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Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a metastatic castration-resistant prostate cancer (mCRPC) subtype. It remains unclear, however, whether CDK12 loss drives prostate cancer (PCa) development or uncovers pharmacologic vulnerabilities. Here, we show Cdk12 ablation in murine prostate epithelium is sufficient to induce preneoplastic lesions with lymphocytic infiltration. In allograft-based CRISPR screening, Cdk12 loss associates positively with Trp53 inactivation but negatively with Pten inactivation. Moreover, concurrent Cdk12/Trp53 ablation promotes proliferation of prostate-derived organoids, while Cdk12 knockout in Pten-null mice abrogates prostate tumor growth. In syngeneic systems, Cdk12/Trp53-null allografts exhibit luminal morphology and immune checkpoint blockade sensitivity. Mechanistically, Cdk12 inactivation mediates genomic instability by inducing transcription-replication conflicts. Strikingly, CDK12-mutant organoids and patient-derived xenografts are sensitive to inhibition or degradation of the paralog kinase, CDK13. We therein establish CDK12 as a bona fide tumor suppressor, mechanistically define how CDK12 inactivation causes genomic instability, and advance a therapeutic strategy for CDK12-mutant mCRPC.

The insecticide dichlorodiphenyltrichloroethane (DDT) and its persistent metabolite, dichlorodiphenyldichloroethylene (DDE), have been associated with increased adiposity and obesity in multiple generations of rodents and humans. These lipophilic pollutants accumulate in adipose tissue and appear to decrease energy expenditure through the impairment of thermogenesis in brown adipose tissue (BAT). We hypothesized that impaired thermogenesis is due to persistent epigenetic modifications of BAT. To address this, we exposed C57BL/6 J mice to DDT or DDE from gestational day (GD) 11.5 to postnatal day (PND) 5, evaluated longitudinal body temperature, and performed reduced representation bisulfite sequencing and RNA sequencing of BAT from infant and adult offspring. Exposure to DDT or DDE reduced core body temperature in adult mice, and differential methylation at the pathway and gene level was persistent from infancy to adulthood. Furthermore, thermogenesis and biological pathways essential for thermogenic function, such as oxidative phosphorylation and mechanistic target of rapamycin kinase (mTOR) signaling, were enriched with differential methylation and RNA transcription in adult mice exposed to DDT or DDE. PAZ6 human brown preadipocytes were differentiated in the presence of DDT or DDE to understand the brown adipocyte-autonomous effect of these pollutants. In vitro exposure led to limited changes in RNA expression; however, mitochondrial membrane potential was decreased in vitro with 0.1 µM and 1 µM doses of DDT or DDE. These results demonstrate that concentrations of DDT and DDE relevant to human exposure have a significant effect on thermogenesis, the transcriptome, and DNA methylome of mouse BAT and the mitochondrial function of human brown adipocytes.

Transcriptional silencing in Saccharomyces cerevisiae is a persistent and highly stable form of gene repression. It involves DNA silencers and repressor proteins that bind nucleosomes. The silenced state is influenced by numerous factors including the concentration of repressors, nature of activators, architecture of regulatory elements, modifying enzymes and the dynamics of chromatin.Silencers function to increase the residence time of repressor Sir proteins at silenced domains while clustering of silenced domains enables increased concentrations of repressors and helps facilitate long-range interactions. The presence of an accessible NDR at the regulatory regions of silenced genes, the cycling of chromatin configurations at regulatory sites, the mobility of Sir proteins, and the non-uniform distribution of the Sir proteins across the silenced domain, all result in silenced chromatin that only stably silences weak promoters and enhancers via changes in transcription burst duration and frequency.These data collectively suggest that silencing is probabilistic and the robustness of silencing is achieved through sub-optimization of many different nodes of action such that a stable expression state is generated and maintained even though individual constituents are in constant flux.

Fapy•dG (N6-(2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2-deoxyguanosine) are major products of 2-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer-dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to 2-deoxyguanosine. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G, or U. Fitting of reaction data indicates that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast- or slow-reacting base pairs vary less than twofold as a function of the nucleotide opposite the lesion. Promoter-dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side by side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.

Stroke causes death of brain tissue leading to long-term deficits. Behavioral evidence from neurorehabilitative therapies suggest learning-induced neuroplasticity can lead to beneficial outcomes. However, molecular and cellular mechanisms that link learning and stroke recovery are unknown. We show that in a mouse model of stroke, which exhibits enhanced recovery of function due to genetic perturbations of learning and memory genes, animals display activity-dependent transcriptional programs that are normally active during formation or storage of new memories. The expression of neuronal activity-dependent genes are predictive of recovery and occupy a molecular latent space unique to motor recovery. With motor recovery, networks of activity-dependent genes are co-expressed with their transcription factor targets forming gene regulatory networks that support activity-dependent transcription, that are normally diminished after stroke. Neuronal activity-dependent changes at the circuit level are influenced by interactions with microglia. At the molecular level, we show that enrichment of activity-dependent programs in neurons lead to transcriptional changes in microglia where they differentially interact to support intercellular signaling pathways for axon guidance, growth and synaptogenesis. Together, these studies identify activity-dependent transcriptional programs as a fundamental mechanism for neural repair post-stroke.

