Gene Expression Events Research Articles

An unexpected role of neurite outgrowth inhibitor A as regulator of tooth enamel formation

Neurite outgrowth inhibitor A (Nogo-A) is a major player in neural development and regeneration and the target of clinical trials aiming at promoting the regeneration of the central nervous system upon traumatic and ischemic injury. In this work, we investigated the functions of Nogo-A during tooth development to determine its role in dental physiology and pathology. Using immunohistochemistry and in situ hybridization techniques, we showed that Nogo-A is highly expressed in the developing mouse teeth and, most specifically, in the ameloblasts that are responsible for the formation of enamel. Using both Nogo-A knockout and K14-Cre;Nogo-A fl/fl transgenic mice, we showed that Nogo-A deletion in the dental epithelium leads to the formation of defective enamel. This phenotype is associated with overexpression of a set of specific genes involved in ameloblast differentiation and enamel matrix production, such as amelogenin, ameloblastin and enamelin. By characterising the interactome of Nogo-A in the dental epithelium of wild-type and mutant animals, we found that Nogo-A directly interacts with molecules important for regulating gene expression, and its deletion disturbs their cellular localisation. Furthermore, we demonstrated that inhibition of the intracellular, but not cell-surface, Nogo-A is responsible for gene expression modulation in ameloblasts. Taken together, these results reveal an unexpected function for Nogo-A in tooth enamel formation by regulating gene expression and cytodifferentiation events.

PerturbAtlas: a comprehensive atlas of public genetic perturbation bulk RNA-seq datasets.

Manipulating gene expression is crucial for understanding gene function, with high-throughput sequencing techniques such as RNA-seq elucidating the downstream mechanisms involved. However, the lack of a standardized metadata format for small-scale perturbation expression datasets in public repositories hinders their reuse. To address this issue, we developed PerturbAtlas, an add-value resource that re-analyzes publicly archived RNA-seq libraries to provide quantitative data on gene expression, transcript profiles, and alternative splicing events following genetic perturbation. PerturbAtlas assists users in identifying trends at the gene and isoform levels in perturbation assays by re-analyzing a curated set of 122801 RNA-seq libraries across 13 species. This resource is freely available at https://perturbatlas.kratoss.site as both raw data tables and an interactive browser, allowing searches by species, tissueor genomic features. The results provide detailed information on alterations following perturbations, accessible through both forward and reverse approaches, thereby enabling the exploration of perturbation consequences and the identification of potential causal perturbations.

Ex vivo disease modelling of Rett syndrome: the transcriptomic and metabolomic implications of direct neuronal conversion.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that primarily affects females and is characterized by a period of normal development followed by severe cognitive, motor, and communication impairment. The syndrome is predominantly caused by mutations in the MECP2. This study aimed to use comprehensive multi-omic analysis to identify the molecular and metabolic alterations associated with Rett syndrome. Transcriptomic and metabolomic profiling was performed using neuron-like cells derived from the fibroblasts of 3 Rett syndrome patients with different MECP2 mutations (R168X, P152R, and R133C) and 1 healthy control. Differential gene expression, alternative splicing events, and metabolite changes were analyzed to identify the key pathways and processes affected in patients with Rett syndrome. Transcriptomic analysis showed there was significant down-regulation of genes associated with the extracellular matrix (ECM) and cytoskeletal components, which was particularly notable in patient P3 (R133C mutation), who had non-random X chromosome inactivation. Additionally, significant changes in microtubule-related gene expression and alternative splicing events were observed, especially in patient P2 (P152R mutation). Metabolomic profiling showed that there were alterations in metabolic pathways, particularly up-regulation of ketone body synthesis and degradation pathways, in addition to an increase in free fatty acid levels. Integrated analysis highlighted the interplay between structural gene down-regulation and metabolic shifts, underscoring the adaptive responses to cellular stress in Rett neurons. The present findings provide valuable insights into the molecular and metabolic landscape of Rett syndrome, emphasizing the importance of combining omic data to enlighten the molecular pathophysiology of this syndrome.

