Transcriptional Regulation Research Articles

Storm: Incorporating transient stochastic dynamics to infer the RNA velocity with metabolic labeling information

The time-resolved scRNA-seq (tscRNA-seq) provides the possibility to infer physically meaningful kinetic parameters, e.g., the transcription, splicing or RNA degradation rate constants with correct magnitudes, and RNA velocities by incorporating temporal information. Previous approaches utilizing the deterministic dynamics and steady-state assumption on gene expression states are insufficient to achieve favorable results for the data involving transient process. We present a dynamical approach, Storm (Stochastic models of RNA metabolic-labeling), to overcome these limitations by solving stochastic differential equations of gene expression dynamics. The derivation reveals that the new mRNA sequencing data obeys different types of cell-specific Poisson distributions when jointly considering both biological and cell-specific technical noise. Storm deals with measured counts data directly and extends the RNA velocity methodology based on metabolic labeling scRNA-seq data to transient stochastic systems. Furthermore, we relax the constant parameter assumption over genes/cells to obtain gene-cell-specific transcription/splicing rates and gene-specific degradation rates, thus revealing time-dependent and cell-state-specific transcriptional regulations. Storm will facilitate the study of the statistical properties of tscRNA-seq data, eventually advancing our understanding of the dynamic transcription regulation during development and disease.

Comparative and Spatial Transcriptome Analysis of Rhododendron decorum Franch. During the Flowering Period and Revelation of the Plant Defense Mechanism

Background: Rhododendron is a globally distributed and extensive genus, comprising over 1000 species. In the southwestern mountains of China, there exists a remarkable diversity of Rhododendron, with Yunnan Province alone harboring more than 600 species. R. decorum Franch. has long been utilized by local communities for its medicinal and edible properties. However, the transcriptional regulation function, medicinal properties, and edibility characteristics of R. decorum Franch. currently lack a solid theoretical basis. Methods: Total RNA was extracted from leaves, corollas and androecium/gynoecium of R. decorum Franch. in Heqing county, followed by the construction of cDNA libraries and the de novo assembly of transcriptomes. Results: A total of 63,050 unigenes were extracted from the flowers and leaf organs of R. decorum Franch. Among these unigenes, 43,517 were predicted to be coding sequences, with 32,690 being effectively annotated. Differential gene expression enrichment was observed among different organs within their respective transcriptomes; notably floral organs exhibited significant defense against plant diseases along with signal transduction functions. Furthermore, during the flower harvesting period, all floral organs exhibited gene enrichment pathways associated with carbohydrate metabolism. Additionally, the stamen and pistil displayed flavonoid metabolism pathways, suggesting their potential applications as functional food or medicine. Conclusions: Our results shed light on plant–pathogen defense mechanisms and the molecular bias of flavonoids biosynthesis on flower organs during the flowering period, which might help to understand the consumption of R. decorum Franch. corollas by the Bai nationality of Heqing county.

Abstract A020: Elevated extracellular glucose level in tumor microenvironment by hyperglycemia increases glycolysis rate, stiffness, contractility, and motility of breast cancer cells through the cAMP-RhoA-Rock and AMPK-YAP-RhoA axes to promote metastasis

