PhoB-regulated phosphate assimilation of Ralstonia solanacearum is cross-activated by VsrB in Pi-abundant rich medium.

Ralstonia solanacearum is a devastating phytopathogen infecting a broad range of economically important crops. Phosphate (Pi) homeostasis and assimilation play a critical role in the environmental adaptation and pathogenicity of many bacteria. However, the Pi assimilation regulatory mechanism of R. solanacearum remains unknown. This study revealed that R. solanacearum pstSCAB-phoU-phoBR operon expression is sensitive to extracellular Pi concentration, with higher expression under Pi-limiting conditions. The PhoB-PhoR fine-tunes the Pi-responsive expression of the Pho regulon genes, demonstrating its pivotal role in Pi assimilation. By contrast, neither PhoB, PhoR, PhoU, nor PstS was found to be essential for virulence on tomato plants. Surprisingly, the PhoB regulon is activated in a Pi-abundant rich medium. Results showed that histidine kinase VsrB, which is known for the exopolysaccharide production regulation, partially mediates PhoB activation in the Pi-abundant rich medium. The 271 histidine of VsrB is vital for this activation. This cross-activation mechanism between the VsrB and PhoB-PhoR systems suggests the carbohydrate-Pi metabolism coordination in R. solanacearum. Overall, this research provides new insights into the complex regulatory interplay between Pi metabolism and growth in R. solanacearum.

The Streptococcus pyogenes stand-alone regulator RofA exhibits characteristics of a PRD-containing virulence regulator.

Streptococcus pyogenes [group A streptococcus (GAS)] is a human pathogen capable of infecting diverse tissues. To successfully infect these sites, GAS must detect available nutrients and adapt accordingly. The phosphoenolpyruvate transferase system (PTS) mediates carbohydrate uptake and metabolic gene regulation to adapt to the nutritional environment. Regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several PRD-containing stand-alone regulators with regulons encoding both metabolic genes and virulence factors [PRD-containing virulence regulators (PCVRs)]. One is RofA, which regulates the expression of virulence genes in multiple GAS serotypes. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate virulence gene expression. In this study, the RofA regulon of M1T1 strain 5448 was determined using RNA sequencing. Two operons were consistently differentially expressed across growth in the absence of RofA; the pilus operon was downregulated, and the capsule operon was upregulated. This correlated with increased capsule production and decreased adherence to keratinocytes. Purified RofA-His was phosphorylated in vitro by PTS proteins EI and HPr, and phosphorylated RofA-FLAG was detected in vivo when GAS was grown in low-glucose C medium. Phosphorylated RofA was not observed when C medium was supplemented 10-fold with glucose. Mutations of select histidine residues within the putative PRDs contributed to the in vivo phosphorylation of RofA, although phosphorylation of RofA was still observed, suggesting other phosphorylation sites exist in the protein. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar metabolism with virulence regulation.

Integrated analysis of single-cell and bulk RNA sequencing data reveals a myeloid cell-related regulon predicting neoadjuvant immunotherapy response across cancers.

BACKGROUND: Immunotherapy has brought about a paradigm shift in the treatment of cancer. However, the majority of patients exhibit resistance or become refractory to immunotherapy, and the underlying mechanisms remain to be explored. METHODS: Sing-cell RNA sequencing (scRNA­seq) datasets derived from 1 pretreatment and 1 posttreatment achieving pathological complete response (pCR) patient with lung adenocarcinoma (LUAD) who received neoadjuvant immunotherapy were collected, and pySCENIC was used to find the gene regulatory network (GRN) between cell types and immune checkpoint inhibitor (ICI) response. A regulon predicting ICI response was identified and validated using large­scale pan-cancer data, including a colorectal cancer scRNA­seq dataset, a breast cancer scRNA­seq dataset, The Cancer Genome Atlas (TCGA) pan-cancer cohort, and 5 ICI transcriptomic cohorts. Symphony reference mapping was performed to construct the myeloid cell map. RESULTS: Thirteen major cluster cell types were identified by comparing pretreatment and posttreatment patients, and the fraction of myeloid cells was higher in the posttreatment group (19.0% vs. 11.8%). A PPARG regulon (containing 23 target genes) was associated with ICI response, and its function was validated by a colorectal cancer scRNA­seq dataset, a breast cancer scRNA­seq dataset, TCGA pan-cancer cohort, and 5 ICI transcriptomic cohorts. Additionally, a myeloid cell map was developed, and cluster I, II, and III myeloid cells with high expression of PPARG were identified. Moreover, we constructed a website called PPARG ( https://pparg.online/PPARG/ or http://43.134.20.130:3838/PPARG/ ), which provides a powerful discovery tool and resource value for researchers. CONCLUSIONS: The PPARG regulon is a predictor of ICI response. The myeloid cell map enables the identification of PPARG subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