A plant virus differentially alters DNA methylation in two cryptic species of a hemipteran vector

Epigenetic patterns including DNA methylation are known to vary between distantly related species, but it is not clear how these patterns differ at an intraspecific level. The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Aleyrodidae; Hemiptera), encompasses several cryptic species. These cryptic species possess highly similar genomes but exhibit substantial biological and physiological differences. B. tabaci cryptic species are invasive, highly polyphagous, and transmit an array of plant infecting single stranded DNA viruses (ssDNA) –begomoviruses. In this study, DNA methylation patterns around genes and genomic features of two prominent B. tabaci cryptic species were investigated following acquisition of a monopartite ssDNA virus –tomato yellow curl virus. The cryptic species investigated included: B (also known as Middle East Asia Minor 1) and Q (also known as Mediterranean). Genomic features, such as promoters, gene bodies, and transposable elements were assessed for methylation levels in both B and Q cryptic species. Despite overall similar trends, both cryptic species showed differences in methylation levels between these genomic features. Virus induced differentially methylated regions were associated with predominantly distinct genes in B and Q cryptic species. All differentially methylated regions were assessed for differential gene expression and alternative splicing events with and without virus acquisition. DNA methylation levels were found to have a negative correlation with differential gene expression in both B and Q cryptic species. The differentially expressed genes were further grouped into hyper- and hypomethylated clusters. These clusters included genes with implications for virus-vector interactions including immune functions and xenobiotics’ detoxification. The observed DNA methylation pattern differences within each cryptic species could, in part, explain some of the biological and physiological differences between them.

Quantum modeling simulates nutrient effect of bioplastic polyhydroxyalkanoate (PHA) production in Pseudomonas putida

Polyhydroxyalkanoates (PHAs) could be used to make sustainable, biodegradable plastics. However, the precise and accurate mechanistic modeling of PHA biosynthesis, especially medium-chain-length PHA (mcl-PHA), for yield improvement remains a challenge to biology. PHA biosynthesis is typically triggered by nitrogen limitation and tends to peak at an optimal carbon-to-nitrogen (C/N) ratio. Specifically, simulation of the underlying dynamic regulation mechanisms for PHA bioprocess is a bottleneck owing to surfeit model complexity and current modeling philosophies for uncertainty. To address this issue, we proposed a quantum-like decision-making model to encode gene expression and regulation events as hidden layers by the general transformation of a density matrix, which uses the interference of probability amplitudes to provide an empirical-level description for PHA biosynthesis. We implemented our framework modeling the biosynthesis of mcl-PHA in Pseudomonas putida with respect to external C/N ratios, showing its optimization production at maximum PHA production of 13.81% cell dry mass (CDM) at the C/N ratio of 40:1. The results also suggest the degree of P. putida’s preference in channeling carbon towards PHA production as part of the bacterium’s adaptative behavior to nutrient stress using quantum formalism. Generic parameters (kD, kN and theta θ) obtained based on such quantum formulation, representing P. putida’s PHA biosynthesis with respect to external C/N ratios, was discussed. This work offers a new perspective on the use of quantum theory for PHA production, demonstrating its application potential for other bioprocesses.

Abstract Mo088: RBFOX2 haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.

Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

Transcriptomic Analysis of the Spatiotemporal Axis of Oogenesis and Fertilization in C. elegans.

The oocyte germline of the C. elegans hermaphrodite presents a unique model to study the formation of oocytes. However, the size of the model animal and difficulties in retrieval of specific stages of the germline have obviated closer systematic studies of this process throughout the years. Here, we present a transcriptomic level analysis into the oogenesis of C. elegans hermaphrodites. We dissected a hermaphrodite gonad into seven sections corresponding to the mitotic distal region, the pachytene, the diplotene, the early diakinesis region and the 3 most proximal oocytes, and deeply sequenced the transcriptome of each of them along with that of the fertilized egg using a single-cell RNA-seq protocol. We identified specific gene expression events as well as gene splicing events in finer detail along the oocyte germline and provided novel insights into underlying mechanisms of the oogenesis process. Furthermore, through careful review of relevant research literature coupled with patterns observed in our analysis, we attempt to delineate transcripts that may serve functions in the interaction between the germline and cells of the somatic gonad. These results expand our knowledge of the transcriptomic space of the C. elegans germline and lay a foundation on which future studies of the germline can be based upon.

