Combined RNA Sequencing Research Articles

M5C related-regulator-mediated methylation modification patterns and prognostic significance in breast cancer

5-Methylcytosine (m5C) is closely associated with cancer. However, the role of m5C in breast cancer(BC)remains unclear. This study combined single-cell RNA sequencing (scRNA-Seq) and transcriptomics datasets to screen m5C regulators associated with BC progression and analyze their clinical values. Firstly, This study elucidates the mechanisms of the m5C landscape and the specific roles of m5C regulators in BC patients. we found that the dysregulation of m5C regulators with m5Cscore play the essential role of the carcinogenesis and progression in epithelial cells and myeloid cells of BC at single cell level. External validation was conducted using an independent scRNA-Seq datasets. Then, three distinct m5C modification patterns were identified by transcriptomics datasets. Based on the m5C differentially expressed regulators, the m5Cscore was constructed, and used to divide patients with BC into high and low m5Cscore groups. Patients with a high m5Cscore had more abundant immune cell infiltration, stronger antitumor immunity, and better prognoses. Finally, Quantitative real-time (PCR) and immunohistochemistry were used for the in vitro experimental validation, which had extensive prognostic value. In this study, we aimed to assess the expression of m5C regulators involved in BC and investigate their correlation with the tumor microenvironment, clinicopathological characteristics, and prognosis of BC. The m5C regulators could be used to effectively assess the cell specific regulation prognosis of patients with BC and develop more effective immunotherapy strategies.

Decoding the multiple functions of ZBP1 in the mechanism of sepsis-induced acute lung injury.

Sepsis-induced acute lung injury (ALI), characterized by severe hypoxemia and pulmonary leakage, remains a leading cause of mortality in intensive care units. The exacerbation of ALI during sepsis is largely attributed to uncontrolled inflammatory responses and endothelial dysfunction. Emerging evidence suggests an important role of Z-DNA binding protein 1 (ZBP1) as a sensor in innate immune to drive inflammatory signaling and cell death during infections. However, the role of ZBP1 in sepsis-induced ALI has yet to be defined. We utilized ZBP1 knockout mice and combined single-cell RNA sequencing with experimental validation to investigate ZBP1's roles in the regulation of macrophages and lung endothelial cells during sepsis. We demonstrate that in sepsis, ZBP1 deficiency in macrophages reduces mitochondrial damage and inhibits glycolysis, thereby altering the metabolic status of macrophages. Consequently, this metabolic shift leads to a reduction in the differentiation of macrophages into pro-inflammatory states and decreases macrophage pyroptosis triggered by activation of the NLRP3 inflammasome. These changes significantly weaken the inflammatory signaling pathways between macrophages and endothelial cells and alleviate endothelial dysfunction and cellular damage. These findings reveal important roles for ZBP1 in mediating multiple pathological processes involved in sepsis-induced ALI by modulating the functional states of macrophages and endothelial cells, thereby highlighting its potential as a promising therapeutic target.

Neuregulin1 Nuclear Signaling Influences Adult Neurogenesis and Regulates a Schizophrenia Susceptibility Gene Network within the Mouse Dentate Gyrus.

Neuregulin1 (Nrg1) signaling is critical for neuronal development and function from fate specification to synaptic plasticity. Type III Nrg1 is a synaptic protein which engages in bidirectional signaling with its receptor ErbB4. Forward signaling engages ErbB4 phosphorylation, whereas back signaling engages two known mechanisms: (1) local axonal PI3K-AKT signaling and (2) cleavage by γ-secretase resulting in cytosolic release of the intracellular domain (ICD), which can traffic to the nucleus (Bao et al., 2003; Hancock et al., 2008). To dissect the contribution of these alternate signaling strategies to neuronal development, we generated a transgenic mouse with a missense mutation (V321L) in the Nrg1 transmembrane domain that disrupts nuclear back signaling with minimal effects on forward signaling or local back signaling and was previously found to be associated with psychosis (Walss-Bass et al., 2006). We combined RNA sequencing, retroviral fate mapping of neural stem cells, behavioral analyses, and various network analyses of transcriptomic data to investigate the effect of disrupting Nrg1 nuclear back signaling in the dentate gyrus (DG) of male and female mice. The V321L mutation impairs nuclear translocation of the Nrg1 ICD and alters gene expression in the DG. V321L mice show reduced stem cell proliferation, altered cell cycle dynamics, fate specification defects, and dendritic dysmorphogenesis. Orthologs of known schizophrenia (SCZ)-susceptibility genes were dysregulated in the V321L DG. These genes coordinated a larger network with other dysregulated genes. Weighted gene correlation network analysis and protein interaction network analyses revealed striking similarity between DG transcriptomes of V321L mouse and humans with SCZ.