(Re)-definition of the holo- and apo-Fur direct regulons of Helicobacter pylori.

Iron homeostasis is a critical process for living organisms because this metal is an essential co-factor for fundamental biochemical activities, like energy production and detoxification, albeit its excess quickly leads to cell intoxication. The protein Fur (ferric uptake regulator) controls iron homeostasis in bacteria by switching from its apo- to holo-form as a function of the cytoplasmic level of ferrous ions, thereby modulating gene expression. The Helicobacter pylori HpFur protein has the rare ability to operate as a transcriptional commutator; apo- and holo-HpFur function as two different repressors with distinct DNA binding recognition properties for specific sets of target genes. Although the regulation of apo- and holo-HpFur in this bacterium has been extensively investigated, we propose a genome-wide redefinition of holo-HpFur direct regulon in H. pylori by integration of RNA-seq and ChIP-seq data, and a large extension of the apo-HpFur direct regulon. We show that in response to iron availability, new coding sequences, non-coding RNAs, toxin-antitoxin systems, and transcripts within open reading frames are directly regulated by apo- or holo-HpFur. These new targets and the more thorough validation and deeper characterization of those already known provide a complete and updated picture of the direct regulons of this two-faced transcriptional regulator.

Redox active plant phenolic, acetosyringone, for electrogenetic signaling.

Redox is a unique, programmable modality capable of bridging communication between biology and electronics. Previous studies have shown that the E. coli redox-responsive OxyRS regulon can be re-wired to accept electrochemically generated hydrogen peroxide (H2O2) as an inducer of gene expression. Here we report that the redox-active phenolic plant signaling molecule acetosyringone (AS) can also induce gene expression from the OxyRS regulon. AS must be oxidized, however, as the reduced state present under normal conditions cannot induce gene expression. Thus, AS serves as a "pro-signaling molecule" that can be activated by its oxidation-in our case by application of oxidizing potential to an electrode. We show that the OxyRS regulon is not induced electrochemically if the imposed electrode potential is in the mid-physiological range. Electronically sliding the applied potential to either oxidative or reductive extremes induces this regulon but through different mechanisms: reduction of O2 to form H2O2 or oxidation of AS. Fundamentally, this work reinforces the emerging concept that redox signaling depends more on molecular activities than molecular structure. From an applications perspective, the creation of an electronically programmed "pro-signal" dramatically expands the toolbox for electronic control of biological responses in microbes, including in complex environments, cell-based materials, and biomanufacturing.

Combined transcriptomic and ChIPseq analyses of the Bordetella pertussis RisA regulon.

