Identification of a Master Regulator of Differentiation in Toxoplasma.

Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Acute-stage tachyzoites differentiate into chronic-stage bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. The molecular basis of this differentiation is unknown, despite being efficiently triggered by stresses in culture. Through Cas9-mediated screening and single-cell profiling, we identify a Myb-like transcription factor (BFD1) necessary for differentiation in cell culture and in mice. BFD1 accumulates during stress and its synthetic expression is sufficient to drive differentiation. Consistent with its function as a transcription factor, BFD1 binds the promoters of many stage-specific genes and represents a counterpoint to the ApiAP2 factors that dominate our current view of parasite gene regulation. BFD1 provides a genetic switch to study and control Toxoplasma differentiation and will inform prevention and treatment of chronic infections.

A Prion Epigenetic Switch Establishes an Active Chromatin State.

Covalent modifications to histones are essential for development, establishing distinct and functional chromatin domains from a common genetic sequence. Whereas repressed chromatin is robustly inherited, no mechanism that facilitates inheritance of an activated domain has been described. Here, we report that the Set3C histone deacetylase scaffold Snt1 can act as a prion that drives the emergence and transgenerational inheritance of an activated chromatin state. This prion, which we term [ESI+] for expressed sub-telomeric information, is triggered by transient Snt1 phosphorylation upon cell cycle arrest. Once engaged, the prion reshapes the activity of Snt1 and the Set3C complex, recruiting RNA pol II and interfering with Rap1 binding to activate genes in otherwise repressed sub-telomeric domains. This transcriptional state confers broad resistance to environmental stress, including antifungal drugs. Altogether, our results establish a robust means by which a prion can facilitate inheritance of an activated chromatin state to provide adaptive benefit.

Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription.

Phages express anti-CRISPR (Acr) proteins to inhibit CRISPR-Cas systems that would otherwise destroy their genomes. Most acr genes are located adjacent to anti-CRISPR-associated (aca) genes, which encode proteins with a helix-turn-helix DNA-binding motif. The conservation of aca genes has served as a signpost for the identification of acr genes, but the function of the proteins encoded by these genes has not been investigated. Here we reveal that an acr-associated promoter drives high levels of acr transcription immediately after phage DNA injection and that Aca proteins subsequently repress this transcription. Without Aca activity, this strong transcription is lethal to a phage. Our results demonstrate how sufficient levels of Acr proteins accumulate early in the infection process to inhibit existing CRISPR-Cas complexes in the host cell. They also imply that the conserved role of Aca proteins is to mitigate the deleterious effects of strong constitutive transcription from acr promoters.

A Repeat-Associated Small RNA Controls the Major Virulence Factors of Helicobacter pylori.

Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

FrzS acts as a polar beacon to recruit SgmX, a central activator of type IV pili during Myxococcus xanthus motility.

In rod-shaped bacteria, type IV pili (Tfp) promote twitching motility by assembling and retracting at the cell pole. In Myxococcus xanthus, a bacterium that moves in highly coordinated cell groups, Tfp are activated by a polar activator protein, SgmX. However, while it is known that the Ras-like protein MglA is required for unipolar targeting, how SgmX accesses the cell pole to activate Tfp is unknown. Here, we demonstrate that a polar beacon protein, FrzS, recruits SgmX at the cell pole. We identified two main functional domains, including a Tfp-activating domain and a polar-binding domain. Within the latter, we show that the direct binding of MglA-GTP unveils a hidden motif that binds directly to the FrzS N-terminal response regulator (CheY). Structural analyses reveal that this binding occurs through a novel binding interface for response regulator domains. In conclusion, the findings unveil the protein interaction network leading to the spatial activation of Tfp at the cell pole. This tripartite system is at the root of complex collective behaviours in this predatory bacterium.

Regulation of Biofilm Exopolysaccharide Biosynthesis and Degradation in Pseudomonas aeruginosa.

Microbial communities enmeshed in a matrix of macromolecules, termed as biofilms, are the natural setting of bacteria. Exopolysaccharide is a critical matrix component of biofilms. Here, we focus on biofilm matrix exopolysaccharides in Pseudomonas aeruginosa. This opportunistic pathogen can adapt to a wide range of environments and can form biofilms or aggregates in a variety of surfaces or environments, such as the lungs of people with cystic fibrosis, catheters, wounds, and contact lenses. The ability to synthesize multiple exopolysaccharides is one of the advantages that facilitate bacterial survival in different environments. P. aeruginosa can produce several exopolysaccharides, including alginate, Psl, Pel, and lipopolysaccharide. In this review, we highlight the roles of each exopolysaccharide in P. aeruginosa biofilm development and how bacteria coordinate the biosynthesis of multiple exopolysaccharides and bacterial motility. In addition, we present advances in antibiofilm strategies targeting matrix exopolysaccharides, with a focus on glycoside hydrolases.

Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes.

Proteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions.

APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

The splicing factor U2AF1 contributes to cancer progression through a noncanonical role in translation regulation.

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias, and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid-binding domain, recognition of the 3' splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we uncovered a noncanonical function of U2AF1, showing that it directly binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysome profiling indicates that the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8, which contributes to metastasis, inflammation, and cancer progression in mice and humans.

Assessing next-generation sequencing-based computational methods for predicting transcriptional regulators with query gene sets.

This article provides an in-depth review of computational methods for predicting transcriptional regulators (TRs) with query gene sets. Identification of TRs is of utmost importance in many biological applications, including but not limited to elucidating biological development mechanisms, identifying key disease genes, and predicting therapeutic targets. Various computational methods based on next-generation sequencing (NGS) data have been developed in the past decade, yet no systematic evaluation of NGS-based methods has been offered. We classified these methods into two categories based on shared characteristics, namely library-based and region-based methods. We further conducted benchmark studies to evaluate the accuracy, sensitivity, coverage, and usability of NGS-based methods with molecular experimental datasets. Results show that BART, ChIP-Atlas, and Lisa have relatively better performance. Besides, we point out the limitations of NGS-based methods and explore potential directions for further improvement.

Canalizing kernel for cell fate determination.

The tendency for cell fate to be robust to most perturbations, yet sensitive to certain perturbations raises intriguing questions about the existence of a key path within the underlying molecular network that critically determines distinct cell fates. Reprogramming and trans-differentiation clearly show examples of cell fate change by regulating only a few or even a single molecular switch. However, it is still unknown how to identify such a switch, called a master regulator, and how cell fate is determined by its regulation. Here, we present CAESAR, a computational framework that can systematically identify master regulators and unravel the resulting canalizing kernel, a key substructure of interconnected feedbacks that is critical for cell fate determination. We demonstrate that CAESAR can successfully predict reprogramming factors for de-differentiation into mouse embryonic stem cells and trans-differentiation of hematopoietic stem cells, while unveiling the underlying essential mechanism through the canalizing kernel. CAESAR provides a system-level understanding of how complex molecular networks determine cell fates.

A variegated model of transcription factor function in the immune system.

Specific combinations of transcription factors (TFs) control the gene expression programs that underlie specialized immune responses. Previous models of TF function in immunocytes had restricted each TF to a single functional categorization [e.g., lineage-defining (LDTFs) vs. signal-dependent TFs (SDTFs)] within one cell type. Synthesizing recent results, we instead propose a variegated model of immunological TF function, whereby many TFs have flexible and different roles across distinct cell states, contributing to cell phenotypic diversity. We discuss evidence in support of this variegated model, describe contextual inputs that enable TF diversification, and look to the future to imagine warranted experimental and computational tools to build quantitative and predictive models of immunocyte gene regulatory networks.

Identification of Natural Regulatory T Cell Epitopes Reveals Convergence on a Dominant Autoantigen.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are critical for the prevention of autoimmunity and the suppression of anti-tumor immunity. The major self-antigens recognized by Treg cells remain undefined, representing a substantial barrier to the understanding of immune regulation. Here, we have identified natural Treg cell ligands in mice. We found that two recurrent Treg cell clones, one prevalent in prostate tumors and the other associated with prostatic autoimmune lesions, recognized distinct non-overlapping MHC-class-II-restricted peptides derived from the same prostate-specific protein. Notably, this protein is frequently targeted by autoantibodies in experimental models of prostatic autoimmunity. On the basis of these findings, we propose a model in which Treg cell responses at peripheral sites converge on those self-proteins that are most susceptible to autoimmune attack, and we suggest that this link could be exploited as a generalizable strategy for identifying the Treg cell antigens relevant to human autoimmunity.

Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

Hfq-licensed RNA-RNA interactome in Pseudomonas aeruginosa reveals a keystone sRNA.

The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.

POU2F3 is a master regulator of a tuft cell-like variant of small cell lung cancer.