Abstract Glucose is a preferred nutrient for most cells, including cancer cells. The tumor microenvironment is both hypoxic and hypoglycemic. Glucose levels in tumors are ten times lower than in normal tissues due to poor vascularization and high glucose consumption by cancer cells, which adapt to hypoglycemia. Obesity, often accompanied by hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, is linked to increased breast cancer metastasis. High extracellular glucose levels have been shown to promote cancer cell motility, though the mechanisms remain unclear. Given cancer cells' reliance on aerobic glycolysis (the Warburg effect), we hypothesized that hyperglycemia in obesity promotes cancer metastasis by modulating glucose metabolism and related cell mechanics pathways. Using Triple-Negative Breast Cancer (TNBC) cells, we found that elevated extracellular glucose levels increase glycolysis, activating the RhoA GTPase signaling pathway. This results in greater cell stiffness and contractility, correlating with increased motility. Our unpublished data, which I will present, validate these findings in obese-TNBC mouse models and further elucidate the glycolysis-mediated activation of the RhoA-ROCK axis. We also identified a novel long-term effect of hyperglycemia in promoting metastasis via transcriptional regulation of the AMPK-YAP-RhoA axis. To confirm hyperglycemia's pro-metastatic effects, we developed two obese-TNBC mouse models using diet-induced obesity (DIO) in NSG and C57BL/6J mice with orthotopic implantation of human and mouse TNBC cells, respectively. RhoA GTPase activity was measured with a GTP-RhoA FRET sensor or pull-down assay. Cell stiffness was assessed using our innovative parallel microfiltration (PMF) assay or Atomic Force Microscopy (AFM). We disrupted glucose uptake by knocking out glucose transporter 3 (GLUT3) via CRISPR/Cas9 or using a GLUT3-selective inhibitor, G3iA. Cell motility was measured by scratch wound-healing and invasion assays. Bulk RNA-seq identified differentially expressed genes under high glucose conditions in human breast cancer cells. Our results show increased lung metastasis of breast cancer cells under hyperglycemic conditions in vivo. Besides the previously reported cAMP-RhoA-ROCK axis, we discovered the glycolysis-AMPK-YAP axis also regulates RhoA-ROCK activity transcriptionally. GLUT3 inhibition activated AMPK, reduced F-actin levels via the AMPK-VASP axis, and decreased non-muscle myosin activity through the AMPK-YAP-RhoA axis. These changes in cell mechanics correlated with reduced motility. Our study suggests that targeting glucose metabolism and cell mechanics could be a novel approach to inhibit cancer metastasis, particularly in obese patients. Citation Format: Tae-Hyung Kim, Aadil Q Bhat, Jun-Yong Choe, Donghoon Yoon. Elevated extracellular glucose level in tumor microenvironment by hyperglycemia increases glycolysis rate, stiffness, contractility, and motility of breast cancer cells through the cAMP-RhoA-Rock and AMPK-YAP-RhoA axes to promote metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A020.

TSG101 depletion dysregulates mitochondria and PML NBs, triggering MAD2-overexpressing interphase cell death (MOID) through AIFM1-PML-DAXX pathway

AbstractOverexpression of mitotic arrest deficiency 2 (MAD2/MAD2L1), a pivotal component of the spindle assembly checkpoint (SAC), resulted in many types of cancer. Here we show that the depletion of tumor susceptibility gene 101 (TSG101), causes synthetic dosage lethality (SDL) in MAD2-overexpressing cells, and we term this cell death MAD2-overexpressing interphase cell death (MOID). The induction of MOID depends on PML and DAXX mediating mitochondrial AIFM1-release. MAD2, TSG101, and AIF-PML-DAXX axis regulate mitochondria, PML nuclear bodies (NBs), and autophagy with close inter-dependent protein stability in survival cells. Loss of C-terminal phosphorylation(s) of TSG101 and closed (C-)MAD2-overexpression contribute to induce MOID. In survival cells, both MAD2 and TSG101 localize at PML NBs in interphase, and TSG101 Y390 phosphorylation is required for localization of TSG101 to PML NBs. PML release from PML NBs through PML deSUMOylation contributes to induce MOID. The post-transcriptional/translational cell death machinery and the non-canonical transcriptional regulation are intricately linked to MOID, and ER-MAM, may serve as a crucial intersection for MOID signaling.

Direct reprogramming of fibroblasts into spiral ganglion neurons by defined transcription factors

AbstractDegeneration of the cochlear spiral ganglion neurons (SGNs) is one of the major causes of sensorineural hearing loss and significantly impacts the outcomes of cochlear implantation. Functional regeneration of SGNs holds great promise for treating sensorineural hearing loss. In this study, we systematically screened 33 transcriptional regulators implicated in neuronal and SGN fate. Using gene expression array and principal component analyses, we identified a sequential combination of Ascl1, Pou4f1 and Myt1l (APM) in promoting functional reprogramming of SGNs. The neurons induced by APM expressed mature neuronal and SGN lineage‐specific markers, displayed mature SGN‐like electrophysiological characteristics and exhibited single‐cell transcriptomes resembling the endogenous SGNs. Thus, transcription factors APM may serve as novel candidates for direct reprogramming of SGNs and hearing recovery due to SGN damages.

