Genomic structure predicts metabolite dynamics in microbial communities.

The metabolic activities of microbial communities play a defining role in the evolution and persistence of life on Earth, driving redox reactions that give rise to global biogeochemical cycles. Community metabolism emerges from a hierarchy of processes, including gene expression, ecological interactions, and environmental factors. In wild communities, gene content is correlated with environmental context, but predicting metabolite dynamics from genomes remains elusive. Here, we show, for the process of denitrification, that metabolite dynamics of a community are predictable from the genes each member of the community possesses. A simple linear regression reveals a sparse and generalizable mapping from gene content to metabolite dynamics for genomically diverse bacteria. A consumer-resource model correctly predicts community metabolite dynamics from single-strain phenotypes. Our results demonstrate that the conserved impacts of metabolic genes can predict community metabolite dynamics, enabling the prediction of metabolite dynamics from metagenomes, designing denitrifying communities, and discovering how genome evolution impacts metabolism.

Zinc mediates control of nitrogen fixation via transcription factor filamentation.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Anaerobic endosymbiont generates energy for ciliate host by denitrification.

Mitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis1,2. Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution3. As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation4. Here we describe 'Candidatus Azoamicus ciliaticola', which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. 'Candidatus A. ciliaticola' contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron-sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. 'Candidatus A. ciliaticola' and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.

Allosteric regulation of nitrate transporter NRT via the signaling protein PII.

Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.

The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply.

Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Whereas several transcriptional regulators of nitrate signaling have been identified, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN7 (NLP7), additional regulators are expected to fine-tune this pivotal physiological response. Here, we characterized Arabidopsis NLP2 as a top-tier transcriptional regulator of the early nitrate response gene regulatory network. NLP2 interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, DNA-binding motif, and some target genes with NLP7. Genetic, genomic, and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multitiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

The small peptide CEP1 and the NIN-like protein NLP1 regulate NRT2.1 to mediate root nodule formation across nitrate concentrations.

Legumes acquire fixed nitrogen (N) from the soil and through endosymbiotic association with diazotrophic bacteria. However, establishing and maintaining N2-fixing nodules are expensive for the host plant, relative to taking up N from the soil. Therefore, plants suppress symbiosis when N is plentiful and enhance symbiosis when N is sparse. Here, we show that the nitrate transporter MtNRT2.1 is required for optimal nodule establishment in Medicago truncatula under low-nitrate conditions and the repression of nodulation under high-nitrate conditions. The NIN-like protein (NLP) MtNLP1 is required for MtNRT2.1 expression and regulation of nitrate uptake/transport under low- and high-nitrate conditions. Under low nitrate, the gene encoding the C-terminally encoded peptide (CEP) MtCEP1 was more highly expressed, and the exogenous application of MtCEP1 systemically promoted MtNRT2.1 expression in a compact root architecture 2 (MtCRA2)-dependent manner. The enhancement of nodulation by MtCEP1 and nitrate uptake were both impaired in the Mtnrt2.1 mutant under low nitrate. Our study demonstrates that nitrate uptake by MtNRT2.1 differentially affects nodulation at low- and high-nitrate conditions through the actions of MtCEP1 and MtNLP1.

Proton exchange by the vacuolar nitrate transporter CLCa is required for plant growth and nitrogen use efficiency.

Nitrate is a major nutrient and osmoticum for plants. To deal with fluctuating nitrate availability in soils, plants store this nutrient in their vacuoles. Chloride channel a (CLCa), a 2NO3-/1H+ exchanger localized to the vacuole in Arabidopsis (Arabidopsis thaliana), ensures this storage process. CLCa belongs to the CLC family, which includes anion/proton exchangers and anion channels. A mutation in a glutamate residue conserved across CLC exchangers is likely responsible for the conversion of exchangers to channels. Here, we show that CLCa with a mutation in glutamate 203 (E203) behaves as an anion channel in its native membrane. We introduced the CLCaE203A point mutation to investigate its physiological importance into the Arabidopsis clca knockout mutant. These CLCaE203A mutants displayed a growth deficit linked to the disruption of water homeostasis. Additionally, CLCaE203A expression failed to complement the defect in nitrate accumulation of clca and favored higher N-assimilation at the vegetative stage. Further analyses at the post-flowering stages indicated that CLCaE203A expression results in an increase in N uptake allocation to seeds, leading to a higher nitrogen use efficiency compared to the wild-type. Altogether, these results point to the critical function of the CLCa exchanger on the vacuole for plant metabolism and development.

Maximizing US nitrate removal through wetland protection and restoration.

