinical specimens. Recently developed techniques, such as single-cell DNase sequencing (scDNase-seq) 4 , indexing-first ChIP-Seq (iChIP) 5 , ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) 6 , and ChIPmentation 7 , allow for the epigenomic investigation of small number of cells or even single cells without requiring microfluidic devices. However, these ...

In nature, microbes often need to "decide" which of several available nutrients to utilize, a choice that depends on a cell’s inherent preference and external nutrient levels. While natural environments can have mixtures of different nutrients, phenotypic variation in microbes’ decisions of which nutrient to utilize is poorly studied. Here, we quantified differences in the concentration of glucose and galactose required to induce galactose-responsive (GAL) genes across 36 wild S. cerevisiae strains. Using bulk segregant analysis, we found that a locus containing the galactose sensor GAL3 was associated with differences in GAL signaling in eight different crosses. Using allele replacements, we confirmed that GAL3 is the major driver of GAL induction variation, and that GAL3 allelic variation alone can explain as much as 90% of the variation in GAL induction in a cross. The GAL3 variants we found modulate the diauxic lag, a selectable trait. These results suggest that ecological constraints on the galactose pathway may have led to variation in a single protein, allowing cells to quantitatively tune their response to nutrient changes in the environment.

1Maximizing growth and survival in the face of a complex, time-varying environment is a common 2 problem for single-celled organisms in the wild. When offered two different sugars as carbon 3 sources, microorganisms first consume the preferred sugar, then undergo a transient growth 4 delay, the "diauxic lag", while inducing genes to metabolize the less preferred sugar. This delay 5 is commonly assumed to be an inevitable consequence of selection to maximize use of the 6 preferred sugar. Contrary to this view, we found that many natural isolates of Saccharomyces 7 cerevisiae display short or non-existent diauxic lags when grown in mixtures of glucose 8 (preferred) and galactose. These strains induce galactose-utilization (GAL) genes hours before 9 glucose exhaustion, thereby "preparing" for the transition from glucose to galactose metabolism. 10The extent of preparation varies across strains, and seems to be determined by the steady-state 11 response of GAL genes to mixtures of glucose and galactose rather than by induction kinetics. 12Although early GAL induction gives strains a competitive advantage once glucose runs out, it 13 comes at a cost while glucose is still present. Costs and benefits correlate with the degree of 14 preparation: strains with higher expression of GAL genes prior to glucose exhaustion 15 experience a larger upfront growth cost but also a shorter diauxic lag. Our results show that 16 classical diauxic growth is only one extreme on a continuum of growth strategies constrained by 17 a cost-benefit tradeoff. This type of continuum is likely to be common in nature, as similar 18 tradeoffs can arise whenever cells evolve to use mixtures of nutrients.