A novel mechanism of ferroptosis inhibition-enhanced atherosclerotic plaque stability: YAP1 suppresses vascular smooth muscle cell ferroptosis through GLS1.

Atherosclerosis is a leading cause of cardiovascular diseases (CVDs), often resulting in major adverse cardiovascular events (MACEs), such as myocardial infarction and stroke due to the rupture or erosion of vulnerable plaques. Ferroptosis, an iron-dependent form of cell death, has been implicated in the development of atherosclerosis. Despite its involvement in CVDs, the specific role of ferroptosis in atherosclerotic plaque stability remains unclear. In this study, we confirmed the presence of ferroptosis in unstable atherosclerotic plaques and demonstrated that the ferroptosis inhibitor ferrostatin-1 (Fer-1) stabilizes atherosclerotic plaques in apolipoprotein E knockout (Apoe-/-) mice. Using bioinformatic analysis combining RNA sequencing (RNA-seq) with single-cell RNA sequencing (scRNA-seq), we identified Yes-associated protein 1 (YAP1) as a potential key regulator of ferroptosis in vascular smooth muscle cells (VSMCs) of unstable plaques. Invitro, we found that YAP1 protects against oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in VSMCs. Mechanistically, YAP1 exerts its anti-ferroptosis effects by regulating the expression of glutaminase 1 (GLS1) to promote the synthesis of glutamate (Glu) and glutathione (GSH). These findings establish a novel mechanism where the inhibition of ferroptosis promotes the stabilization of atherosclerotic plaques through the YAP1/GLS1 axis, attenuating VSMC ferroptosis. Thus, targeting the YAP1/GLS1 axis to suppress VSMC ferroptosis may represent a novel strategy for preventing and treating unstable atherosclerotic plaques.

Role of Peptidyl Arginine Deiminase 4-Dependent Macrophage Extracellular Trap Formation in Type 1 Diabetes Pathogenesis.

Excessive macrophage extracellular traps (METs) formation has been implicated in several autoimmune disease pathogenesis; however, its impact on Type 1 Diabetes (T1D) and related mechanisms remains enigmatic. Here, we demonstrated the pivotal role of peptidyl arginine deiminase 4 (PAD4) in driving profuse METs formation and macrophage M1 polarization in intestinal inflammation of non-obese diabetic (NOD) mice. Genetic knockout of PAD4 or adoptive transfer of METs alters the proportion of pro-inflammatory T cells in the intestine, subsequently influencing their migration to the pancreas. Combining RNA sequencing and CUT&Tag analysis we found activated PAD4 transcriptionally regulated CXCL10 expression. This study comprehensively investigated how excessive PAD4-mediated METs formation in the colon increases the aggravation of intestinal inflammation and pro-inflammatory T cells migration, and finally involves T1D progression, suggesting that inhibition METs formation may be a potential therapeutic target for T1D.

Single-cell RNA and T-cell receptor sequencing unveil mycosis fungoides heterogeneity and a possible gene signature.

Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). Comprehensive analysis of MF cells in situ and ex vivo is complicated by the fact that is challenging to distinguish malignant from reactive T cells with certainty. To overcome this limitation, we performed combined single-cell RNA (scRNAseq) and T-cell receptor TCR sequencing (scTCRseq) of skin lesions of cutaneous MF lesions from 12 patients. A sufficient quantity of living T cells was obtained from 9 patients, but 2 had to be excluded due to unclear diagnoses (coexisting CLL or revision to a fixed toxic drug eruption). From the remaining patients we established single-cell mRNA expression profiles and the corresponding TCR repertoire of 18,630 T cells. TCR clonality unequivocally identified 13,592 malignant T cells. Reactive T cells of all patients clustered together, while malignant cells of each patient formed a unique cluster expressing genes typical of naive/memory, such as CD27, CCR7 and IL7R, or cytotoxic T cells, e.g., GZMA, NKG7 and GNLY. Genes encoding classic CTCL markers were not detected in all clusters, consistent with the fact that mRNA expression does not correlate linearly with protein expression. Nevertheless, we successfully pinpointed distinctive gene signatures differentiating reactive malignant from malignant T cells: keratins (KRT81, KRT86), galectins (LGALS1, LGALS3) and S100 genes (S100A4, S100A6) being overexpressed in malignant cells. Combined scRNAseq and scTCRseq not only allows unambiguous identification of MF cells, but also revealed marked heterogeneity between and within patients with unexpected functional phenotypes. While the correlation between mRNA and protein abundance was limited with respect to established MF markers, we were able to identify a single-cell gene expression signature that distinguishes malignant from reactive T cells.

Spatial and temporal gene expression patterns during early human odontogenesis process.

Studies on odontogenesis are of great importance to treat dental abnormalities and tooth loss. However, the odontogenesis process was poorly studied in humans, especially at the early developmental stages. Here, we combined RNA sequencing (RNA-seq) with Laser-capture microdissection (LCM) to establish a spatiotemporal transcriptomic investigation for human deciduous tooth germs at the crucial developmental stage to offer new perspectives to understand tooth development and instruct tooth regeneration. Several hallmark events, including angiogenesis, ossification, axonogenesis, and extracellular matrix (ECM) organization, were identified during odontogenesis in human dental epithelium and mesenchyme from the cap stage to the early bell stage. ECM played an essential role in the shift of tooth-inductive capability. Species comparisons demonstrated these hallmark events both in humans and mice. This study reveals the hallmark events during odontogenesis, enriching the transcriptomic research on human tooth development at the early stage.

Arabidopsis WRKY1 promotes monocarpic senescence by integrative regulation of flowering, leaf senescence, and nitrogen remobilization

Monocarpic senescence, characterized by whole-plant senescence following a single flowering phase, is widespread in seed plants, particularly in crops, determining seed harvest time and quality. However, how external and internal signals are systemically integrated into monocarpic senescence remains largely unknown. Here, we report that the Arabidopsis thaliana transcription factor WRKY1 plays essential roles in multiple key steps of monocarpic senescence. WRKY1 expression is induced by age, salicylic acid (SA), and nitrogen (N) deficiency. Flowering and leaf senescence are accelerated in the WRKY1 overexpression lines but are delayed in the wrky1 mutants. The combined DNA affinity purification sequencing and RNA sequencing analyses uncover the direct target genes of WRKY1. Further studies show that WRKY1 coordinately regulates three processes in monocarpic senescence: (1) suppressing FLOWERING LOCUS C gene expression to initiate flowering, (2) inducing SA biosynthesis genes to promote leaf senescence, and (3) activating the N assimilation and transport genes to trigger N remobilization. In summary, our study reveals how one stress-responsive transcription factor, WRKY1, integrates flowering, leaf senescence, and N remobilization processes into monocarpic senescence, providing important insights into plant lifetime regulation.

CrossMP: Enabling Cross-Modality Translation between Single-Cell RNA-Seq and Single-Cell ATAC-Seq through Web-Based Portal.