Targeting SAT1 prevents osteoporosis through promoting osteoclast apoptosis

Osteoporosis is a systemic bone disease characterized by decreased bone mass that is tightly regulated by the coordinated actions of osteoclasts and osteoblasts. Apoptosis as a precise programmed cell death involves a cascade of gene expression events which are mechanistically linked to the regulation of bone metabolism. Nevertheless, the critical biomolecules involved in regulating cell apoptosis in osteoporosis remain unknown. To gain a deeper insight into the relationship between apoptosis and osteoporosis, this study integrated the sequencing results of human samples and using a machine learning workflow to overcome the limitations of a single study. Among all immune cell populations, we assessed the apoptotic level and portrayed the distinct subtypes and lineage differentiation of monocytic cells in osteoporotic tissues. Osteoclasts expressed a higher level of Spermidine/spermine-N1-Acetyltransferase1 (SAT1) during osteoclastogenesis which prevented osteoclasts apoptosis and facilitate osteoporosis progression. In addition, Berenil, one potent SAT1 inhibitor, increased osteoclast apoptosis and reversed the bone loss in the femurs of a murine ovariectomy model. In summary, Berenil promotes osteoclast apoptosis, inhibits the bone resorption and improves the abnormal bone structure in vitro and in vivo models by targeting SAT1, demonstrating its potential as a precise therapeutic strategy for clinical osteoporosis treatment.

Axon-derived PACSIN1 binds to the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

High-Throughput RNA-HCR-FISH Detection of Endogenous Pre-mRNA Splice Variants.

RNA-fluorescence in situ hybridization (RNA-FISH) is an essential and widely used tool for visualizing RNA molecules in intact cells. Recent advances have increased RNA-FISH sensitivity, signal detection efficiency, and throughput. However, detection of endogenous mRNA splice variants has been challenging due to the limits of visualization of RNA-FISH fluorescence signals and due to the limited number of RNA-FISH probes per target. HiFENS (high-throughput FISH detection of endogenous pre-mRNA splicing isoforms) is a method that enables visualization and relative quantification of mRNA splice variants at single-cell resolution in an automated high-throughput manner. HiFENS incorporates HCR (hybridization chain reaction) signal amplification strategies to enhance the fluorescence signal generated by low abundance transcripts or a small number of FISH probes targeting short stretches of RNA, such as single exons. The technique offers a significant advance in high-throughput FISH-based RNA detection and provides a powerful tool that can be used as a readout in functional genomics screens to discover and dissect cellular pathways regulating gene expression and alternative pre-mRNA splicing events.

Transcriptomic analysis of the spatiotemporal axis of oogenesis and fertilization in C. elegans.

Caenorhabditis elegans hermaphrodite presents a unique model to study the formation of oocytes. However, the size of the model animal and difficulties in retrieval of specific stages of the germline have obviated closer systematic studies of this process throughout the years. Here, we present a transcriptomic level analysis into the oogenesis of C. elegans hermaphrodites. We dissected a hermaphrodite gonad into seven sections corresponding to the mitotic distal region, the pachytene region, the diplotene region, the early diakinesis region and the 3 most proximal oocytes, and deeply sequenced the transcriptome of each of them along with that of the fertilized egg using a single-cell RNA-seq (scRNA-seq) protocol. We identified specific gene expression events as well as gene splicing events in finer detail along the gonad and provided novel insights into underlying mechanisms of the oogenesis process. Furthermore, through careful review of relevant research literature coupled with patterns observed in our analysis, we delineate transcripts that may serve functions in the interactions between the germline and cells of the somatic gonad. These results expand our knowledge of the transcriptomic space of the C. elegans germline and lay a foundation on which future studies of the germline can be based upon.