Growing populations and agricultural intensification have led to raised riverine nitrogen (N) loads, widespread oxygen depletion in coastal zones (coastal hypoxia)1 and increases in the incidence of algal blooms.Although recent work has suggested that individual wetlands have the potential to improve water quality2-9, little is known about the current magnitude of wetland N removal at the landscape scale. Here we use National Wetland Inventory data and 5-kilometre grid-scale estimates of N inputs and outputs to demonstrate that current N removal by US wetlands (about 860 ± 160 kilotonnes of nitrogen per year) is limited by a spatial disconnect between high-density wetland areas and N hotspots. Our model simulations suggest that a spatially targeted increase in US wetland area by 10 per cent (5.1 million hectares) would double wetland N removal. This increase would provide an estimated 54 per cent decrease in N loading in nitrate-affected watersheds such as the Mississippi River Basin. The costs of this increase in area would be approximately 3.3 billion US dollars annually across the USA-nearly twice the cost of wetland restoration on non-agricultural, undeveloped land-but would provide approximately 40 times more N removal. These results suggest that water quality improvements, as well as other types of ecosystem services such as flood control and fish and wildlife habitat, should be considered when creating policy regarding wetland restoration and protection.

Cyclic di-GMP inhibits nitrate assimilation by impairing the antitermination function of NasT in Pseudomonas putida.

The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT.

Structural basis for transcription activation by the nitrate-responsive regulator NarL.

Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation.

Nitrate alleviates ammonium toxicity in Brassica napus by coordinating rhizosphere and cell pH and ammonium assimilation.

In natural and agricultural situations, ammonium ( NH 4 + ) is a preferred nitrogen (N) source for plants, but excessive amounts can be hazardous to them, known as NH 4 + toxicity. Nitrate ( NO 3 - ) has long been recognized to reduce NH 4 + toxicity. However, little is known about Brassica napus, a major oil crop that is sensitive to high NH 4 + . Here, we found that NO 3 - can mitigate NH 4 + toxicity by balancing rhizosphere and intracellular pH and accelerating ammonium assimilation in B. napus. NO 3 - increased the uptake of NO 3 - and NH 4 + under high NH 4 + circumstances by triggering the expression of NO 3 - and NH 4 + transporters, while NO 3 - and H+ efflux from the cytoplasm to the apoplast was enhanced by promoting the expression of NO 3 - efflux transporters and genes encoding plasma membrane H+ -ATPase. In addition, NO 3 - increased pH in the cytosol, vacuole, and rhizosphere, and down-regulated genes induced by acid stress. Root glutamine synthetase (GS) activity was elevated by NO 3 - under high NH 4 + conditions to enhance the assimilation of NH 4 + into amino acids, thereby reducing NH 4 + accumulation and translocation to shoot in rapeseed. In addition, root GS activity was highly dependent on the environmental pH. NO 3 - might induce metabolites involved in amino acid biosynthesis and malate metabolism in the tricarboxylic acid cycle, and inhibit phenylpropanoid metabolism to mitigate NH 4 + toxicity. Collectively, our results indicate that NO 3 - balances both rhizosphere and intracellular pH via effective NO 3 - transmembrane cycling, accelerates NH 4 + assimilation, and up-regulates malate metabolism to mitigate NH 4 + toxicity in oilseed rape.

GmNLP1 and GmNLP4 activate nitrate-induced CLE peptides NIC1a/b to mediate nitrate-regulated root nodulation.

Symbiotic nitrogen fixation is an energy-intensive process, to maintain the balance between growth and nitrogen fixation, high concentrations of nitrate inhibit root nodulation. However, the precise mechanism underlying the nitrate inhibition of nodulation in soybean remains elusive. In this study, CRISPR-Cas9-mediated knockout of GmNLP1 and GmNLP4 unveiled a notable nitrate-tolerant nodulation phenotype. GmNLP1b and GmNLP4a play a significant role in the nitrate-triggered inhibition of nodulation, as the expression of nitrate-responsive genes was largely suppressed in Gmnlp1b and Gmnlp4a mutants. Furthermore, we demonstrated that GmNLP1b and GmNLP4a can bind to the promoters of GmNIC1a and GmNIC1b and activate their expression. Manipulations targeting GmNIC1a and GmNIC1b through knockdown or overexpression strategies resulted in either increased or decreased nodule number in response to nitrate. Additionally, transgenic roots that constitutively express GmNIC1a or GmNIC1b rely on both NARK and hydroxyproline O-arabinosyltransferase RDN1 to prevent the inhibitory effects imposed by nitrate on nodulation. In conclusion, this study highlights the crucial role of the GmNLP1/4-GmNIC1a/b module in mediating high nitrate-induced inhibition of nodulation.

MtNPF6.5 mediates chloride uptake and nitrate preference in Medicago roots.