In recent years, there has been a growing interest in profiling multiomic modalities within individual cells simultaneously. One such example is integrating combined single-cell RNA sequencing (scRNA-seq) data and single-cell transposase-accessible chromatin sequencing (scATAC-seq) data. Integrated analysis of diverse modalities has helped researchers make more accurate predictions and gain a more comprehensive understanding than with single-modality analysis. However, generating such multimodal data is technically challenging and expensive, leading to limited availability of single-cell co-assay data. Here, we propose a model for cross-modal prediction between the transcriptome and chromatin profiles in single cells. Our model is based on a deep neural network architecture that learns the latent representations from the source modality and then predicts the target modality. It demonstrates reliable performance in accurately translating between these modalities across multiple paired human scATAC-seq and scRNA-seq datasets. Additionally, we developed CrossMP, a web-based portal allowing researchers to upload their single-cell modality data through an interactive web interface and predict the other type of modality data, using high-performance computing resources plugged at the backend.

M6A mRNA methylation-mediated MAPK signaling modulates the nasal mucosa inflammatory response in allergic rhinitis.

Allergic rhinitis (AR) is a complex disease in which gene-environment interactions contribute to its pathogenesis. Epigenetic modifications, such as N6-methyladenosine (m6A) modification of mRNA, play important roles in regulating gene expression in multiple physiological and pathological processes. However, the function of m6A modification in AR and the inflammatory response is poorly understood. We used the ovalbumin (OVA) and aluminum hydroxide to induce an AR mouse model. Nasal symptoms, histopathology, and serum cytokines were examined. We performed combined m6A and RNA sequencing to analyze changes in m6A modification profiles. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and methylated RNA immunoprecipitation sequencing qPCR (MeRIP-qPCR) were used to verify differential methylation of mRNAs and the m6A methylation level. Knockdown or inhibition of Alkbh5 in nasal mucosa of mice was mediated by lentiviral infection or IOX1 treatment. We showed that m6A was enriched in a group of genes involved in MAPK signaling pathway. Moreover, we identified a MAPK pathway involving Map3k8, Erk2, and Nfκb1 that may play a role in the disrupted inflammatory response associated with nasal inflammation. The m6A eraser, Alkbh5, was highly expressed in the nasal mucosa of AR model mice. Furthermore, knockdown of Alkbh5 expression by lentiviral infection resulted in high MAPK pathway activity and a significant nasal mucosa inflammatory response. Our findings indicate that ALKBH5-mediated m6A dysregulation likely contributes to a nasal inflammatory response via the MAPK pathway. Together, our data show that m6A dysregulation mediated by ALKBH5, is likely to contribute to inflammation of the nasal mucosa via the MAPK signaling pathway, suggesting that ALKBH5 is a potential biomarker for AR treatment.

PR-SET7 epigenetically restrains uterine interferon response and cell death governing proper postnatal stromal development

The differentiation of the stroma is a hallmark event during postnatal uterine development. However, the spatiotemporal changes that occur during this process and the underlying regulatory mechanisms remain elusive. Here, we comprehensively delineated the dynamic development of the neonatal uterus at single-cell resolution and characterized two distinct stromal subpopulations, inner and outer stroma. Furthermore, single-cell RNA sequencing revealed that uterine ablation of Pr-set7, the sole methyltransferase catalyzing H4K20me1, led to a reduced proportion of the inner stroma due to massive cell death, thus impeding uterine development. By combining RNA sequencing and epigenetic profiling of H4K20me1, we demonstrated that PR-SET7-H4K20me1 either directly repressed the transcription of interferon stimulated genes or indirectly restricted the interferon response via silencing endogenous retroviruses. Declined H4K20me1 level caused viral mimicry responses and ZBP1-mediated apoptosis and necroptosis in stromal cells. Collectively, our study provides insight into the epigenetic machinery governing postnatal uterine stromal development mediated by PR-SET7.