Drugs that inhibit HIV transcription and/or reactivation of latent HIV have been proposed as a strategy to reduce HIV-associated immune activation or to achieve a functional cure, yet comparative studies are lacking. We evaluated 26 drugs, including drugs previously reported to inhibit HIV transcription (inhibitors of Tat-dependent HIV transcription, Rev, HSF-1/PTEF-b, HSP90, Jak/Stat, or SIRT1/Tat deacetylation) and other agents that were not tested before (inhibitors of PKC, NF-κB, SP-1, or histone acetyltransferase; NR2F1 agonists), elongation (inhibitors of CDK9/ PTEF-b), completion (inhibitors of PolyA-polymerase), or splicing (inhibitors of human splice factors). To investigate if those drugs would vary in their ability to affect different blocks to HIV transcription, we measured levels of initiated, elongated, midtranscribed, completed, and multiply spliced HIV RNA in PBMCs from antiretroviral therapy-suppressed individuals following ex vivo treatment with each drug and subsequent T cell activation. We identified new drugs that prevent HIV reactivation, including CDK and splicing inhibitors. While some drugs inhibited 1 or 2 steps, other drugs (CDK inhibitors, splicing inhibitors, tanespimycin, and triptolide) inhibited multiple stages of HIV transcription and blocked the production of supernatant viral RNA. These drugs and targets deserve further study in strategies aimed at reducing HIV-associated immune activation or achieving a functional cure.

Transcription-blocking lesions (TBLs) stall elongating RNA polymerase II (Pol II), which then initiates transcription-coupled repair (TCR) to remove TBLs and allow transcription recovery. In the absence of TCR, eviction of lesion-stalled Pol II is required for alternative pathways to address the damage, but the mechanism is unclear. Using Protein-Associated DNA Damage Sequencing (PADD-seq), this study reveals that the p97-proteasome pathway can evict lesion-stalled Pol II independently of repair. Both TCR and repair-independent eviction require CSA and ubiquitination. However, p97 is dispensable for TCR and Pol II eviction in TCR-proficient cells, highlighting repairs prioritization over repair-independent eviction. Moreover, ubiquitination of RPB1-K1268 is important for both pathways, with USP7s deubiquitinase activity promoting TCR without abolishing repair-independent Pol II release. In summary, this study elucidates the fate of lesion-stalled Pol II, and may shed light on the molecular basis of genetic diseases caused by the defects of TCR genes.

The androgen receptor (AR) is the main driver in the development of castration-resistant prostate cancer, where the emergence of AR splice variants leads to treatment-resistant disease. Through detailed molecular studies of the marine alkaloid manzamine A (MA), we identified transcription factor E2F8 as a previously unknown regulator of AR transcription that prevents AR synthesis in prostate cancer cells. MA significantly inhibited the growth of various prostate cancer cell lines and was highly effective in inhibiting xenograft tumor growth in mice without any pathophysiological perturbations in major organs. MA suppressed the full-length AR (AR-FL), its spliced variant AR-V7, and the AR-regulated prostate-specific antigen (PSA; also known as KLK3) and human kallikrein 2 (hK2; also known as KLK2) genes. RNA sequencing (RNA-seq) analysis and protein modeling studies revealed E2F8 interactions with DNA as a potential novel target of MA, suppressing AR transcription and its synthesis. This novel mechanism of blocking AR biogenesis via E2F8 may provide an opportunity to control therapy-resistant prostate cancer over the currently used AR antagonists designed to target different parts of the AR gene.

UNLABELLED: Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in the different environmental niches they occupy. H-NS, the histone-like nucleoid structuring protein, is the best-studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently discovered HAEs encoding for phage defense systems, we show that a lack of H-NS causes a robust increase in the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity. We also identified few sites that are affected by TsrA independently of H-NS, suggesting TsrA may act with diverse regulatory mechanisms. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently discovered HAEs. IMPORTANCE: New strains of the bacterial pathogen Vibrio cholerae, bearing novel horizontally acquired elements (HAEs), frequently emerge. HAEs provide beneficial traits to the bacterium, such as antibiotic resistance and defense against invading bacteriophages. Xenogeneic silencers are proteins that help bacteria harness new HAEs and silence those HAEs until they are needed. H-NS is the best-studied xenogeneic silencer; it is one of the nucleoid-associated proteins (NAPs) in gamma-proteobacteria and is responsible for the proper regulation of HAEs within the bacterial transcriptional network. We studied the effects of H-NS and other NAPs on the HAEs of a clinical isolate of V. cholerae. Importantly, we found that H-NS partners with a small and poorly characterized protein, TsrA, to help domesticate new HAEs involved in bacterial survival and in causing disease. A proper understanding of the regulatory state in emerging isolates of V. cholerae will provide improved therapies against new isolates of the pathogen.

Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.

Fluctuations in the initiation rate of transcription, the first step in gene expression, ensue from the stochastic behavior of the molecular process that controls transcription. In steady state, the regulatory process is often assumed to operate reversibly, i.e., in equilibrium. However, reversibility imposes fundamental limits to information processing. For instance, the assumption of equilibrium is difficult to square with the precision with which the regulatory process executes its task in eukaryotes. Here we provide evidence - from microscopic analyses of the transcription dynamics at a single gene copy of yeast - that the regulatory process for transcription is cyclic and irreversible (out of equilibrium). The necessary coupling to reservoirs of free energy occurs via sequence-specific transcriptional activators and the recruitment, in part, of ATP-dependent chromatin remodelers. Our findings may help explain how eukaryotic cells reconcile the dual but opposing requirements for fast regulatory kinetics and high regulatory specificity.