MULTIPLE MEASURES OF FITNESS ASSOCIATION WITH MUSCLE MRNA SPLICING VARIANTS IN THE GESTALT STUDY

Abstract Regular physical activity has been associated with higher skeletal muscle mass and strength as well as better mitochondrial function. Previous studies in master octogenarian athletes found a higher content of mitochondrial- and reduced spliceosome proteins compared to non-athlete age/sex-matched controls. These findings suggest that higher mitochondrial function is associated with down-regulation of the spliceosome. In this study we aimed to detect mitochondrial respiration associations with expression of specific genes and differentially spliced isoforms. We performed RNA-seq from vastus lateralis skeletal muscle biopsies in 82 male/female participants in the GESTALT study (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing). We correlated gene expression, transcript usage and splicing events with I) self-reported level of physical activity in daily life, II) peak oxygen consumption assessed during an incremental treadmill stress testing, III) muscle oxidative capacity assessed by p31 MR Spectroscopy and IV) mitochondrial-respirometry assessed on permeabilized muscle fibers, using regression models adjusted for age, gender, and batch. Except for IV), all three mitochondrial measures positively correlated with each other, and were downregulated along the Aging axis. We detected 423, 219, 178 and 226 differentially expressed genes (p-value<0.01), respectively for the 4 mitochondrial fitness measures. Gene-set-enrichment analysis (GSEA) detected up-top TCA cycle and mitochondrial respiration kPCr and mito-respirometry detected the highest number of AS events (448 and 412). Dissimilarities among transcriptomic profile associated to these four bioenergetic outcomes provide insight into the biological mechanisms that connect them to mitochondrial function and suggest that mitochondrial function is not the only correlate of movement behaviors.

MUSCLE UP: WHAT AGING LOOKS LIKE FROM THE INSIDE OUT

Abstract The Longitudinal Studies Section of the Translational Gerontology Branch of the National Institute on Aging Intramural Research Program has the mission and opportunity to examine aging physiology from the cellular to the full body functional level in model systems and human populations. This symposium presents ongoing research that targets pathologic and normative change in muscle with aging and the significance of these changes for understanding the aging process. Data come from the Baltimore Longitudinal Study of Aging (BLSA) and the Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing (GESTALT) study. In nearly 500 BLSA participants aged 50+, Zenobia Moore evaluates the association between multiple epigenetic age metrics and change over 6 years in mid-femur areas of muscle and subcutaneous and intramuscular fat derived from computed tomography. Qu (Teresa) Tian investigates the relationship between skeletal muscle mitochondrial function and risk of MCI or dementia over 3.3 years in 460 initially cognitively normal BLSA participants and PET and blood biomarkers of AD in available subsamples. In 82 men and women from GESTALT, Stefano Donega examines relationships between physical activity, peak oxygen consumption and muscle oxidative capacity using p31 MR spectroscopy and gene expression, transcript usage and splicing events derived from vastus lateralis biopsies. Using volumetric Focused Ion Beam Scanning Electron Microscopy, Lisa Hartnell shares 3D images of muscle samples at the level of the mitochondrial reticulum and sarcomere from young and old healthy and frail GESTALT participants. Together, these findings highlight the value of muscle as a window on aging.

Genomic Landscape Comparison of Cardiac versus Extra-Cardiac Angiosarcomas.

Angiosarcomas (ASs) are rare malignant vascular entities that can affect several regions in our body, including the heart. Cardiac ASs comprise 25-40% of cardiac sarcomas and can cause death within months of diagnosis. Thus, our aim was to identify potential differences and/or similarities between cardiac and extra-cardiac ASs to enhance targeted therapies and, consequently, patients' prognosis. Whole-transcriptome analysis of three cardiac and eleven extra-cardiac non-cutaneous samples was performed to investigate differential gene expression and mutational events between the two groups. The gene signature of cardiac and extra-cardiac non-cutaneous ASs was also compared to that of cutaneous angiosarcomas (n = 9). H/N/K-RAS and TP53 alterations were more recurrent in extra-cardiac ASs, while POTE-gene family overexpression was peculiar to cardiac ASs. Additionally, in vitro functional analyses showed that POTEH upregulation conferred a growth advantage to recipient cells, partly supporting the cardiac AS aggressive phenotype and patients' scarce survival rate. These features should be considered when investigating alternative treatments.