The jasmonate pathway promotes nodule symbiosis and suppresses host plant defense in Medicago truncatula

Root nodule symbiosis (RNS) between legumes and rhizobia is a major source of nitrogen in agricultural systems. Effective symbiosis requires precise regulation of plant defense responses. The role of the defense hormone jasmonic acid (JA) in the immune response has been extensively studied. Current research shows that JA can play either a positive or negative regulatory role in RNS depending on its concentration, but the molecular mechanisms remain to be elucidated. In this study, we found that inoculation with the rhizobia Sm1021 induces the JA pathway in Medicago truncatula, and blocking the JA pathway significantly reduces the number of infection threads. Mutations in the MtMYC2 gene, which encodes a JA signaling master transcription factor, significantly inhibited rhizobia infection, terminal differentiation, and symbiotic cell formation. Combining RNA sequencing and chromatin immunoprecipitation sequencing, we discovered that MtMYC2 regulates the expression of nodule-specific MtDNF2, MtNAD1, and MtSymCRK to suppress host defense, while it activates MtDNF1 expression to regulate the maturation of MtNCRs, which in turn promotes bacteroid formation. More importantly, MtMYC2 participates in symbiotic signal transduction by promoting the expression of MtIPD3. Notably, the MtMYC2-MtIPD3 transcriptional regulatory module is specifically present in legumes, and the Mtmyc2 mutants are susceptible to the infection by the pathogen Rhizoctonia solani. Collectively, these findings reveal the molecular mechanisms of how the JA pathway regulates RNS, broadening our understanding of the roles of JA in plant-microbe interactions.

Neoantigen landscape supports feasibility of personalized cancer vaccine for follicular lymphoma

AbstractPersonalized cancer vaccines designed to target neoantigens represent a promising new treatment paradigm in oncology. In contrast to classical idiotype vaccines, we hypothesized that “polyvalent” vaccines could be engineered for the personalized treatment of follicular lymphoma (FL) using neoantigen discovery by combined whole-exome sequencing (WES) and RNA sequencing (RNA-seq). Fifty-eight tumor samples from 57 patients with FL underwent WES and RNA-seq. Somatic and B-cell clonotype neoantigens were predicted and filtered to identify high-quality neoantigens. B-cell clonality was determined by the alignment of B-cell receptor (BCR) CDR3 regions from RNA-seq data, grouping at the protein level, and comparison with the BCR repertoire from healthy individuals using RNA-seq data. An average of 52 somatic mutations per patient (range, 2-172) were identified, and ≥2 (median, 15) high-quality neoantigens were predicted for 56 of 58 FL samples. The predicted neoantigen peptides were composed of missense mutations (77%), indels (9%), gene fusions (3%), and BCR sequences (11%). Building off of these preclinical analyses, we initiated a pilot clinical trial using personalized neoantigen vaccination combined with PD-1 blockade in patients with relapsed or refractory FL (#NCT03121677). Synthetic long peptide vaccines targeting predicted high-quality neoantigens were successfully synthesized for and administered to all 4 patients enrolled. Initial results demonstrate feasibility, safety, and potential immunologic and clinical responses. Our study suggests that a genomics-driven personalized cancer vaccine strategy is feasible for patients with FL, and this may overcome prior challenges in the field. This trial was registered at www.ClinicalTrials.gov as #NCT03121677.

Identification of potential candidate genes associated with milk protein differences in Holstein cows: a meta-analysis integrating GWAS and RNA-Seq transcriptome

