Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data, yet have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We apply this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells (DCs) to pathogens. Our approach revealed the regulatory functions of 125 transcription factors, chromatin modifiers, and RNA binding proteins and constructed a network model consisting of two dozen core regulators and 76 fine-tuners that help 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.

Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in-vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.

Protein expression is regulated by production and degradation of mRNAs and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics to build a quantitative genomic model of the differential regulation of gene expression in LPS-stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for over half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction of novel cellular functions and remodeling of preexisting functions through the protein life cycle.

Salmonella sneaks past security C ertain gut cells can leave resident bacteria alone but respond selectively to invaders. Satoshi Uematsu, Shizuo Akira, and colleagues (Osaka University, Japan) suggest that gut cells achieve this differentiation by using a special, pathogen-specifi c receptor called the Toll-like receptor 5 (TLR5). But the pathogenic Salmonella typhimurium turns the situation around: events triggered by the special receptor help the bug to invade its host. TLRs, which are expressed on professional antigen-presenting cells, recognize common pathogen-associated molecules and trigger innate immunity. TLR5 on dendritic cells recognizes bacterial fl agellin protein in vitro, but its function in vivo was previously unknown. Akira's team found that TLR5 mRNA was highly expressed in the mouse intestine particularly in a specifi c subpopulation of antigen-presenting lamina propria cells (CD11c + LPCs). In these cells, TLR5 was necessary for bacterial fl agellin to induce infl ammatory cytokines, yet when the team infected TLR5 −/− mice with Salmonella, a fl agellated bacterium, these mice were unexpectedly resistant to the bug. It was not, however, invasion of the CD11c + LPCs that showed a difference. In the gut, Salmonella invaded the CD11c + LPCs in both TLR5 +/+ and TLR5 −/− mice. However, in the TLR5 −/− mice, fewer bacteria-laden CD11c + LPCs moved from the intestinal tract to the mesenteric lymph nodes, probably because the LPCs failed to be activated by the bacteria. These mice had more resistance to systemic infection-fewer bacteria reached their livers and spleens-but it is not yet clear whether a similar tactic of TLR5 blocking would work in humans.

SUMMARY Type-1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical “Interferon Signature Genes” which encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN, and revealed underlying changes in chromatin configuration. We combined 1398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry.

A circuit level understanding of immune cells and hematological cancers has been severely impeded by a lack of techniques that enable intracellular perturbation without significantly altering cell viability and function. Here, we demonstrate that vertical silicon nanowires (NWs) enable gene-specific manipulation of diverse murine and human immune cells with negligible toxicity. To illustrate the power of the technique, we then apply NW-mediated gene silencing to investigate the role of the Wnt signaling pathway in chronic lymphocytic leukemia (CLL). Remarkably, CLL-B cells from different patients exhibit tremendous heterogeneity in their response to the knockdown of a single gene, LEF1. This functional heterogeneity defines three distinct patient groups not discernible by conventional CLL cytogenetic markers and provides a prognostic indicator for patients’ time to first therapy. Analyses of gene expression signatures associated with these functional patient subgroups reveal unique insights into the underlying molecular basis for disease heterogeneity. Overall, our findings suggest a functional classification that can potentially guide the selection of patient-specific therapies in CLL and highlight the opportunities for nanotechnology to drive biological inquiry.

T follicular regulatory (TFR) cells inhibit T follicular helper (TFH) cell-mediated antibody production. The mechanisms by which TFR cells exert their key immunoregulatory functions are largely unknown. Here we show that TFR cells induce a distinct suppressive state in TFH and B cells, in which effector transcriptional signatures are maintained, but key effector molecules and metabolic pathways are suppressed. TFR cell suppression of B cell antibody production and metabolism is durable, and persists even in the absence of TFR cells. This durable suppression is due, in part, to epigenetic changes. IL-21 can overcome TFR cell-mediated suppression, inhibiting TFR cells and stimulating B cells. By determining mechanisms of TFR cell-mediated suppression, we have identified methods for modulating TFR cell function and antibody production.

SUMMARY 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.