The five NF-κB family members and three nuclear IκB proteins play important biological roles, but the mechanisms by which distinct members of these protein families contribute to selective gene transcription remain poorly understood, especially at a genome-wide scale. Using nascent transcript RNA-seq, we observed considerable overlap between p50-dependent and IκBζ-dependent genes in Toll-like receptor 4 (TLR4)-activated macrophages. Key immunoregulatory genes, including Il6, Il1b, Nos2, Lcn2, and Batf, are among the p50-IκBζ-codependent genes. IκBζ-bound genomic sites are occupied at earlier time points by NF-κB dimers. However, p50-IκBζ codependence does not coincide with preferential binding of either p50 or IκBζ, as RelA co-occupies hundreds of genomic sites with the two proteins. A common feature of p50-IκBζ-codependent genes is a nearby p50/RelA/IκBζ-cobound site exhibiting p50-dependent binding of both RelA and IκBζ. This and other results suggest that IκBζ acts in concert with RelA:p50 heterodimers. Notably, p50-IκBζ-codependent genes comprise a high percentage of genes exhibiting the greatest differential expression between TLR4-stimulated and tumor necrosis factor receptor (TNFR)-stimulated macrophages. Thus, our genome-centric analysis reveals a defined p50-IκBζ pathway that selectively activates a set of key immunoregulatory genes and serves as an important contributor to differential TNFR and TLR4 responses.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood. We examined gene expression patterns of 870 tumors of varied histological types, to identify transcriptional correlates of ecDNA. Here, we show that ecDNA-containing tumors impact four major biological processes. Specifically, ecDNA-containing tumors up-regulate DNA damage and repair, cell cycle control, and mitotic processes, but down-regulate global immune regulation pathways. Taken together, these results suggest profound alterations in gene regulation in ecDNA-containing tumors, shedding light on molecular processes that give rise to their development and progression.

It has proved challenging to quantitatively relate the proteome to the transcriptome on a per-gene basis. Recent advances in data analytics have enabled a biologically meaningful modularization of the bacterial transcriptome. We thus investigate whether matched datasets of transcriptomes and proteomes from bacteria under diverse conditions can be modularized in the same way to reveal novel relationships between their compositions. We find that; (1) the modules of the proteome and the transcriptome are comprised of a similar list of gene products, (2) the modules in the proteome often represent combinations of modules from the transcriptome, (3) known transcriptional and post-translational regulation is reflected in differences between two sets of modules, allowing for knowledge-mapping when interpreting module functions, and (4) through statistical modeling, absolute proteome allocation can be inferred from the transcriptome alone. Quantitative and knowledge-based relationships can thus be found at the genome-scale between the proteome and transcriptome in bacteria.

Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Transcriptional silencing in Saccharomyces cerevisiae involves the generation of a chromatin state that stably represses transcription. Using multiple reporter assays, a diverse set of upstream activating sequence enhancers and core promoters were investigated for their susceptibility to silencing. We show that heterochromatin stably silences only weak and stress-induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements, and the partial repression of these elements did not result in bistable expression states. Permutation analysis of enhancers and promoters indicates that both elements are targets of repression. Chromatin remodelers help specific regulatory elements to resist repression, most probably by altering nucleosome mobility and changing transcription burst duration. The strong enhancers/promoters can be repressed if silencer-bound Sir1 is increased. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating-type gene regulatory elements but not strong housekeeping gene regulatory sequences.

Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues such as age, resource availability, and biotic and abiotic stresses allow plants to abscise organs in a timely manner. How these signals are integrated into the molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene is one of the main drivers of floral organ abscission in Arabidopsis and is known to transcriptionally respond to most abscission-regulating cues. By interrogating the IDA promoter in silico and in vitro, we identified transcription factors that could potentially modulate IDA expression. We probed the importance of ERF- and WRKY-binding sites for IDA expression during floral organ abscission, with WRKYs being of special relevance to mediate IDA up-regulation in response to biotic stress in tissues destined for separation. We further characterized WRKY57 as a positive regulator of IDA and IDA-like gene expression in abscission zones. Our findings highlight the promise of promoter element-targeted approaches to modulate the responsiveness of the IDA signaling pathway to harness controlled abscission timing for improved crop productivity.

Although often located at a distance from their target gene promoters, enhancers are the primary genomic determinants of temporal and spatial transcriptional specificity in metazoans. Since the discovery of the first enhancer element in simian virus 40, there has been substantial interest in unraveling the mechanism(s) by which enhancers communicate with their partner promoters to ensure proper gene expression. These research efforts have benefited considerably from the application of increasingly sophisticated sequencing- and imaging-based approaches in conjunction with innovative (epi)genome-editing technologies; however, despite various proposed models, the principles of enhancer-promoter interaction have still not been fully elucidated. In this review, we provide an overview of recent progress in the eukaryotic gene transcription field pertaining to enhancer-promoter specificity. A better understanding of the mechanistic basis of lineage- and context-dependent enhancer-promoter engagement, along with the continued identification of functional enhancers, will provide key insights into the spatiotemporal control of gene expression that can reveal therapeutic opportunities for a range of enhancer-related diseases.

Dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA1-3. Chromatin complex compositions change during cellular differentiation and ageing, and are expected to be highly heterogeneous among terminally differentiated single cells4-7. Here we introduce the multinucleic acid interaction mapping in single cells (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression and RNA-chromatin associations within individual nuclei. When applied to 14 human frontal cortex samples from older donors, MUSIC delineated diverse cortical cell types and states. We observed that nuclei exhibiting fewer short-range chromatin interactions were correlated with both an older transcriptomic signature and Alzheimers disease pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci and a promoter tends to be that in which these cis expression quantitative trait loci specifically affect the expression of their target gene. In addition, female cortical cells exhibit highly heterogeneous interactions between XIST non-coding RNA and chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploration of chromatin architecture and transcription at cellular resolution in complex tissues.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

Androgen receptor (AR) drives prostate cancer (PC) growth and progression, and targeting AR signaling is the mainstay of pharmacological therapies for PC. Resistance develops relatively fast as a result of refueled AR activity. A major gap in the field is the lack of understanding of targetable mechanisms that induce persistent AR expression in castrate-resistant PC (CRPC). This study uncovers an unexpected function of active Stat5 signaling, a known promoter of PC growth and clinical progression, as a potent inducer of AR gene transcription. Stat5 suppression inhibited AR gene transcription in preclinical PC models and reduced the levels of wild-type, mutated, and truncated AR proteins. Pharmacological Stat5 inhibition by a specific small-molecule Stat5 inhibitor down-regulated Stat5-inducible genes as well as AR and AR-regulated genes and suppressed PC growth. This work introduces the concept of Stat5 as an inducer of AR gene transcription in PC. Pharmacological Stat5 inhibitors may represent a new strategy for suppressing AR and CRPC growth.