Despite the identification of candidate genes influencing milk protein, the connections between genes and regulatory pathways remains elusive. This study aimed integrate findings from genome-wide association studies (GWAS) and RNA sequencing (RNA-Seq) through meta-analysis to pinpoint single nucleotide polymorphisms (SNPs) and genes responsible for high and low protein yield in cows. Previous GWAS and RNA-Seq analyses had identified 663 SNPs and 1106 genes ( P < 0.05). Twenty SNPs from GWAS, 10 genes from RNA-Seq, and 49 SNP/gene associations from both datasets, were identified using meta-analysis. Meta-analysis validated several SNPs previously identified through GWAS, such as rs135549651 ( P = 2.6 × 10−256), rs109146371 ( P = 3.1 × 10−208), rs109350371 ( P = 4.0 × 10−207), and rs109774038 ( P = 8.6 × 10−587). Genes identified in RNA-Seq experiments, including NR4A1 ( P = 3.2 × 10−7), ATF3 ( P = 9.6 × 10−7), CDH16 ( P = 9.9 × 10−7), VEGFA ( P = 1.0 × 10−6), and SAA3 ( P = 7.3 × 10−11), were confirmed. The combined GWAS and RNA-Seq datasets highlighted CCND2 ( P = 8.9 × 10−111), MAPK15 ( P = 1.3 × 10−151), and CPSF1 ( P = 1.2 × 10−306) as the most significant genes. Additionally, significant gene ontology (GO) terms, including ionizing radiation ( P = 1.5 × 10−4), nuclear pore cytoplasmic filaments ( P = 9.4 × 10−5), and phenylalanine 4-monooxygenase activity ( P = 1.4 × 10−5), were identified. In conclusion, the integration of GWAS and RNA-Seq, coupled with GO enrichment, allowed identification of candidate SNPs and genes with higher accuracy. These findings improve our knowledge about genomic architecture of milk protein and enhance evaluation of Holstein cows.

Activation of ERβ hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor β (ERβ) in TNBC, but the detailed molecular mechanisms downstream ERβ activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERβ agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERβ were critical for the binding between U2AF1 and ERβ. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERβ-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERβ activation in TNBC, which provides rationale for ERβ activation-based single or combined therapy for patients with TNBC.

Insight into the relationship between metabolic enzymes and oxadiazon degradation in Oryza sativa for reducing environmental risks

Oxadiazon (ODZ) is extensively utilized in agricultural fields for weed control owing to its strong effectiveness. However, excessive loading of ODZ in water bodies and agricultural soils can lead to various environmental concerns. Therefore, it is crucial to understand the ODZ metabolic process and associated mechanisms in crops to assess the likelihood of ODZ contamination in the environment. This study aimed to assess the effects of ODZ on the growth and toxicological responses of rice (Oryza sativa). The growth of rice tissues was notably compromised with the increase in ODZ concentrations. RNA sequencing in combination with liquid chromatography–quadrupole-time-of-flight–high-resolution mass spectrometry/mass spectrometry (LC–Q-TOF–HRMS/MS) analysis allowed for the identification of numerous transcriptional components associated with ODZ metabolism. Four libraries comprising rice roots and shoots exposed to ODZ were RNA-sequenced in triplicate. The application of environmentally realistic ODZ concentrations upregulated the expression of 844 genes in shoots and 1476 genes in roots. Gene enrichment analysis revealed the presence of multiple enzymes involved in ODZ metabolism and detoxification. These enzymes play a critical role in mitigating environmental stress and facilitating xenobiotic metabolism. Notably, among differentially expressed genes, several key enzymes were identified, including cytochrome P450s, protein kinases, aminotransferases, and ATP-binding cassette transporters involved in the metabolic process. Using LC–Q-TOF–HRMS/MS, 3 metabolites and 13 conjugates were identified in multiple metabolic pathways involving oxidation, hydrolysis, glycosylation, acetylation, and methylation. This study successfully established a potential link between the specific metabolic products of ODZ and increased activities of their corresponding enzymes. Moreover, this study considerably elucidates the detailed pathways and mechanisms involved in ODZ metabolism. The study findings provide valuable insights into the development of genotypes for reducing ODZ residues in paddy fields and minimizing their accumulation in rice crops.

Transcriptional chronology reveals conserved genes involved in pennate diatom sexual reproduction.