The regulation of Bordetella pertussis virulence is mediated by the two-component system BvgA/S, which activates the transcription of virulence-activated genes (vags). In the avirulent phase, the vags are not expressed, but instead, virulence-repressed genes (vrgs) are expressed, under the control of another two-component system, RisA/K. Here, we combined transcriptomic and chromatin immunoprecipitation sequencing (ChIPseq) data to examine the RisA/K regulon. We performed RNAseq analyses of RisA-deficient and RisA-phosphoablative B. pertussis mutants cultivated in virulent and avirulent conditions. We confirmed that the expression of most vrgs is regulated by phosphorylated RisA. However, the expression of some, including those involved in flagellum biosynthesis and chemotaxis, requires RisA independently of phosphorylation. Many RisA-regulated genes encode proteins with regulatory functions, suggesting multiple RisA regulation cascades. By ChIPseq analyses, we identified 430 RisA-binding sites, 208 within promoter regions, 201 within open reading frames, and 21 in non-coding regions. RisA binding was demonstrated in the promoter regions of most vrgs and, surprisingly, of some vags, as well as for other genes not identified as vags or vrgs. Unexpectedly, many genes, including some vags, like prn, brpL, bipA, and cyaA, contain a BvgA-binding site and a RisA-binding site, which increases the complexity of the RisAK/BvgAS network in B. pertussis virulence regulation.IMPORTANCEThe expression of virulence-activated genes (vags) of Bordetella pertussis, the etiological agent of whooping cough, is under the transcriptional control of the two-component system BvgA/S, which allows the bacterium to switch between virulent and avirulent phases. In addition, the more recently identified two-component system RisA/K is required for the expression of B. pertussis genes, collectively named vrgs, that are repressed during the virulent phase but activated during the avirulent phase. We have characterized the RisA/K regulon by combined transcriptomic and chromatin immunoprecipitation sequencing analyses. We identified more than 400 RisA-binding sites. Many of them are localized in promoter regions, especially vrgs, but some were found within open reading frames and in non-coding regions. Surprisingly, RisA-binding sites were also found in promoter regions of some vags, illustrating the previously underappreciated complexity of virulence regulation in B. pertussis.

Concentration dependent exposure of vancomycin and teicoplanin induces vanG regulon in Staphylococcus aureus.

Therapeutic options for staphylococcus infections have been raised due to the emergence of VISA and VRSA. Six isolates of Staphylococcus aureus of clinical origin which were previously confirmed to carry vanG were selected for this study. Antimicrobial susceptibility was performed by disc diffusion method. Transcriptional expression of vanG and vanSG showed down regulation against vancomycin and teicoplanin but expression was increased with increasing concentration of antibiotics. vanUG, vanRG showed up regulation against glycopeptide exposure. The present study underscored that expression of vanG and its regulatory gene operons are dependent on concentration of vancomycin and teicoplanin exposure in S.aureus.

Inferred regulons are consistent with regulator binding sequences in E. coli.

The transcriptional regulatory network (TRN) of E. coli consists of thousands of interactions between regulators and DNA sequences. Regulons are typically determined either from resource-intensive experimental measurement of functional binding sites, or inferred from analysis of high-throughput gene expression datasets. Recently, independent component analysis (ICA) of RNA-seq compendia has shown to be a powerful method for inferring bacterial regulons. However, it remains unclear to what extent regulons predicted by ICA structure have a biochemical basis in promoter sequences. Here, we address this question by developing machine learning models that predict inferred regulon structures in E. coli based on promoter sequence features. Models were constructed successfully (cross-validation AUROC > = 0.8) for 85% (40/47) of ICA-inferred E. coli regulons. We found that: 1) The presence of a high scoring regulator motif in the promoter region was sufficient to specify regulatory activity in 40% (19/47) of the regulons, 2) Additional features, such as DNA shape and extended motifs that can account for regulator multimeric binding, helped to specify regulon structure for the remaining 60% of regulons (28/47); 3) investigating regulons where initial machine learning models failed revealed new regulator-specific sequence features that improved model accuracy. Finally, we found that strong regulatory binding sequences underlie both the genes shared between ICA-inferred and experimental regulons as well as genes in the E. coli core pan-regulon of Fur. This work demonstrates that the structure of ICA-inferred regulons largely can be understood through the strength of regulator binding sites in promoter regions, reinforcing the utility of top-down inference for regulon discovery.

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane ß-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic ß-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate ß-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Multilayered regulation of amino acid metabolism in Escherichia coli.