Light‐Activated Gene Expression System Using a Caging‐Group‐Free Photoactivatable Dye

AbstractOptical regulation of transcription using chemical compounds is an effective strategy to manipulate gene expression spatiotemporally. Conventional caging approaches with photoremovable protecting groups may require intense UV‐light exposure and release potentially toxic byproducts. To address these problems, here we developed a light‐mediated transcriptional regulation system by combining a caging‐group‐free photoactivatable dye PaX560 and a multidrug‐binding transcriptional regulator QacR. The cationic dye generated from PaX560 through traceless photoconversion bound QacR and reduced its repressor function, resulting in transcriptional activation. Importantly, this system allowed transcriptional activation with a large dynamic range under mild visible light exposure and simultaneous detection of the state of the photoactivated effector. This module was integrated into the T7 RNA polymerase expression system to demonstrate light‐activated transcription in vitro and in living cells.

Phytochrome-interacting factors PIF4 and PIF5 directly regulate autophagy during leaf senescence in Arabidopsis.

During leaf senescence, autophagy plays a critical role by removing damaged cellular components and participating in nutrient remobilization to sink organs. However, how AUTOPHGAY (ATG) genes are regulated during natural leaf senescence remains largely unknown. In this study, we attempted to identify upstream transcriptional regulator(s) of ATGs and their molecular basis during leaf senescence in Arabidopsis through the combined analyses of promoter binding, autophagy flux, and genetic interactions. We found that PIF4 and PIF5 (PIF4/PIF5) directly bind to the promoters of the ATG5, ATG12a, ATG12b, ATG8a, ATG8e, ATG8f, and ATG8g, inducing their transcription. These target ATGs are downregulated in pif4, pif5, and pif4pif5 mutants, resulting in decreased autophagic activity and slower degradation of chloroplast proteins and chlorophyll. Conversely, overexpression of ATG8s accelerated protein degradation with early leaf senescence. Moreover, our data suggests partial suppression of the pif4pif5 phenotype by ATG8a overexpression. PIF4/PIF5 also influences senescence induced by nutrient starvation, another hallmark of the autophagy pathway. Furthermore, we observed that the PIF4/PIF5-ATG regulatory module may contribute to seed maturation. Our study not only unveils transcriptional regulators of autophagy in natural leaf senescence but also underscores the potential role of PIF4/PIF5 as functional regulators in leaf senescence and nutrient remobilization.

EZH2 inhibition sensitizes retinoic acid-driven senescence in synovial sarcoma.

Synovial sarcoma (SS) is driven by a unique t(18;X) chromosomal translocation resulting in expression of the SS18-SSX fusion oncoprotein, a transcriptional regulator with both activating and repressing functions. However, the manner in which SS18-SSX contributes to the development of SS is not entirely known. Here, we show that SS18-SSX drives the expression of Preferentially Expressed Antigen in Melanoma (PRAME), which is highly expressed in SS but whose function remains poorly understood. The fusion protein directly binds and activates the PRAME promoter and we found that expression of SS18-SSX and PRAME are positively correlated. We provide evidence that PRAME modulates retinoic acid (RA) signaling, forming a ternary complex with the RA receptor α (RARα) and the Enhancer of Zeste Homolog 2 (EZH2). Knockdown of PRAME suppressed the response to all-trans retinoic acid (ATRA) supporting PRAME's role in modulating RA-signaling. Notably, we demonstrate that combined pharmacological inhibition of EZH2 and treatment with ATRA reconstituted RA signaling followed by reduced proliferation and induction of cellular senescence. In conclusion, our data provides new insights on the role of the SS18-SSX fusion protein in regulation of PRAME expression and RA signaling, highlighting the therapeutic potential of disrupting the RARα-PRAME-EZH2 complex in SS. Schematic presentation of the proposed model. A The RARα-PRAME-EZH2 ternary complex in SS. The fusion SS18-SSX oncoprotein binds to the PRAME promoter and activates its expression. PRAME in turn interacts with RARα-RXR heterodimers as well as with EZH2, and the complex binds to retinoic acid response elements (RAREs) in the DNA. This results in transcriptional repression of retinoic acid (RA) responsive genes and thus inhibition of RA-signaling, allowing tumor cell proliferation. B Therapeutic strategy. Treatment with an EZH2 inhibitor, such as GSK343, or activation of RAR receptors via all-trans retinoic acid (ATRA), disrupts the RARα-PRAME-EZH2 ternary complex and restores RA-signaling. Exposure to GSK343 or ATRA results in inhibition of cell proliferation and induction of cellular senescence, where GSK343 shows a dominant effect. The Figure was created with Biorender.com.