Sexual reproduction is a major driver of adaptation and speciation in eukaryotes. In diatoms, siliceous microalgae with a unique cell size reduction-restitution life cycle and among the world's most prolific primary producers, sex also acts as the main mechanism for cell size restoration through the formation of an expanding auxospore. However, the molecular regulators of the different stages of sexual reproduction and size restoration are poorly explored. Here, we combined RNA sequencing with the assembly of a 55 Mbp reference genome for Cylindrotheca closterium to identify patterns of gene expression during different stages of sexual reproduction. These were compared with a corresponding transcriptomic time series of Seminavis robusta to assess the degree of expression conservation. Integrative orthology analysis revealed 138 one-to-one orthologues that are upregulated during sex in both species, among which 56 genes consistently upregulated during cell pairing and gametogenesis, and 11 genes induced when auxospores are present. Several early, sex-specific transcription factors and B-type cyclins were also upregulated during sex in other pennate and centric diatoms, pointing towards a conserved core regulatory machinery for meiosis and gametogenesis across diatoms. Furthermore, we find molecular evidence that the pheromone-induced cell cycle arrest is short-lived in benthic diatoms, which may be linked to their active mode of mate finding through gliding. Finally, we exploit the temporal resolution of our comparative analysis to report the first marker genes for auxospore identity called AAE1-3 ("Auxospore-Associated Expression"). Altogether, we introduce a multi-species model of the transcriptional dynamics during size restoration in diatoms and highlight conserved gene expression dynamics during different stages of sexual reproduction.

Noradrenaline release from the locus coeruleus shapes stress-induced hippocampal gene expression.

Exposure to an acute stressor triggers a complex cascade of neurochemical events in the brain. However, deciphering their individual impact on stress-induced molecular changes remains a major challenge. Here, we combine RNA sequencing with selective pharmacological, chemogenetic, and optogenetic manipulations to isolate the contribution of the locus coeruleus-noradrenaline (LC-NA) system to the acute stress response in mice. We reveal that NA release during stress exposure regulates a large and reproducible set of genes in the dorsal and ventral hippocampus via β-adrenergic receptors. For a smaller subset of these genes, we show that NA release triggered by LC stimulation is sufficient to mimic the stress-induced transcriptional response. We observe these effects in both sexes, and independent of the pattern and frequency of LC activation. Using a retrograde optogenetic approach, we demonstrate that hippocampus-projecting LC neurons directly regulate hippocampal gene expression. Overall, a highly selective set of astrocyte-enriched genes emerges as key targets of LC-NA activation, most prominently several subunits of protein phosphatase 1 (Ppp1r3c, Ppp1r3d, Ppp1r3g) and type II iodothyronine deiodinase (Dio2). These results highlight the importance of astrocytic energy metabolism and thyroid hormone signaling in LC-mediated hippocampal function and offer new molecular targets for understanding how NA impacts brain function in health and disease.

MUCIN MRNA ISOFORM SIGNATURES AS POTENTIAL NOVEL BIOMARKERS TO EVALUATE DISEASE STATUS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES

Abstract Mucosal barrier dysfunction and aberrant mucin expression are major hallmarks in the pathophysiology of IBD. Mucins are highly polymorphic, and the presence of genetic differences can alter gene expression, resulting in several mRNA isoforms via alternative splicing. While most isoforms encode similar biological functions, others alter protein function, potentially resulting in progression towards disease. Currently, little attention has been given to the importance of mucin mRNA isoforms in IBD. The aim of our study is to investigate the potential of mucin mRNA isoforms as novel biomarkers for the evaluation of IBD activity and subtypes. To obtain this goal, RNA was extracted from colonic and terminal ileal biopsies of IBD patients that underwent an endoscopy at the Antwerp University Hospital (UZA). Additionally, patients without a history of IBD undergoing an endoscopy due to a positive iFOBT which show no endoscopic abnormalities, were included as controls. Library preparation was performed with the PacBio Iso-Seq multiplex protocol adapted for targeted transcriptome sequencing. Targeted capture was accomplished by using a custom-designed pool of probes, developed for the capture of all mucin gene transcripts. Samples were sequenced on the PacBio Sequel platform at the University of Antwerp. The data was analyzed by using the isoseq3-pipeline and additional filtering with SQANTI3 was performed. In total 106 biopsies were sequenced on the PacBio platform. The resulting intestinal mucin transcriptome was merged with the human reference transcriptome. On this combined mucin transcriptome Illumina bulk RNA sequencing data from over 2000 intestinal biopsies (GEO dataset GSE193677) were mapped to determine mucin isoform expression. An external dataset (GEO dataset GSE165512) was used for additional validation. A classification random forest was trained on this data to distinguish inflamed IBD from non-inflamed control patients based on the mucin isoform expression alone. The model performed well on train and test datasets but decreased in the external validation (Table 1). Dividing the samples based on disease phenotype greatly increases performance on the external validation dataset (Table 1). When only training on ileal biopsies, the model proved to be excellent in distinguishing Crohn’s disease patients from controls (Table 1). Classification of inflamed ulcerative colitis from control patients based on only the biopsies from the distal colon was similar to the latter (Table 1). Our machine learning model was able to distinguish Crohn’s disease from control patients and ulcerative colitis from control patients based on mucin mRNA isoform expression. In addition, our data suggests that the mucin isoform expression may vary between different regions within the colon. Table 1 Area under the receiver operator curve (AUCROC) from a random forest classifier trained on mucin isoform expression of inflamed biopsies of IBD and non-inflamed control patients.

MYH1G-AS is a chromatin-associated lncRNA that regulates skeletal muscle development in chicken

BackgroundSkeletal muscle development is pivotal for animal growth and health. Recently, long noncoding RNAs (lncRNAs) were found to interact with chromatin through diverse roles. However, little is known about how lncRNAs act as chromatin-associated RNAs to regulate skeletal muscle development. Here, we aim to investigate the regulation of chromatin-associated RNA (MYH1G-AS) during skeletal muscle development.MethodsWe provided comprehensive insight into the RNA profile and chromatin accessibility of different myofibers, combining RNA sequencing (RNA-seq) with an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). The dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the transcriptional regulation mechanism of MYH1G-AS. ALKBH5-mediated MYH1G-AS N6-methyladenosine (m6A) demethylation was assessed by a single-base elongation and ligation-based qPCR amplification method (SELECT) assay. Functions of MYH1G-AS were investigated through a primary myoblast and lentivirus/cholesterol-modified antisense oligonucleotide (ASO)-mediated animal model. To validate the interaction of MYH1G-AS with fibroblast growth factor 18 (FGF18) protein, RNA pull down and an RNA immunoprecipitation (RIP) assay were performed. Specifically, the interaction between FGF18 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) protein was analyzed by coimmunoprecipitation (Co-IP) and a yeast two-hybrid assay.ResultsA total of 45 differentially expressed (DE) lncRNAs, with DE ATAC-seq peaks in their promoter region, were classified as open chromatin-associated lncRNAs. A skeletal muscle-specific lncRNA (MSTRG.15576.9; MYH1G-AS), which is one of the open chromatin-associated lncRNA, was identified. MYH1G-AS transcription is coordinately regulated by transcription factors (TF) SMAD3 and SP2. Moreover, SP2 represses ALKBH5 transcription to weaken ALKBH5-mediated m6A demethylation of MYH1G-AS, thus destroying MYH1G-AS RNA stability. MYH1G-AS accelerates myoblast proliferation but restrains myoblast differentiation. Moreover, MYH1G-AS drives a switch from slow-twitch to fast-twitch fibers and causes muscle atrophy. Mechanistically, MYH1G-AS inhibits FGF18 protein stabilization to reduce the interaction of FGF18 to SMARCA5, thus repressing chromatin accessibility of the SMAD4 promoter to activate the SMAD4-dependent pathway.ConclusionsOur results reveal a new pattern of the regulation of lncRNA expression at diverse levels and help expound the regulation of m6A methylation on chromatin status.Graphical