Altered CELF4 splicing factor enhances pancreatic neuroendocrine tumors aggressiveness influencing mTOR and everolimus response

Pancreatic neuroendocrine tumors (PanNETs) comprise a heterogeneous group of tumors with growing incidence. Recent molecular analyses provided a precise picture of their genomic and epigenomic landscape. Splicing dysregulation is increasingly regarded as a novel cancer hallmark influencing key tumor features. We have previously demonstrated that splicing machinery is markedly dysregulated in PanNETs. Here, we aimed to elucidate the molecular and functional implications of CELF4, one of the most altered splicing factors in PanNETs. CELF4 expression was determined in 20 PanNETs, comparing tumor and non-tumoral adjacent tissue. RNA-Seq dataset was analyzed to explore CELF4-linked interrelations among clinical features, gene expression, and splicing events. Two PanNETs cell lines were employed to assess CELF4 function in vitro and in vivo. PanNETs display markedly upregulated CELF4 expression, which is closely associated to malignancy features, altered expression of key tumor players, and distinct splicing event profiles. Modulation of CELF4 influenced proliferation in vitro and reduced in vivo xenograft tumor growth. Interestingly, functional assays and RNA-Seq analysis revealed that CELF4 silencing altered mTOR signaling pathway, enhancing the effect of everolimus. We demonstrate that CELF4 is dysregulated in PanNETs, where it influences tumor development and aggressiveness, likely by modulating mTOR pathway, suggesting its potential as therapeutic target.

Identification of Chromatin Regulators Required for Enucleation

Erythropoiesis involves several distinct steps as precursor cells to differentiate and mature into erythrocytes which follows differentiation to proerythroblasts, transformation into basophilic, polychromatic, and orthochromatic erythroblasts, followed by enucleation to form reticulocytes and erythrocytes. Indeed, erythropoiesis and enucleation are complex processes that involve the coordinated action of multiple regulators. Various transcription factors, signaling pathways, and epigenetic modifiers play significant roles in controlling gene expression, cell differentiation, and cellular events during erythropoiesis. As an example, BCL11A is necessary for nucleus expulsion and the progression of erythropoiesis. Similarly, other transcription factors, histone deacetylases, DNA methylation enzymes, and chromatin remodeling complexes also contribute to the enucleation process. However, the exact role of chromatin regulators and how they are involved in the enucleation process is still not fully understood. To identify novel chromatin factors that regulate both enucleation and proliferation of erythroid cells, we conducted a pooled epigenome-wide CRISPR-Cas9 knockout screen using the human erythroid cell line BEL-A. The epigenetic knockout library consists of 719 genes associated with chromatin-related pathways and 35 essential genes, which include those encoding ribosomal proteins. Each gene is targeted by 10 single guide RNAs (sgRNAs). The primary objective of this screen was to determine essential chromatin regulators that play a critical role in the process of enucleation. To achieve this, we planned the screening groups as followed: (i) cells collected immediately after antibiotic selection (time zero), (ii) undifferentiated, expanded cells grown for 13 days, (iii) expelled nuclei (pyrenocytes), and (iv) orthochromatic erythroblasts that have not undergone enucleation. To isolate the distinct cellular stages mentioned above, we utilized fluorescence-activated cell sorting (FACS). The pyrenocyte group was compared with all other groups to identify depleted gRNAs, which represent factors that are necessary for enucleation. Setd8, which is an important epigenetic factor that is required for enucleation was determined to be depleted only in pyrenocytes (β score -0,433), validating our screen approach. In addition, we identified more than 15 other genes that showed even greater depletion than Setd8, and could be essential for enucleation. Among the identified genes, EP300, PCGF1, Setd1B, CDY2B, and TLK2 were found to be the most depleted in pyrenocytes. Additionally, TRIM28, KAT5, and EXOSC7 were identified as enriched genes in the study. On the other hand, we identified a group of genes that exhibited significant depletion in pyrenocytes compared to orthochromatic erythroblasts. Among these genes, UHRF1, DPF2, KEAP1, and TAF10 were found to be most depleted.To better understand the role of KEAP1 in enucleation, KEAP1 knockout (KO) BEL-A cells were generated. The enucleation ratio of the KEAP1 KO cells decreased significantly, and their survival also dramatically decreased after differentiation on day 12 compared to the wild-type cells. Live cell imaging of enucleation of the KEAP1 KO cells further confirmed the decrease of the enucleation rate. The significant decrease of enucleation observed in KEAP1 KO cells has triggered questions about the specific pathways and processes required for enucleation. Given that KEAP1 is the main regulator of NRF2, our future studies will mainly focus on understanding the importance of oxidative stress in enucleation.