LPS-induced neuroinflammation induces changes in the transcriptional profile of members of the CoRest repressive complex in the hippocampus.

The action of Corepressor for Element Silencing Transcription Factor 1 (CoREST) is primarily related to neural fate decisions. However, the molecular mechanisms linking neuroinflammation to the histone modifying complex remain unclear. CoREST is a hub for several cofactors that play important roles in epigenetic remodeling and transcriptional regulation. It allows us to question their functions during the inflammatory response in the Central Nervous System. The impact of LPS-induced neuroinflammation on the transcriptional epigenetic control of members with CoRest repressive complex in the hippocampus was investigated. Characterizing the basal transcriptional profile in the hippocampus of members with CoREST repressive complex showed that the Rcor3 is the most expressed gene, and the Rcor2 is the least expressed one. It was also demonstrated that the levels of Lsd1, CoREST1 (Rcor1), and CoREST2 (Rcor2) transcripts increased in the hippocampus after LPS i.p. administration, while for CoREST3 (Rcor3), no significant difference was observed. A significant increase was noticed in the percentages of the 5-meC mark (Hypermethylation) for the Rcor1 and Lsd1 genes with a positive Pearson correlation between methylation and expression. However, the correlation was directly proportional, ruling out DNA methylation as the main mechanism in transcriptional control.

Integration of Single-Cell and Spatial Transcriptomic Data Reveals Spatial Architecture and Potential Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by the gradual loss of neurons and the accumulation of amyloid plaques and neurofibrillary tangles. Despite advancements in the understanding of AD's pathophysiology, the cellular organization and interactions in the prefrontal cortex (PFC) remain elusive. Eight single-cell RNA sequencing (scRNA-seq) datasets from both normal controls and individuals with AD were harmonized. Stringent preprocessing protocols were implemented to uphold dataset integrity. Unsupervised clustering and annotation revealed 22 distinct cell clusters corresponding to 19 unique cell types. The spatial architecture of the PFC region was constructed using the CARD tool. Further analyses encompassed trajectory examination of Oligodendrocyte subtypes, evaluation of regulon activity scores, and spot clustering within white matter regions (WM). Differential expression analysis and functional enrichment assays unveiled molecular signatures linked to AD progression and were validated using microarray data sourced from neurodegenerative disorder patients. Our investigation employs scRNA-seq and spatial transcriptomics to uncover the cellular atlas and spatial architecture of the human PFC in AD. Moreover, our results indicate that Oligodendrocytes are more prevalent in AD patients, showcasing diverse subtypes and spatial organization within WM regions. Each subtype appears to be associated with distinct biological processes and transcriptional regulators, shedding light on their involvement in AD pathology. Notably, the Oligodendrocyte_C6 subtype is linked to neurological damage in AD patients, characterized by heightened expression of genes involved in cell-cell connections, cell membrane stability, and myelination. Additionally, 12 target genes regulated by NFIA were identified, which are upregulated in AD patients and associated with disease progression. Elevated PLXDC2 expression in peripheral blood was also identified, suggesting its potential as a non-invasive biomarker for early AD detection. Our study provides novel insights into the role of Oligodendrocytes in AD and highlights the potential of PLXDC2 as a blood biomarker for non-invasive diagnosis and monitoring of AD patients.

Transcription factor PbrERF114 is involved in the regulation of ethylene synthesis during pear fruit ripening.

The plant hormone ethylene is indispensable to the ripening of climacteric fruits. Although extensive studies have been conducted on ethylene signaling, the ethylene response factor (ERF)-mediated transcriptional regulation of ethylene biosynthesis in pear fruits remains to be fully elucidated. We here constructed, sequenced, and analyzed transcriptome libraries in ethephon-treated and 1-MCP-treated Cuiguan pear fruits. In total, 721 fruit ripening-associated differentially expressed genes were identified. Among them, two key genes exhibited positive correlations: the 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-encoding gene PbrACS3 and the ERF-encoding gene named PbrERF114. PbrERF114 overexpression increased ethylene production as well as the PbrACS3 expression level. Conversely, virus-induced gene silencing downregulated PbrERF114, thereby decreasing ethylene production and reducing PbrACS3 expression levels. Notably, PbrERF114 could directly interact with PbrACS3 and PbrERF24 promoters, thus inducing their expression. However, it did not result in an enhancement in luciferase activity, which is regulated by the PbrACS1b or PbrACO1 promoter. PbrERF24 could directly bind to PbrACO1 as well as PbrACS3 to promote ethylene synthesis. In conclusion, PbrERF114 can directly and indirectly mediate ethylene biosynthesis by transcriptionally regulating PbrACS3 and PbrERF24, respectively, thereby triggering a signaling cascade that induces the expression of both PbrACS3 and PbrACO1.