Eukaryotic transcription factors activate gene expression with their DNA-binding domains and activation domains. DNA-binding domains bind the genome by recognizing structurally related DNA sequences; they are structured, conserved, and predictable from protein sequences. Activation domains recruit chromatin modifiers, coactivator complexes, or basal transcriptional machinery via structurally diverse protein-protein interactions. Activation domains and DNA-binding domains have been called independent, modular units, but there are many departures from modularity, including interactions between these regions and overlap in function. Compared to DNA-binding domains, activation domains are poorly understood because they are poorly conserved, intrinsically disordered, and difficult to predict from protein sequences. This review, organized around commonly asked questions, describes recent progress that the field has made in understanding the sequence features that control activation domains and predicting them from sequence.

SUMMARY The ability of seeds to restore their amino acid content and composition after the elimination of the most abundant seed storage proteins (SSPs) is well‐documented, yet the underlying mechanisms remain unclear. To better understand how seeds compensate for major proteomic disruptions, we conducted a comprehensive analysis on an Arabidopsis mutant lacking the three most abundant SSPs, the cruciferins. Our initial findings indicated that carbon, nitrogen, and sulfur levels, as well as total protein and oil content, remained unchanged in these mutants suggesting rebalanced seeds. Transcriptomics and proteomics performed during seed maturation of Col‐0 and the triple mutant revealed significant modulation in many components of the translational machinery, especially ribosomal proteins (RPs), and in the antioxidation response in the mutant. These findings suggest that RPs play a critical role in facilitating proteomic homeostasis during seed maturation when proteomic perturbation occurs. Biochemical and metabolic analyses of the triple mutant dry seeds revealed increased protein carbonylation and elevated glutathione levels further supporting the link between SSP accumulation and seed redox homeostasis. Overall, we propose that in response to significant proteomic perturbations, changes in the proteome and amino acid composition of seeds are accompanied by a broad remodeling of the translation apparatus. We postulate that these alterations are key elements in seed adaptability and robustness to large proteomic perturbations during seed maturation. [ABSTRACT FROM AUTHOR]

Background: Dietary restriction (DR) has multiple beneficial effects on health and longevity and can also improve the efficacy of certain therapies. Diets used to instigate DR are diverse and the corresponding response is not uniformly measured. We compared the systemic and liver-specific transcriptional response to intermittent fasting (IF) and commercially available fasting-mimicking diet (FMD) after short- and long-term use in C57BL/6 J mice. Results: We show that neither DR regimen causes observable adverse effects in mice. The weight loss was limited to 20% and was quickly compensated during refeeding days. The slightly higher weight loss upon FMD versus IF correlated with stronger fasting response assessed by lower glucose levels and higher ketone body, free fatty acids and especially FGF21 concentrations in blood. RNA sequencing demonstrated similar transcriptional programs in the liver after both regimens, with PPARα signalling as top enriched pathway, while on individual gene level FMD more potently increased gluconeogenesis-related, and PPARα and p53 target gene expression compared to IF. Repeated IF induced similar transcriptional responses as acute IF. However, repeated cycles of FMD resulted in blunted expression of genes involved in ketogenesis and fatty acid oxidation. Conclusions: Short-term FMD causes more pronounced changes in blood parameters and slightly higher weight loss than IF, while both activate similar pathways (particularly PPARα signalling) in the liver. On individual gene level FMD induces a stronger transcriptional response, whereas cyclic application blunts transcriptional upregulation of fatty acid oxidation and ketogenesis only in FMD. Hence, our comparative characterization of IF and FMD protocols renders both as effective DR regimens and serves as resource in the fasting research field. [ABSTRACT FROM AUTHOR]

The flavonoid pathway is characteristic of land plants and a central biosynthetic component enabling life in a terrestrial environment. Flavonoids provide tolerance to both abiotic and biotic stresses and facilitate beneficial relationships, such as signalling to symbiont microorganisms, or attracting pollinators and seed dispersal agents. The biosynthetic pathway shows great diversity across species, resulting principally from repeated biosynthetic gene duplication and neofunctionalization events during evolution. Such events may reflect a selection for new flavonoid structures with novel functions that enable occupancy of varied ecological niches. However, the biochemical and genetic diversity of the pathway also likely resulted from evolution along parallel trends across land plant lineages, producing variant compounds with similar biological functions. Analyses of the wide range of whole-plant genome sequences now available, particularly for archegoniate plants, have enabled proposals on which genes were ancestral to land plants and which arose within the land plant lineages. In this review, we discuss the emerging proposals for how the flavonoid pathway may have evolved and diversified. This article is part of the theme issue 'The evolution of plant metabolism'. [ABSTRACT FROM AUTHOR]

Cells adapt to environments and tune gene expression by controlling the concentrations of proteins and their kinetics in regulatory networks. In both eukaryotes and prokaryotes, experiments and theory increasingly attest that these networks can and do consume biochemical energy. How does this dissipation enable cellular behaviors forbidden in equilibrium? This open question demands quantitative models that transcend thermodynamic equilibrium. Here, we study the control of simple, ubiquitous gene regulatory networks to explore the consequences of departing equilibrium in transcription. Employing graph theory to model a set of especially common regulatory motifs, we find that dissipation unlocks nonmonotonicity and enhanced sensitivity of gene expression with respect to a transcription factor's concentration. These features allow a single transcription factor to act as both a repressor and activator at different concentrations or achieve outputs with multiple concentration regimes of locally enhanced sensitivity. We systematically dissect how energetically driving individual transitions within regulatory networks, or pairs of transitions, generates a wide range of more adjustable and sensitive phenotypic responses than in equilibrium. These results generalize to more complex regulatory scenarios, including combinatorial control by multiple transcription factors, which we relate and often find collapse to simple mathematical behaviors. Our findings quantify necessary conditions and detectable consequences of energy expenditure. These richer mathematical behaviors--feasibly accessed using biological energy budgets and rates--may empower cells to accomplish sophisticated regulation with simpler architectures than those required at equilibrium. [ABSTRACT FROM AUTHOR]