The preference for nitrate over chloride through regulation of transporters is a fundamental feature of plant ion homeostasis. We show that Medicago truncatula MtNPF6.5, an ortholog of Arabidopsis thaliana AtNPF6.3/NRT1.1, can mediate nitrate and chloride uptake in Xenopus oocytes but is chloride selective and that its close homologue, MtNPF6.7, can transport nitrate and chloride but is nitrate selective. The MtNPF6.5 mutant showed greatly reduced chloride content relative to wild type, and MtNPF6.5 expression was repressed by high chloride, indicating a primary role for MtNPF6.5 in root chloride uptake. MtNPF6.5 and MtNPF6.7 were repressed and induced by nitrate, respectively, and these responses required the transcription factor MtNLP1. Moreover, loss of MtNLP1 prevented the rapid switch from chloride to nitrate as the main anion in nitrate-starved plants after nitrate provision, providing insight into the underlying mechanism for nitrate preference. Sequence analysis revealed three sub-types of AtNPF6.3 orthologs based on their predicted substrate-binding residues: A (chloride selective), B (nitrate selective), and C (legume specific). The absence of B-type AtNPF6.3 homologues in early diverged plant lineages suggests that they evolved from a chloride-selective MtNPF6.5-like protein.

Genetic variation underlying differential ammonium and nitrate responses in Arabidopsis thaliana.

Nitrogen is an essential element required for plant growth and productivity. Understanding the mechanisms and natural genetic variation underlying nitrogen use in plants will facilitate the engineering of plant nitrogen use to maximize crop productivity while minimizing environmental costs. To understand the scope of natural variation that may influence nitrogen use, we grew 1,135 Arabidopsis thaliana natural genotypes on two nitrogen sources, nitrate and ammonium, and measured both developmental and defense metabolite traits. By using different environments and focusing on multiple traits, we identified a wide array of different nitrogen responses. These responses are associated with numerous genes, most of which were not previously associated with nitrogen responses. Only a small portion of these genes appear to be shared between environments or traits, while most are predominantly specific to a developmental or defense trait under a specific nitrogen source. Finally, by using a large population, we were able to identify unique nitrogen responses, such as preferring ammonium or nitrate, which appear to be generated by combinations of loci rather than a few large-effect loci. This suggests that it may be possible to obtain novel phenotypes in complex nitrogen responses by manipulating sets of genes with small effects rather than solely focusing on large-effect single gene manipulations.

Nitrate transport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus.

Legumes have adaptive mechanisms that regulate nodulation in response to the amount of nitrogen in the soil. In Lotus japonicus, two NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors, LjNLP4 and LjNLP1, play pivotal roles in the negative regulation of nodulation by controlling the expression of symbiotic genes in high nitrate conditions. Despite an improved understanding of the molecular basis for regulating nodulation, how nitrate plays a role in the signaling pathway to negatively regulate this process is largely unknown. Here, we show that nitrate transport via NITRATE TRANSPORTER 2.1 (LjNRT2.1) is a key step in the NLP signaling pathway to control nodulation. A mutation in the LjNRT2.1 gene attenuates the nitrate-induced control of nodulation. LjNLP1 is necessary and sufficient to induce LjNRT2.1 expression, thereby regulating nitrate uptake/transport. Our data suggest that LjNRT2.1-mediated nitrate uptake/transport is required for LjNLP4 nuclear localization and induction/repression of symbiotic genes. We further show that LjNIN, a positive regulator of nodulation, counteracts the LjNLP1-dependent induction of LjNRT2.1 expression, which is linked to a reduction in nitrate uptake. These findings suggest a plant strategy in which nitrogen acquisition switches from obtaining nitrogen from the soil to symbiotic nitrogen fixation.

A platform for distributed production of synthetic nitrated proteins in live bacteria.

The incorporation of the nonstandard amino acid para-nitro-L-phenylalanine (pN-Phe) within proteins has been used for diverse applications, including the termination of immune self-tolerance. However, the requirement for the provision of chemically synthesized pN-Phe to cells limits the contexts where this technology can be harnessed. Here we report the construction of a live bacterial producer of synthetic nitrated proteins by coupling metabolic engineering and genetic code expansion. We achieved the biosynthesis of pN-Phe in Escherichia coli by creating a pathway that features a previously uncharacterized nonheme diiron N-monooxygenase, which resulted in pN-Phe titers of 820 ± 130 µM after optimization. After we identified an orthogonal translation system that exhibited selectivity toward pN-Phe rather than a precursor metabolite, we constructed a single strain that incorporated biosynthesized pN-Phe within a specific site of a reporter protein. Overall, our study has created a foundational technology platform for distributed and autonomous production of nitrated proteins.

The nitrate-inducible NAC transcription factor NAC056 controls nitrate assimilation and promotes lateral root growth in Arabidopsis thaliana.