Transcriptional Responses of Lacticaseibacillus rhamnosus to TNFα, IL-6, IL-8, and IL-10 Cytokines

The interaction between gut microbiota and the host immune system is a complex and understudied field, with cytokines like TNFα, IL-6, IL-8, and IL-10 playing pivotal roles. Commensal bacteria, including lactobacilli, respond to these cytokines through adaptive mechanisms that support their survival and function within the gut. While the influence of cytokines on pathogenic bacteria is well documented, their impact on commensal bacteria, particularly lactobacilli, remains underexplored. This study investigates the transcriptional responses of Lacticaseibacillus rhamnosus strains K32 and R19-3 to various cytokines using next-generation RNA sequencing (RNA-seq). Our findings reveal that cytokines, especially IL-8 and IL-10, significantly alter the L. rhamnosus transcriptome, affecting genes involved in carbohydrate metabolism, stress response, and transcriptional regulation. Notably, IL-8 and IL-10 induce a significant downregulation of genes related to the phosphotransferase system, suggesting a reduction in metabolic activity in response to inflammatory signals. This study unveils a previously unexplored aspect of L. rhamnosus adaptation, highlighting its intricate response to cytokine signals. By modulating gene expression, L. rhamnosus may mitigate the adverse effects of inflammation and promote gut health. These insights could inform the development of targeted probiotic therapies for inflammatory bowel disease (IBD) and other conditions with altered cytokine levels. Our results suggest that co-evolution between a host and gut microbiota enables bacteria to respond to specific cytokines through gene expression changes, revealing a unique and underexplored facet of the interaction between commensal bacteria and the host organism.

CDH2 and CDH13 as potential prognostic and therapeutic targets for adrenocortical carcinoma

ABSTRACT Cadherin 2 (CDH2, N-cadherin) and cadherin 13 (CDH13, T-cadherin, H-cadherin) affect the progress and prognoses of many cancers. However, their roles in adrenocortical carcinoma (ACC), a rare endocrine cancer, remain unclear. To decipher the roles of these proteins in ACC and to identify their regulatory targets, we analyzed their expression levels, gene regulatory networks, prognostic value, and targets in ACC, using various bioinformatic analyses. CDH2 was strongly downregulated and CDH13 was strongly upregulated in patients with ACC; the expression levels of these genes affected the prognosis. In 75 patients, the expression of CDH2 and CDH13 was altered by 8% and 5%, respectively. CDH2 and CDH13, as well as their neighboring genes, were predicted to form a complex network of interactions, mainly through coexpression and physical and genetic interactions. CDH2 and its altered neighboring genes (ANGs) mainly affect tumor-related gene expression, cell cycle, and energy metabolism. The regulation of tumor-related integrin function, gene transcription, metabolism, and amide and phospholipid metabolism are the main functions of CDH13 and its ANGs. MiRNA and kinase targets of CDH2 and CDH13 in ACC were identified. CDH13 expression in patients with ACC was positively associated with immune cell infiltration. Anti-PD1/CTLA-4/PD-L1 immunotherapy significantly downregulated the expression of CDH13 in patients with ACC. Foretinib and elesclomol were predicted to exert strong inhibitory effects on SW13 cells by inhibiting the expression of CDH2 and CDH13. These data indicate that CDH2 and CDH13 are promising targets for precise treatment of ACC and may serve as new biomarkers for ACC prognosis.

Time-dependent changes in genome-wide gene expression and post-transcriptional regulation across the post death process in silkworm.