Experience- and activity-dependent transcription is a candidate mechanism to mediate development and refinement of specific cortical circuits. Here, we demonstrate that the activity-dependent transcription factor myocyte enhancer factor 2C (MEF2C) is required in both presynaptic layer (L) 4 and postsynaptic L2/3 mouse (male and female) somatosensory (S1) cortical neurons for development of this specific synaptic connection. While postsynaptic deletion of Mef2c weakens L4 synaptic inputs, it has no effect on inputs from local L2/3, contralateral S1, or the ipsilateral frontal/motor cortex. Similarly, homozygous or heterozygous deletion of Mef2c in presynaptic L4 neurons weakens L4 to L2/3 excitatory synaptic inputs by decreasing presynaptic release probability. Postsynaptic MEF2C is specifically required during an early postnatal, experience-dependent, period for L4 to L2/3 synapse function, and expression of transcriptionally active MEF2C (MEF2C-VP16) rescues weak L4 to L2/3 synaptic strength in sensory-deprived mice. Together, these results suggest that experience- and/or activity-dependent transcriptional activation of MEF2C promotes development of L4 to L2/3 synapses. Additionally, MEF2C regulates the expression of many pre- and postsynaptic genes in postnatal cortical neurons. Interestingly, MEF2C was necessary for activity-dependent expression of many presynaptic genes, including those that function in transsynaptic adhesion and neurotransmitter release. This work provides mechanistic insight into the experiencedependent development of specific cortical circuits. [ABSTRACT FROM AUTHOR]

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA n) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA 4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA 4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity. [Display omitted] • CalpL filament formation is obligatory for its protease and ring nuclease activities • Within the CalpL filament, CalpT is cleaved by the adjacent CalpL subunit • CalpL cleavage exposes the CalpT C-degron, which targets it for further degradation • CalpL SAVED domain is a ring nuclease cleaving cA 4 via a sequential three-step mechanism Smalakyte et al. report the molecular mechanism of the type III CRISPR-Cas tripartite effector system. cA 4 induces filament formation and activation of Lon-protease CalpL to cleave CalpT, releasing σ-factor CalpS, which binds RNA polymerase, arresting E. coli growth. Signaling cascade is regulated by ring nuclease activity of the CalpL SAVED domain. [ABSTRACT FROM AUTHOR]

Fibroblast Growth Factor signalling via ERK exerts diverse roles in development and disease. In mammalian preimplantation embryos and naïve pluripotent stem cells ERK promotes differentiation, whereas in primed pluripotent states closer to somatic differentiation ERK sustains self-renewal. How can the same pathway produce different outcomes in two related cell types? To explore context-dependent ERK signalling we generated cell and mouse lines that allow for tissue- and time-specific ERK activation. Using these tools, we find that specificity in ERK response is mostly mediated by repression of transcriptional targets that occur in tandem with reductions in chromatin accessibility at regulatory regions. Furthermore, immediate early ERK responses are largely shared by different cell types but produce cell-specific programmes as these responses interface with emergent networks in the responding cells. Induction in naïve pluripotency is accompanied by chromatin changes, whereas in later stages it is not, suggesting that chromatin context does not shape signalling response. Altogether, our data suggest that cell-type-specific responses to ERK signalling exploit the same immediate early response, but then sculpt it to specific lineages via repression of distinct cellular programmes. [ABSTRACT FROM AUTHOR]

Cyclin‐dependent kinase 9 (CDK9), a catalytic subunit of the positive transcription elongation factor b (P‐TEFb) complex, is a global transcriptional elongation factor associated with cell proliferation. CDK9 activity is regulated by certain histone acetyltransferases, such as p300, GCN5 and P/CAF. However, the impact of males absent on the first (MOF) (also known as KAT8 or MYST1) on CDK9 activity has not been reported. Therefore, the present study aimed to elucidate the regulatory role of MOF on CDK9. We present evidence from systematic biochemical assays and molecular biology approaches arguing that MOF interacts with and acetylates CDK9 at the lysine 35 (i.e. K35) site, and that this acetyl‐group can be removed by histone deacetylase HDAC1. Notably, MOF‐mediated acetylation of CDK9 at K35 promotes the formation of the P‐TEFb complex through stabilizing CDK9 protein and enhancing its association with cyclin T1, which further increases RNA polymerase II serine 2 residues levels and global transcription. Our study reveals for the first time that MOF promotes global transcription by acetylating CDK9, providing a new strategy for exploring the comprehensive mechanism of the MOF–CDK9 axis in cellular processes. [ABSTRACT FROM AUTHOR]

Histone 3 lysine 4 methylation (H3K4me) is a highly evolutionary conserved chromatin modification associated with active transcription, and its three methylation states—mono, di, and trimethylation—mark distinct regulatory elements. However, whether H3K4me plays functional roles in transcriptional regulation or is merely a by-product of histone methyltransferases recruited to actively transcribed loci is still under debate. Here, we outline the studies that have addressed this question in yeast, Drosophila, and mammalian systems. We review evidence from histone residue mutation, histone modifier manipulation, and epigenetic editing, focusing on the relative roles of H3K4me1 and H3K4me3. We conclude that H3K4me1 and H3K4me3 may have convergent functions in establishing open chromatin and promoting transcriptional activation during cell differentiation. [ABSTRACT FROM AUTHOR]