Amino acid metabolism in Escherichia coli has long been studied and has established the basis for regulatory mechanisms at the transcriptional, posttranscriptional, and posttranslational levels. In addition to the classical signal transduction cascade involving posttranslational modifications (PTMs), novel PTMs in the two primary nitrogen assimilation pathways have recently been uncovered. The regulon of the master transcriptional regulator NtrC is further expanded by a small RNA derived from the 3´UTR of glutamine synthetase mRNA, which coordinates central carbon and nitrogen metabolism. Furthermore, recent advances in sequencing technologies have revealed the global regulatory networks of transcriptional and posttranscriptional regulators, Lrp and GcvB. This review provides an update of the multilayered and interconnected regulatory networks governing amino acid metabolism in E. coli.

Characterization of the RofA regulon in the pandemic M1global and emergent M1UK lineages of Streptococcus pyogenes.

The standalone regulator RofA is a positive regulator of the pilus locus in Streptococcus pyogenes. Found in only certain emm genotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominant emm1 S. pyogenes is unclear. Given the recent emergence of a new emm1 (M1UK) toxigenic lineage that is distinguished by three non-synonymous SNPs in rofA, we characterized the rofA regulon in six emm1 strains that are representative of the two contemporary major emm1 lineages (M1global and M1UK) using RNAseq analysis, and then determined the specific role of the M1UK-specific rofA SNPs. Deletion of rofA in three M1global strains led to altered expression of 14 genes, including six non-pilus locus genes. In M1UK strains, deletion of rofA led to altered expression of 16 genes, including nine genes that were unique to M1UK. Only the pilus locus genes were common to the RofA regulons of both lineages, while transcriptomic changes varied between strains even within the same lineage. Although introduction of the three SNPs into rofA did not impact gene expression in an M1global strain, reversal of three SNPs in an M1UK strain led to an unexpected number of transcriptomic changes that in part recapitulated transcriptomic changes seen when deleting RofA in the same strain. Computational analysis predicted that interactions with a key histidine residue in the PRD domain of RofA would differ between M1UK and M1global. RofA is a positive regulator of the pilus locus in all emm1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs in rofA that characterize M1UK may impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation.

ppGpp accumulation reduces the expression of the global nitrogen homeostasis-modulating NtcA regulon by affecting 2-oxoglutarate levels.

The cyanobacterium Synechococcus elongatus PCC 7942 accumulates alarmone guanosine tetraphosphate (ppGpp) under stress conditions, such as darkness. A previous study observed that artificial ppGpp accumulation under photosynthetic conditions led to the downregulation of genes involved in the nitrogen assimilation system, which is activated by the global nitrogen regulator NtcA, suggesting that ppGpp regulates NtcA activity. However, the details of this mechanism have not been elucidated. Here, we investigate the metabolic responses associated with ppGpp accumulation by heterologous expression of the ppGpp synthetase RelQ. The pool size of 2-oxoglutarate (2-OG), which activates NtcA, is significantly decreased upon ppGpp accumulation. De novo 13C-labeled CO2 assimilation into the Calvin-Benson-Bassham cycle and glycolytic intermediates continues irrespective of ppGpp accumulation, whereas the labeling of 2-OG is significantly decreased under ppGpp accumulation. The low 2-OG levels in the RelQ overexpression cells could be because of the inhibition of metabolic enzymes, including aconitase, which are responsible for 2-OG biosynthesis. We propose a metabolic rearrangement by ppGpp accumulation, which negatively regulates 2-OG levels to maintain carbon and nitrogen balance.

Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by different mutations. Previously, we showed that each mutational subtype develops its specific gene regulatory network (GRN) with transcription factors interacting within multiple gene modules, many of which are transcription factor genes themselves. Here, we hypothesize that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We test this hypothesis using FLT3-ITD-mutated AML as a model and conduct an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict crucial regulatory modules required for AML growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD+ AML and that its removal leads to GRN collapse and cell death.