Despite death marking the end of life, several gene expression and miRNA-mediated post-transcriptional regulation events may persist or be initiated. The silkworm (Bombyx mori) is a valuable model for exploring life processes, including death. In this study, we combined transcriptomics and miRNAomics analyses of young, old, and postmortem silkworms across the entire process after death to unravel the dynamics of gene expression and miRNA-mediated post-transcriptional regulation. In total, 171 genes exhibited sustained differential expression in postmortem silkworms compared to the pre-death state, which are primarily involved in nerve signaling, transport, and immune response. Postmortem time-specific genes were associated with cell cycle regulation, thermogenesis, immunity, and zinc ion homeostasis. We found that the down-regulated expression of 36 genes related to transcription, epigenetic modification, and homeostasis resulted in a significant shift in global gene expression patterns at 2h post-death. We also identified five mRNA-miRNA pairs (i.e., bmo-miR-2795-mhca, 2784-achi, 2762-oa1, 277-5p-creb, and 1000-tcb1) associated with stress hormone regulation, transcription activity, and signal transduction. The roles of these pairs were validated through in vivo experiments using miRNA mimics in silkworms. The findings provide valuable insights into the intricate mechanisms underlying the transcriptional and miRNA-mediated post-transcriptional regulation events in animals after death.

MCRS1 sensitizes T cell-dependent immunotherapy by augmenting MHC-I expression in solid tumors.

Dampened antigen presentation underscores the resistance of pancreatic cancer to T cell-mediated anti-tumor immunity, rendering immunotherapy largely ineffective. By high-throughput CRISPR activation perturbation, we discovered that the transcriptional regulator MCRS1 significantly augmented the sensitivity of mouse pancreatic cancer cells to T cell immunity in vitro and in vivo. Mechanistically, MCRS1 interacted with the transcription factor and genome organizer YY1 to coordinately increase the chromatin accessibility and expression of MHC-I genes. Elevated MCRS1 subverted MHC-I suppression and activated anti-tumor T cells, which sensitized mouse pancreatic cancer to α-PD-1 therapy. Remarkably, high MCRS1 expression was associated with increased T cell infiltration and extended survival of patients with pancreatic cancer and was predictive of favorable responses to α-PD-1 therapy in patients with lung cancer. Together, our study uncovers that MCRS1 sensitizes cancer cells to T cell immunity by transcriptionally subverting MHC-I suppression, which enhances the effectiveness of α-PD-1 therapy in mice and humans, paving the way to further improve immunotherapy against solid tumors.

The effects of PstR, a PadR family transcriptional regulatory factor, in Plesiomonas shigelloides are revealed by transcriptomics.

Plesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors. In this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides' capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides. Overall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria.

Maleylpyruvic Acid-Inducible Gene Expression System and Its Application for the Development of Gentisic Acid Biosensors.

Gentisic acid is a secondary plant metabolite, known for its health benefits, not only widely used as a supplement but also implicated as a potential biomarker for cancer-associated metabolism alterations. To advance bioproduction and detection of this compound or its derivatives, cell-based approaches have become of interest in recent years. However, the lack of tools for high-throughput gentisic acid monitoring and compound-metabolizing organism screening limits the progress in this area. Here, we analyzed the gene cluster responsible for gentisic acid metabolism in Cupriavidus necator H16. The transcriptional regulator GtdR-based inducible gene expression system CnGtdR/PgtdA was elucidated, showing that it was activated when C. necator cells were subjected to gentisic acid. Subsequently, a 3-maleylpyruvic acid was identified as a primary inducer for this inducible system. Furthermore, genes gtdA and gtdT, encoding for gentisate 1,2-dioxygenase and MFS transporter, were shown to be essential for inducible system activation in the presence of gentisic acid with GtdA enabling conversion of this phenolic acid into the inducer. The CnGtdRAT/PgtdA-based inducible system was employed to develop a whole-cell biosensor for the intracellular and extracellular detection of gentisic acid. The potential of the 3-maleylpyruvic acid-inducible system was demonstrated by its application in metabolic pathway research, detection of highly unstable 3-maleylpyruvic acid, and development of biosensors for the intracellular or extracellular determination of gentisic acid. In addition, the utility of the biosensor was emphasized by its application for detection of gentisic acid as a potential biomarker for cancer in urine samples.

RAEPI: Predicting Enhancer-Promoter Interactions Based on Restricted Attention Mechanism.