The three‐dimensional architecture of the bacterial chromosome is intertwined with genome processes such as transcription and replication. Conspicuously so, that the structure of the chromosome permits accurate prediction of active genome processes. Although appreciation of this interplay has developed rapidly in the past two decades, our understanding of this subject is still in its infancy, with research primarily focusing on how the process of transcription regulates and is regulated by chromosome structure. Here, we summarize the latest developments in the field with a focus on the interplay between chromosome structure and transcription in Escherichia coli (E. coli) as mediated by H‐NS—a model nucleoid structuring protein. We describe how the organization of chromosomes at the global and local scales is dependent on transcription, and how transcription is regulated by chromosome structure. Finally, we take note of studies that highlight our limited knowledge of structure‐function relationships in the chromosome, and we point out research tracks that will improve our insight in the topic. [ABSTRACT FROM AUTHOR]

Background and Aims: During fasting, bodily homeostasis is maintained due to hepatic production of glucose (gluconeogenesis) and ketone bodies (ketogenesis). The main hormones governing hepatic fuel production are glucagon and glucocorticoids that initiate transcriptional programs aimed at supporting gluconeogenesis and ketogenesis. Methods: Using primary mouse hepatocytes as an ex vivo model, we employed transcriptomic analysis (RNA‐seq), genome‐wide profiling of enhancer dynamics (ChIP‐seq), perturbation experiments (inhibitors, shRNA), hepatic glucose production measurements and computational analyses. Results: We found that in addition to the known metabolic genes transcriptionally induced by glucagon and glucocorticoids, these hormones induce a set of genes encoding transcription factors (TFs) thereby initiating transcriptional cascades. Upon activation by glucocorticoids, the glucocorticoid receptor (GR) induced the genes encoding two TFs: CCAAT/enhancer‐binding protein beta (C/EBPβ) and peroxisome proliferator‐activated receptor alpha (PPARα). We found that the GR‐C/EBPβ cascade mainly serves as a secondary amplifier of primary hormone‐induced gene programs. C/EBPβ augmented gluconeogenic gene expression and hepatic glucose production. Conversely, the GR‐PPARα cascade initiated a secondary transcriptional wave of genes supporting ketogenesis. The cascade led to synergistic induction of ketogenic genes which is dependent on protein synthesis. Genome‐wide analysis of enhancer dynamics revealed numerous enhancers activated by the GR‐PPARα cascade. These enhancers were proximal to ketogenic genes, enriched for the PPARα response element and showed increased PPARα binding. Conclusion: This study reveals abundant transcriptional cascades occurring during fasting. These cascades serve two separated purposes: the amplification of the gluconeogenic transcriptional program and the induction of a gene program aimed at enhancing ketogenesis. [ABSTRACT FROM AUTHOR]

Ferroptosis, a key factor in tumor progression, is poorly understood at the molecular level. This study investigates how ELK4 and CHMP6 regulate skin cutaneous melanoma (SKCM) cell proliferation and ferroptosis. Analysis of TCGA data reveals high expression of ELK4 and CHMP6 in SKCM. Overexpression of ELK4 or CHMP6 enhances cell proliferation, invasion, and migration while reducing ROS and Fe2 + levels. It also increases GPX4 and xCT expression and decreases ACSL4 levels in SKCM cells. The opposite effects are observed with ELK4 or CHMP6 knockdown. ELK4 binds to the CHMP6 promoter, promoting CHMP6 transcription. Knockdown of CHMP6 reverses the oncogenic effects of ELK4 overexpression. In conclusion, ELK4 enhances proliferation, invasion, and migration while inhibiting ferroptosis in SKCM cells by upregulating CHMP6 transcription. This study sheds light on the intricate mechanisms involved in SKCM progression and identifies potential therapeutic targets in melanoma treatment. [ABSTRACT FROM AUTHOR]

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The β and β′ subunits of the RNA polymerase (RNAP) are large proteins with complex multi‐domain architectures that include several insertional domains. Here, we analyze the domain organizations of RNAP‐β and RNAP‐β′ using sequence, experimentally determined structures and AlphaFold structure predictions. We observe that lineage‐specific insertional domains in bacterial RNAP‐β belong to a group that we call BEAN (broadly embedded annex). We observe that lineage‐specific insertional domains in bacterial RNAP‐β′ belong to a group that we call HABAS (hammerhead/barrel‐sandwich hybrid). The BEAN domain has a characteristic three‐dimensional structure composed of two square bracket‐like elements that are antiparallel relative to each other. The HABAS domain contains a four‐stranded open β‐sheet with a GD‐box‐like motif in one of the β‐strands and the adjoining loop. The BEAN domain is inserted not only in the bacterial RNAP‐β′, but also in the archaeal version of universal ribosomal protein L10. The HABAS domain is inserted in several metabolic proteins. The phylogenetic distributions of bacterial lineage‐specific insertional domains of β and β′ subunits of RNAP follow the Tree of Life. The presence of insertional domains can help establish a relative timeline of events in the evolution of a protein because insertion is inferred to post‐date the base domain. We discuss mechanisms that might account for the discovery of homologous insertional domains in non‐equivalent locations in bacteria and archaea. [ABSTRACT FROM AUTHOR]

The ability of cancer cells to change and adapt poses a critical challenge to identifying curative solutions. Tumor evolution has been extensively studied from a genetic perspective, to guide clinicians in selecting the most appropriate therapeutic option based on a patient's mutational profile. However, several studies reported that tumors can evolve toward more aggressive stages or become resistant to therapies without changing their genetic makeup. Indeed, several cell‐intrinsic and cell‐extrinsic mechanisms contribute to tumor evolution. In this viewpoint, I focus on how chromatin, epigenetic, and transcriptional changes contribute to tumor evolution, allowing cancer cells to transition to different cell states and bypass response to therapies. Although tumor nongenetic evolution is harder to trace and predict, understanding its principles might open new therapeutic opportunities. [ABSTRACT FROM AUTHOR]