Interdependence of Molecular Lesions That Drive Uveal Melanoma Metastasis.

The metastatic risk of uveal melanoma (UM) is defined by a limited number of molecular lesions, somatic mutations (SF3B1 and BAP1), and copy number alterations (CNA): monosomy of chromosome 3 (M3), chr8q gain (8q), chr6p gain (6p), yet the sequence of events is not clear. We analyzed data from three datasets (TCGA-UVM, GSE27831, GSE51880) with information regarding M3, 8q, 6p, SF3B1, and BAP1 status. We confirm that BAP1 mutations are always associated with M3 in high-risk patients. All other features (6p, 8q, M3, SF3B1 mutation) were present independently from each other. Chr8q gain was frequently associated with chr3 disomy. Hierarchical clustering of gene expression data of samples with different binary combinations of aggressivity factors shows that patients with 8q|M3, BAP1|M3 form one cluster enriched in samples that developed metastases. Patients with 6p combined with either 8q or SF3B1 are mainly represented in the other, low-risk cluster. Several gene expression events that show a non-significant association with outcome when considering single features become significant when analyzing combinations of risk features indicating additive action. The independence of risk factors is consistent with a random risk model of UM metastasis without an obligatory sequence.

FoxM1 coordinates cell division, protein synthesis, and mitochondrial activity in a subset of β cells during acute metabolic stress

Pancreatic β cells display functional and transcriptional heterogeneity in health and disease. The sequence of events leading to β cell heterogeneity during metabolic stress is poorly understood. Here, we characterize β cell responses to early metabolic stress invivo by employing RNA sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing(ATAC-seq), single-cell RNA-seq (scRNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and real-time imaging to decipher temporal events of chromatin remodeling and gene expression regulating the unfolded protein response (UPR), protein synthesis, mitochondrial function, and cell-cycle progression. We demonstrate that a subpopulation of β cells with active UPR, decreased protein synthesis, and insulin secretary capacities is more susceptible to proliferation after insulin depletion. Alleviation of endoplasmic reticulum (ER) stress precedes the progression of the cell cycle and mitosis and ensures appropriate insulin synthesis. Furthermore, metabolic stress rapidly activates key transcription factors including FoxM1, which impacts on proliferative and quiescent β cells by regulating protein synthesis, ER stress, and mitochondrial activity via direct repression of mitochondrial-encoded genes.

Dietary Implications of Polyunsaturated Fatty Acids during Pregnancy and in Neonates.

Certain limitations exist for animals to modify fatty acid changes. Besides the role of arachidonic acid (AA), docosahexaenoic acid (DHA) and other 20-carbon long-chain polyunsaturated fatty acids (LCPUFAs) for the synthesis of inflammatory mediators as eicosanoids, different LCPUFAs have many other effects, including their abilities to regulate gene expression and downstream events. LCPUFAs are susceptible to autoxidation, which is prevented by the action of antioxidants in the form of enzymes like superoxide dismutases, catalases and peroxidases, as well as antioxidant compounds that protect against oxidation or repair the damage caused. Under normal conditions, the fetus needs both essential fatty acids (EFAs) and LCPUFAs, which are obtained from its mother by placental transfer. In early pregnancy, dietary derived fatty acids are accumulated in maternal adipose tissue. However, during late pregnancy, corresponding to the period of the highest fetal growth, maternal adipose tissue becomes catabolic and LCPUFAs are released into the circulation by adipose lipolytic activity. The released LCPUFAs are taken up by maternal liver to be esterified and released back to the circulation as triacylglycerides (TAGs) in very-low-density lipoprotein (VLDL) that become available to the placenta to be transferred to the fetus in the form of non-esterified fatty acids (NEFAs). An enhanced adipose tissue lipolysis is maintained around parturition and esterified LCPUFAs are diverted to mammary glands thanks to an increased activity of lipoprotein lipase for milk production. Throughout this process, LCPUFAs become available to the newborn during suckling. The important role of both DHA and AA for the development of the nervous system and for growth has motivated their dietary supplement during different postnatal stages. This has been especially important in preterm infants both because under normal conditions, the fetus acquires most of these fatty acids during late pregnancy, and because the immaturity of the enzyme systems for the synthesis of AA and DHA from their respective EFAs.