Enhancer-promoter interactions (EPIs) are crucial in gene transcription regulation and cell differentiation. Traditional biological experiments are costly and time-consuming, motivating the development of computational prediction methods. However, existing EPI prediction methods inadequately capture the intricate direct interactions between enhancer and promoter sequences, which limits their prediction performance to some extent. In this work, we propose an innovative attention-based approach RAEPI, which uses convolutional neural networks to extract initial features of enhancers and promoters, combined with a specially designed Restricted Attention mechanism with Query-Key-Value constrained to simulate the interactions between them for further feature extraction. To improve cross-cell line prediction, we employ a transfer learning strategy for pre-training. Furthermore, we extracted sequence motifs to evaluate the RAEPI's effectiveness from a visualization perspective. Experimental results show that RAEPI achieves competitive prediction performance to existing methods on the benchmark dataset.

Identification of two transcription factors that work coordinately to regulate early development in Entamoeba.

The protozoan parasite Entamoeba has a life cycle that switches between infective cysts and invasive trophozoites. Encystation, a crucial process in parasite biology, is controlled by different mechanisms including transcriptional control. We identified two nuclear proteins in Entamoeba invadens, EIN_066100 and EIN_085620, that regulate parasite development by binding to a DNA motif (TCACTTTC) in the promoter regions of genes upregulated in the first 8 h of stage conversion. Overexpression of EIN_066100, a homolog of MAK16 protein, resulted in reduced amoebic proliferation without affecting encystation efficiency. Overexpression of EIN_085620, a protein with an RNA-recognition motif (RRM), led to increased encystation efficiency. Glutathione S-transferase (GST) pull down assays revealed that EIN_066100 interacts with EIN_085620 both in vivo and in vitro, and this interaction is mediated by the EIN_085620 RRM domain. By evaluating truncated proteins with deletions at either the N-terminal or C-terminal regions of EIN_066100, we elucidated the importance of its N-terminal region in proper protein localization, proliferation, encystation, and interaction with EIN_085620. Taken together, these results indicate a coordinated role of EIN_066100 and EIN_085620 in regulating Entamoeba development. This work sheds light on the molecular mechanisms in the earliest stages of Entamoeba encystation.IMPORTANCEAn important biological process in the biology of Entamoeba is stage conversion, which plays a crucial role in disease propagation, facilitating parasite survival outside the host and spreading to new hosts. Multiple mechanisms contribute to controlling the expression of amebic stage-specific genes such as epigenetic and transcriptional control. Identification of early transcriptional control regulators is crucial to understanding the initiation of the encystation cascade. We identified two nuclear proteins, EIN_066100 and EIN_085620, involved in the proliferation and developmental regulation of E. invadens. These proteins work by direct binding to each other and mediating encystation efficiency. Study of new regulators involved in Entamoeba development represents an important advance in a critical aspect of parasite biology.

MicroRNA 223 Enhances ABCA1 Protein Stability and Supports Efflux in Cholesterol-Burdened Macrophages.

Macrophages are present in all vertebrates as part of the innate immune system, which protects from pathogens and scavenges sterol rich, cellular debris and modified lipoproteins. Thus, resident macrophages are prone to excessive levels of intracellular cholesterol esters. Intramacrophage cholesterol esters can efflux via cell surface transporters, ABCA1 and ABCG1, to lipoprotein carriers such as apo-AI and HDL. Systemically, Apo-AI and HDL facilitate trafficking of cholesterol back to the liver, in a process called reverse cholesterol transport. Impaired macrophage cholesterol efflux is a primary factor in the etiology of atherosclerosis. We hypothesized that microRNA 223 (miR-223) regulated macrophage LDL metabolism, due to predicted binding to Sp1 and Sp3 mRNA, transcriptional regulators of ABCA1 expression. Primary mouse (WT, miR-223 KO) macrophages were loaded with acetylated LDL and stimulated with LPS to form an inflammatory foam cell phenotype. miR-223 KO foam cells demonstrated impaired efflux to both apo-AI and HDL. While transcriptional regulation was intact in miR-223 KO foam cells, ABCA1 protein degradation was greatly accelerated. Blockade of both proteasomal and lysosomal degradation pathways rescued miR-223 deficiency-mediated ABCA1 degradation to the WT levels. Our findings demonstrate that miR-223 expression in macrophages is required for maintenance of ABCA1 and ABCG1 proteins.