Small cell lung cancer (SCLC) is widely considered to be a tumor of pulmonary neuroendocrine cells; however, a variant form of this disease has been described that lacks neuroendocrine features. Here, we applied domain-focused CRISPR screening to human cancer cell lines to identify the transcription factor (TF) POU2F3 (POU class 2 homeobox 3; also known as SKN-1a/OCT-11) as a powerful dependency in a subset of SCLC lines. An analysis of human SCLC specimens revealed that POU2F3 is expressed exclusively in variant SCLC tumors that lack expression of neuroendocrine markers and instead express markers of a chemosensory lineage known as tuft cells. Using chromatin- and RNA-profiling experiments, we provide evidence that POU2F3 is a master regulator of tuft cell identity in a variant form of SCLC. Moreover, we show that most SCLC tumors can be classified into one of three lineages based on the expression of POU2F3, ASCL1, or NEUROD1. Our CRISPR screens exposed other unique dependencies in POU2F3-expressing SCLC lines, including the lineage TFs SOX9 and ASCL2 and the receptor tyrosine kinase IGF1R (insulin-like growth factor 1 receptor). These data reveal POU2F3 as a cell identity determinant and a dependency in a tuft cell-like variant of SCLC, which may reflect a previously unrecognized cell of origin or a trans-differentiation event in this disease.

Identifying a missing lineage driver in a subset of lung neuroendocrine tumors.

Tumor heterogeneity of a primary histologic cancer type has major implications for cancer research and therapeutics. An important and understudied aspect of this heterogeneity is the role of transcription factors that serve as "lineage oncogenes" in a tumor type. A demonstration that different subgroups have distinct dependencies on lineage-specific transcription factors is highlighted in a relatively homogenous cancer type: the pulmonary neuroendocrine cancer small cell lung carcinoma (SCLC). Identification of these factors is providing new insights into the origin of the heterogeneity and subtype-specific vulnerabilities in SCLC and provides a template for studying heterogeneity in other cancer types.

Experimental validation and characterization of putative targets of Escargot and STAT, two master regulators of the intestinal stem cells in Drosophila melanogaster.

Many organs contain adult stem cells (ASCs) to replace cells due to damage, disease, or normal tissue turnover. ASCs can divide asymmetrically, giving rise to a new copy of themselves (self-renewal) and a sister that commits to a specific cell type (differentiation). Decades of research have led to the identification of pleiotropic genes whose loss or gain of function affect diverse aspects of normal ASC biology. Genome-wide screens of these so-called genetic "master regulator" (MR) genes, have pointed to hundreds of putative targets that could serve as their downstream effectors. Here, we experimentally validate and characterize the regulation of several putative targets of Escargot (Esg) and the Signal Transducer and Activator of Transcription (Stat92E, a.k.a. STAT), two known MRs in Drosophila intestinal stem cells (ISCs). Our results indicate that regardless of bioinformatic predictions, most experimentally validated targets show a profile of gene expression that is consistent with co-regulation by both Esg and STAT, fitting a rather limited set of co-regulatory modalities. A bioinformatic analysis of proximal regulatory sequences in specific subsets of co-regulated targets identified additional transcription factors that might cooperate with Esg and STAT in modulating their transcription. Lastly, in vivo manipulations of validated targets rarely phenocopied the effects of manipulating Esg and STAT, suggesting the existence of complex genetic interactions among downstream targets of these two MR genes during ISC homeostasis.

JNK Kinase regulates cachexia like syndrome in scribble knockdown tumor model of Drosophila melanogaster.

Cachexia and systemic organ wasting are metabolic syndrome often associated with cancer. However, the exact mechanism of cancer associated cachexia like syndrome still remain elusive. In this study, we utilized a scribble (scrib) knockdown induced hindgut tumor to investigate the role of JNK kinase in cachexia like syndrome. Scrib, a cell polarity regulator, also acts as a tumor suppressor gene. Its loss and mis-localization are reported in various type of malignant cancer-like breast, colon and prostate cancer. The scrib knockdown flies exhibited male lethality, reduced life span, systemic organ wasting and increased pJNK level in hindgut of female flies. Interestingly, knocking down of human JNK Kinase analogue, hep, in scrib knockdown background in hindgut leads to restoration of loss of scrib mediated lethality and systemic organ wasting. Our data showed that scrib loss in hindgut is capable of inducing cancer associated cachexia like syndrome. Here, we firstly report that blocking the JNK signaling pathway effectively rescued the cancer cachexia induced by scrib knockdown, along with its associated gut barrier disruption. These findings have significantly advanced our understanding of cancer cachexia and have potential implications for the development of therapeutic strategies. However, more research is needed to fully understand the complex mechanisms underlying this condition.