The Azotobacter vinelandii AlgU regulon during vegetative growth and encysting conditions: A proteomic approach.

In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.

Developing multispecies quorum-sensing modulators based on the Streptococcus mitis competence-stimulating peptide.

Bacteria utilize quorum sensing (QS) to coordinate many group behaviors. As such, QS has attracted significant attention as a potential mean to attenuate bacterial infectivity without introducing selective pressure for resistance development. Streptococcus mitis, a human commensal, acts as a genetic diversity reservoir for Streptococcus pneumoniae, a prevalent human pathogen. S. mitis possesses a typical comABCDE competence regulon QS circuitry; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon QS circuitry in S. mitis are yet to be explored. We set out to delineate the competence regulon QS circuitry in S. mitis, including confirming the identity of the native CSP signal, evaluating the molecular mechanism that governs CSP interactions with histidine kinase receptor ComD leading to ComD activation, and defining the regulatory roles of the competence regulon QS circuitry in initiating various S. mitis phenotypes. Our analysis revealed important structure-activity relationship insights of the CSP signal and facilitated the development of novel CSP-based QS modulators. Our analysis also revealed the involvement of the competence regulon in modulating competence development and biofilm formation. Furthermore, our analysis revealed that the native S. mitis CSP signal can modulate QS response in S. pneumoniae. Capitalizing on this crosstalk, we developed a multispecies QS modulator that activates both the pneumococcus ComD receptors and the S. mitis ComD-2 receptor with high potencies. The novel scaffolds identified herein can be utilized to evaluate the effects temporal QS modulation has on S. mitis as it inhabits its natural niche.

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis.

BACKGROUND: The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-ß-xylanase XynA on the B. subtilis transcriptome using RNA-seq. RESULTS: Using the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%. CONCLUSIONS: Overproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.

Translational control of furina by an RNA regulon is important for left-right patterning, heart morphogenesis and cardiac valve function.

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.

Reshaping the yeast galactose regulon via GPCR signaling cascade.

Dynamically regulated systems are preferable to control metabolic pathways for an improved strain performance with better productivity. Here, we harnessed to the G protein-coupled receptor (GPCR) signaling pathway to reshape the yeast galactose regulon. The galactose-regulated (GAL) system was coupled with the GPCR signaling pathway for mating pheromone via a synthetic transcription factor. In this study, we refabricated the dynamic range, sensitivity, and response time of the GAL system to α factor by modulating the key components of the GPCR signaling cascade. A series of engineered yeasts with self-secretion of α factor were constructed to achieve quorum-sensing behaviors. In addition, we also repurposed the GAL system to make it responsive to heat shock. Taken together, our work showcases the great potential of synthetic biology in creating user-defined metabolic controls. We envision that the plasticity of our genetic design would be of significant interest for the future fabrication of novel gene expression systems.

Expanding the coverage of regulons from high-confidence prior knowledge for accurate estimation of transcription factor activities.

Gene regulation plays a critical role in the cellular processes that underlie human health and disease. The regulatory relationship between transcription factors (TFs), key regulators of gene expression, and their target genes, the so called TF regulons, can be coupled with computational algorithms to estimate the activity of TFs. However, to interpret these findings accurately, regulons of high reliability and coverage are needed. In this study, we present and evaluate a collection of regulons created using the CollecTRI meta-resource containing signed TF-gene interactions for 1186 TFs. In this context, we introduce a workflow to integrate information from multiple resources and assign the sign of regulation to TF-gene interactions that could be applied to other comprehensive knowledge bases. We find that the signed CollecTRI-derived regulons outperform other public collections of regulatory interactions in accurately inferring changes in TF activities in perturbation experiments. Furthermore, we showcase the value of the regulons by examining TF activity profiles in three different cancer types and exploring TF activities at the level of single-cells. Overall, the CollecTRI-derived TF regulons enable the accurate and comprehensive estimation of TF activities and thereby help to interpret transcriptomics data.