Nitrate can affect many aspects of plant growth and development, such as promoting root growth and inhibiting the synthesis of secondary metabolites. However, the mechanisms underlying such effects and how plants can integrate nitrate signals and root growth needs further exploration. Here, we identified a nitrate-inducible NAC family transcription factor (TF) NAC056 which promoted both nitrate assimilation and root growth in Arabidopsis. NAC056 is a nuclear-localized transcription activator, which is predominantly expressed in the root system and hypocotyl. Using the yeast one-hybrid assay, we identified the NAC056-specific binding sequence (NAC56BM), T [T/G/A] NCTTG. We further showed that the nac056 mutant compromised root growth. NAC056 overexpression promotes LR Initiation and nitrate deficiency tolerance. Using RNA sequencing analysis and in vitro biochemical experiment, we found NAC056 regulated the expression of genes required for NO3- assimilation, directly targeting the key nitrate assimilation gene NIA1. In addition, mutation of NIA1 suppresses LR development and nitrate deficiency tolerance in the 35S::NAC056 transgenic plants. Therefore, NAC056 mediates the response of plants to environmental nitrate signals to promote root growth in Arabidopsis.

Molecular framework integrating nitrate sensing in root and auxin-guided shoot adaptive responses.

Mineral nutrition is one of the key environmental factors determining plant development and growth. Nitrate is the major form of macronutrient nitrogen that plants take up from the soil. Fluctuating availability or deficiency of this element severely limits plant growth and negatively affects crop production in the agricultural system. To cope with the heterogeneity of nitrate distribution in soil, plants evolved a complex regulatory mechanism that allows rapid adjustment of physiological and developmental processes to the status of this nutrient. The root, as a major exploitation organ that controls the uptake of nitrate to the plant body, acts as a regulatory hub that, according to nitrate availability, coordinates the growth and development of other plant organs. Here, we identified a regulatory framework, where cytokinin response factors (CRFs) play a central role as a molecular readout of the nitrate status in roots to guide shoot adaptive developmental response. We show that nitrate-driven activation of NLP7, a master regulator of nitrate response in plants, fine tunes biosynthesis of cytokinin in roots and its translocation to shoots where it enhances expression of CRFs. CRFs, through direct transcriptional regulation of PIN auxin transporters, promote the flow of auxin and thereby stimulate the development of shoot organs.

Denitrification in foraminifera has an ancient origin and is complemented by associated bacteria.

Benthic foraminifera are unicellular eukaryotes that inhabit sediments of aquatic environments. Several foraminifera of the order Rotaliida are known to store and use nitrate for denitrification, a unique energy metabolism among eukaryotes. The rotaliid Globobulimina spp. has been shown to encode an incomplete denitrification pathway of bacterial origin. However, the prevalence of denitrification genes in foraminifera remains unknown, and the missing denitrification pathway components are elusive. Analyzing transcriptomes and metagenomes of 10 foraminiferal species from the Peruvian oxygen minimum zone, we show that denitrification genes are highly conserved in foraminifera. We infer the last common ancestor of denitrifying foraminifera, which enables us to predict the ability to denitrify for additional foraminiferal species. Additionally, an examination of the foraminiferal microbiota reveals evidence for a stable interaction with Desulfobacteraceae, which harbor genes that complement the foraminiferal denitrification pathway. Our results provide evidence that foraminiferal denitrification is complemented by the foraminifera-associated microbiome. The interaction of foraminifera with their resident bacteria is at the basis of foraminiferal adaptation to anaerobic environments that manifested in ecological success in oxygen depleted habitats.

Spatiotemporal analysis identifies ABF2 and ABF3 as key hubs of endodermal response to nitrate.

Nitrate is a nutrient and a potent signal that impacts global gene expression in plants. However, the regulatory factors controlling temporal and cell type-specific nitrate responses remain largely unknown. We assayed nitrate-responsive transcriptome changes in five major root cell types of the Arabidopsis thaliana root as a function of time. We found that gene-expression response to nitrate is dynamic and highly localized and predicted cell type-specific transcription factor (TF)-target interactions. Among cell types, the endodermis stands out as having the largest and most connected nitrate-regulatory gene network. ABF2 and ABF3 are major hubs for transcriptional responses in the endodermis cell layer. We experimentally validated TF-target interactions for ABF2 and ABF3 by chromatin immunoprecipitation followed by sequencing and a cell-based system to detect TF regulation genome-wide. Validated targets of ABF2 and ABF3 account for more than 50% of the nitrate-responsive transcriptome in the endodermis. Moreover, ABF2 and ABF3 are involved in nitrate-induced lateral root growth. Our approach offers an unprecedented spatiotemporal resolution of the root response to nitrate and identifies important components of cell-specific gene regulatory networks.