The eukaryotic transcriptional Mediator comprises a large core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (RNA Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein, we report cryoelectron microscopy structures of the complete human Mediator and its CKM. The CKM binds to multiple regions on cMED through both MED12 and MED13, including a large intrinsically disordered region (IDR) in the latter. MED12 and MED13 together anchor the CKM to the cMED hook, positioning CDK8 downstream and proximal to the transcription start site. Notably, the MED13 IDR obstructs the recruitment of RNA Pol II/MED26 onto cMED by direct occlusion of their respective binding sites, leading to functional repression of cMED-dependent transcription. Combined with biochemical and functional analyses, these structures provide a conserved mechanistic framework to explain the basis for CKM-mediated repression of cMED function. [Display omitted] • Structures of the complete human Mediator and its CDK8 kinase module (CKM) are presented • CKM binds cMED through MED12 and the intrinsically disordered region (IDR) of MED13 • MED13 IDR blocks RNA Pol II/MED26 from binding to cMED by occupying their binding sites • Human and yeast MED13 share a conserved mechanism for transcriptional repression Chao and Chen et al. present cryo-EM structures of the complete human Mediator and its CDK8 kinase module (CKM). The CKM uses its MED13 IDR to occupy RNA-polymerase-II- and MED26-binding sites, blocking their interactions with Mediator and repressing Mediator-dependent transcription—a mechanism conserved in humans and yeast. [ABSTRACT FROM AUTHOR]

Chemical modifications of cellular RNAs play key roles in gene expression and host defense. The cap-adjacent N6,2′-O-dimethyladenosine (m6Am) is a prevalent modification of vertebrate and viral mRNAs and is catalyzed by the newly discovered N6 methyltransferase PCIF1. However, its role in gene expression remains unclear due to conflicting reports on its effects on mRNA stability and translation. In this study, we investigated the impact of siRNA-mediated transient suppression of PCIF1 on global mRNA expression in HeLa cells. We identified a subset of differentially expressed genes (DEGs) that exhibited minimal overlap with previously reported DEGs. Subsequent validation revealed that PCIF1 positively and negatively regulates RAB23 and CNOT6 expression, respectively, at both the mRNA and protein levels. Mechanistic analyses demonstrated that PCIF1 regulates the stability of these target mRNAs rather than their transcription, and rescue experiments confirmed the requirement of PCIF1's methyltransferase activity for these regulations. Furthermore, MeRIP-qPCR analysis showed that PCIF1 suppression significantly reduced the m6A levels of RAB23 and CNOT6 mRNAs. These findings suggest that PCIF1 regulates the stability of specific mRNAs in opposite ways through m6A modification, providing new insights into the role of m6Am in the regulation of gene expression. [ABSTRACT FROM AUTHOR]

SUMMARY: Flooding impairs plant growth through oxygen deprivation, which activates plant survival and acclimation responses. Transcriptional responses to low oxygen are generally associated with the activation of group VII ETHYLENE‐RESPONSE FACTOR (ERFVII) transcription factors. However, the exact mechanisms and molecular components by which ERFVII factors initiate gene expression are not fully elucidated. Here, we show that the ERFVII factors RELATED TO APETALA 2.2 (RAP2.2) and RAP2.12 cooperate with the Mediator complex subunit AtMED25 to coordinate gene expression under hypoxia in Arabidopsis thaliana. Respective med25 knock‐out mutants display reduced low‐oxygen stress tolerance. AtMED25 physically associates with a distinct set of hypoxia core genes and its loss partially impairs transcription under hypoxia due to decreased RNA polymerase II recruitment. Association of AtMED25 with target genes requires the presence of ERFVII transcription factors. Next to ERFVII protein stabilisation, also the composition of the Mediator complex including AtMED25 is potentially affected by hypoxia stress as shown by protein‐complex pulldown assays. The dynamic response of the Mediator complex to hypoxia is furthermore supported by the fact that two subunits, AtMED8 and AtMED16, are not involved in the establishment of hypoxia tolerance, whilst both act in coordination with AtMED25 under other environmental conditions. We furthermore show that AtMED25 function under hypoxia is independent of ethylene signalling. Finally, functional conservation at the molecular level was found for the MED25‐ERFVII module between A. thaliana and the monocot species Oryza sativa, pointing to a potentially universal role of MED25 in coordinating ERFVII‐dependent transcript responses to hypoxia in plants. [ABSTRACT FROM AUTHOR]

Summary Neonatal and adult megakaryocytes differ in proliferative capacity and ploidy levels, and neonatal and adult platelets differ in function, gene expression, and protein content. The mechanisms underlying these differences are incompletely understood. CDK8 and CDK19 are transcriptional kinases part of the CDK‐mediator complex, which regulates gene transcription in a cell‐specific manner. We discovered that cortistatin A, a potent highly selective inhibitor of CDK8/CDK19, significantly reduced cell expansion and increased ploidy in cord blood‐derived megakaryocytes. These phenotypic changes were associated with gene expression changes that partially overlapped developmentally regulated genes. These findings might have relevance for the management of developmental megakaryocyte disorders. [ABSTRACT FROM AUTHOR]

Background: The first 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the first 24 h of infection with influenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa. Results: Pseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably affected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (> 100 nt) were represented similarly among the RNAs that were differentially expressed upon infection with the three pathogens (mRNA 77–82%; lncRNA 15–17%; pseudogenes 4–5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifically associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identified airway epithelial cells and macrophages as the predominant IAV host cells, but inflammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identified a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection. Conclusions: Human lung tissue mounted substantially different transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level. [ABSTRACT FROM AUTHOR]

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium. [ABSTRACT FROM AUTHOR]

Background: Protein kinase CK2 is a ubiquitous and highly conserved protein Ser/Thr kinase with diverse cell functions. CK2 is upregulated in various cancers and affects numerous aspects of their underlying pathobiology. The important role of microRNAs (miRNAs) referred to as oncomirs is also recognized in various cancers. Elevation of both CK2 and altered miRNA expression in cancers raised the question whether there was a connection between CK2 function and oncomirs in cancer. Methods: PCR array analysis was used to examine the effects of CK2 siRNA-mediated downregulation on miRNA levels in C4-2 prostate cancer cells. We employed prostate cancer, breast cancer, and head and neck squamous cell carcinoma (HNSCC) cells as well as a prostate cancer xenograft orthotopic tumor model to examine the effects of CK2 siRNA-mediated downregulation or chemical inhibition on oncomir cluster miR-17 ~ 92 and miR-106b ~ 25 constituent miRNAs by quantitative reverse-transcriptase stem-loop PCR. Pri-miRNAs were measured in cancer cell lines by quantitative reverse-transcriptase PCR. Protein levels were assessed by western blot. PC3-LN4 prostate cancer orthotopic xenograft tumors and blood were collected from nude mice following repeated treatments with tenfibgen ligand nanocapsules containing RNAi-CK2 or RNAi-Control cargoes. Results: PCR array analysis demonstrated effect on a subset of miRNAs following CK2 downregulation; we focused our investigation on CK2 regulation of miR-17 ~ 92 and 106b ~ 25 oncomir clusters. Chemical inhibition or molecular downregulation of CK2 greatly reduced expression of miR-17 ~ 92 and 106b ~ 25 in prostate, breast and head and neck cancer cells in vitro. CK2α and CK2α´ protein levels were significantly correlated with many of the miR-17 ~ 92 and some of the miR-106b ~ 25 constituent members in prostate cancer cells. Decreased pri-miRNA levels for the miR-17 ~ 92 gene cluster transcript were observed for 5 of 6 cancer cell lines tested following CK2 downregulation. Nanocapsule-mediated delivery of RNAi-CK2 reduced CK2 protein expression in orthotopic prostate xenograft tumors and decreased intra-tumoral and serum levels of the oncomirs. Conclusions: Targeting CK2 for the development of new cancer therapies is under active investigation in many laboratories and pharmaceutical companies. Our data suggest a new role for CK2 in cell signaling and survival in multiple cancer types through maintenance of miR-17 ~ 92 and 106b ~ 25 biogenesis. [ABSTRACT FROM AUTHOR]

MITF, a basic Helix-Loop-Helix Zipper (bHLHZip) transcription factor, plays vital roles in melanocyte development and functions as an oncogene. We perform a genetic screen for suppressors of the Mitf-associated pigmentation phenotype in mice and identify an intragenic Mitf mutation that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). The resulting protein is more nuclear but less stable than wild-type MITF and retains DNA-binding ability. As a dimer, it can translocate wild-type and mutant MITF partners into the nucleus, improving its own stability thus ensuring nuclear MITF supply. smFRET analysis shows interactions between K316 SUMOylation and S409 phosphorylation sites across monomers; these interactions largely explain the observed effects. The recurrent melanoma-associated E318K mutation in MITF, which affects K316 SUMOylation, also alters protein regulation in concert with S409. This suggests that residues K316 and S409 of MITF are impacted by SUMOylation and phosphorylation, respectively, mediating effects on nuclear localization and stability through conformational changes. Our work provides a novel mechanism of genetic suppression, and an example of how apparently deleterious mutations lead to normal phenotypes. Synopsis: An intragenic Mitf suppressor mutation was identified that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). This mutation provides novel information on how the dynamic IDR mediates MITF localization and stability. The MITF suppressor mutation uncovers a novel mechanism of genetic suppression that opens unexpected insights into MITF subcellular localization and stability. The MITF suppressor mutation provides one explanation for how humans with knockout mutations in essential genes are viable and normal. An intragenic Mitf suppressor mutation was identified that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). This mutation provides novel information on how the dynamic IDR mediates MITF localization and stability. [ABSTRACT FROM AUTHOR]

Background: Batten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common pathogenic variant shared by most patients is the "1-kb" deletion which removes two internal coding exons (7 and 8) in CLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the 1-kb deletion: the 'major' and 'minor' transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigate CLN3 transcription in "healthy" samples without juvenile CLN3 disease. Methods: We leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range of CLN3 transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources. Results: We found that a readthrough gene affects the quantification and annotation of CLN3. After taking this into account, we detected over 100 novel CLN3 transcripts, with no dominantly expressed CLN3 transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated 'major' transcripts are detected. Together, they have median usage of 1.5% across 22 samples. Furthermore, we identified 48 CLN3 ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third of CLN3 transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data. Conclusion: Overall, these findings provide valuable insights into the complexity of CLN3 transcription, highlighting the importance of studying both canonical and non-canonical CLN3 protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) of CLN3. This knowledge is essential for investigating the impact of the 1-kb deletion and rare pathogenic variants on CLN3 transcription and disease pathogenesis. [ABSTRACT FROM AUTHOR]

Asexual replication of Plasmodium falciparum in the human blood results in exponential parasite growth and causes all clinical symptoms of malaria. However, at each round of the replicative cycle, some parasites convert into sexual precursors called gametocytes, which develop through different stages until they become infective to mosquito vectors. The genome-wide distribution of heterochromatin, a type of chromatin generally refractory to gene expression, is identical at all asexual blood stages, but is altered in stage II/III and more mature gametocytes. However, it is not known if these changes occur concomitantly with sexual conversion or at a later time during gametocyte development. Using a transgenic line in which massive sexual conversion can be conditionally induced, we show that the genome-wide distribution of heterochromatin at the initial stages of sexual development (i.e., sexual rings and stage I gametocytes) is almost identical to asexual blood stages, and major changes do not occur until stage II/III. However, we found that at loci with heterochromatin alterations, transcriptional changes associated with sexual development typically precede, rather than follow, changes in heterochromatin occupancy. [ABSTRACT FROM AUTHOR]