Differentiation Events Research Articles

Changes in chromatin accessibility landscape and histone H3 core acetylation during valproic acid-induced differentiation of embryonic stem cells

Directed differentiation of mouse embryonic stem cells (mESCs) or induced pluripotent stem cells (iPSCs) provides powerful models to dissect the molecular mechanisms leading to the formation of specific cell lineages. Treatment with histone deacetylase inhibitors can significantly enhance the efficiency of directed differentiation. However, the mechanisms are not well understood. Here, we use CUT&RUN in combination with ATAC-seq to determine changes in both histone modifications and genome-wide chromatin accessibility following valproic acid (VPA) exposure. VPA induced a significant increase in global histone H3 acetylation (H3K56ac), a core histone modification affecting nucleosome stability, as well as enrichment at loci associated with cytoskeletal organization and cellular morphogenesis. In addition, VPA altered the levels of linker histone H1 subtypes and the total histone H1/nucleosome ratio indicative of initial differentiation events. Notably, ATAC-seq analysis revealed changes in chromatin accessibility of genes involved in regulation of CDK serine/threonine kinase activity and DNA duplex unwinding. Importantly, changes in chromatin accessibility were evident at several key genomic loci, such as the pluripotency factor Lefty, cardiac muscle troponin Tnnt2, and the homeodomain factor Hopx, which play critical roles in cardiomyocyte differentiation. Massive parallel transcription factor (TF) footprinting also indicates an increased occupancy of TFs involved in differentiation toward mesoderm and endoderm lineages and a loss of footprints of POU5F1/SOX2 pluripotency factors following VPA treatment. Our results provide the first genome-wide analysis of the chromatin landscape following VPA-induced differentiation in mESCs and provide new mechanistic insight into the intricate molecular processes that govern departure from pluripotency and early lineage commitment.

Genome-wide Identification and Analysis of Splicing QTLs in Multiple Sclerosis by RNA-Seq Data.

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelinating lesions in the central nervous system. Recently, the dysregulation of alternative splicing (AS) in the brain has been found to significantly influence the progression of MS. Moreover, previous studies demonstrate that many MS-related variants in the genome act as the important regulation factors of AS events and contribute to the pathogenesis of MS. However, by far, no genome-wide research about the effect of genomic variants on AS events in MS has been reported. Here, we first implemented a strategy to obtain genomic variant genotype and AS isoform average percentage spliced-in values from RNA-seq data of 142 individuals (51 MS patients and 91 controls). Then, combing the two sets of data, we performed a cis-splicing quantitative trait loci (sQTLs) analysis to identify the cis-acting loci and the affected differential AS events in MS and further explored the characteristics of these cis-sQTLs. Finally, the weighted gene coexpression network and gene set enrichment analyses were used to investigate gene interaction pattern and functions of the affected AS events in MS. In total, we identified 5835 variants affecting 672 differential AS events. The cis-sQTLs tend to be distributed in proximity of the gene transcription initiation site, and the intronic variants of them are more capable of regulating AS events. The retained intron AS events are more susceptible to influence of genome variants, and their functions are involved in protein kinase and phosphorylation modification. In summary, these findings provide an insight into the mechanism of MS.

Developmental Temporal Patterns and Molecular Network Features in the Transcriptome of Rat Spinal Cord

The molecular network features of spinal cord development that are integral to tissue engineering remain poorly understood in placental mammals, especially in terms of their relationships with vital biological processes such as regeneration. Here, using a large-scale temporal transcriptomic analysis of rat spinal cord from the embryonic stage to adulthood, we show that fluctuating RNA expression levels reflect highly active transcriptional regulation, which may initiate spinal cord patterning. We also demonstrate that microRNAs (miRNAs) and transcriptional factors exhibit a mosaic profile based on their expression patterns, while differential alternative splicing events reveal that alternative splicing may be a driving force for the development of the node of Ranvier. Our study also supports the existence of a negative correlation between innate immunity and intrinsic growth capacity. Epigenetic modifications appear to perform their respective regulatory functions at different stages of development, while guanine nucleotide-binding protein (G protein)-coupled receptors (including olfactory receptors (ORs)) may perform pleiotropic roles in axonal growth. This study provides a valuable resource for investigating spinal cord development and complements the increasing number of single-cell datasets. These findings also provide a genetic basis for the development of novel tissue engineering strategies.

Zinc is an intracellular signal during sperm activation in Caenorhabditis elegans.

Sperm activation is a rapid and dramatic cell differentiation event that does not involve changes in transcription, and the signaling cascades that mediate this process have not been fully defined. zipt-7.1 encodes a zinc transporter, and zipt-7.1(lf) mutants display sperm-activation defects, leading to the hypothesis that zinc signaling mediates sperm activation in Caenorhabditis elegans. Here, we describe the development of a method for dynamic imaging of labile zinc during sperm activation using the zinc-specific fluorescence probe FluoZin-3 AM and time-lapse confocal imaging. Two phases of dynamic changes in labile zinc levels were observed during sperm activation. Forced zinc entry using the zinc ionophore pyrithione activated sperm in vitro, and it suppressed the defects of zipt-7.1(lf) mutants, indicating that high levels of cytosolic zinc are sufficient for sperm activation. We compared activation by zinc pyrithione to activation by extracellular zinc, the Na+/H+ antiporter monensin and the protease cocktail pronase in multiple mutant backgrounds. These results indicate that the protease pathway does not require zinc signaling, suggesting that zinc signaling is sufficient to activate sperm but is not always necessary.

DNA Methylation and Immune Memory Response

The generation of memory is a cardinal feature of the adaptive immune response, involving different factors in a complex process of cellular differentiation. This process is essential for protecting the second encounter with pathogens and is the mechanism by which vaccines work. Epigenetic changes play important roles in the regulation of cell differentiation events. There are three types of epigenetic regulation: DNA methylation, histone modification, and microRNA expression. One of these epigenetic changes, DNA methylation, occurs in cytosine residues, mainly in CpG dinucleotides. This brief review aimed to analyse the literature to verify the involvement of DNA methylation during memory T and B cell development. Several studies have highlighted the importance of the DNA methyltransferases, enzymes that catalyse the methylation of DNA, during memory differentiation, maintenance, and function. The methylation profile within different subsets of naïve activated and memory cells could be an interesting tool to help monitor immune memory response.

The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle.

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

Transcriptomic effects of rs4845604, an IBD and allergy-associated RORC variant, in stimulated ex vivo CD4+ T cells.

RORγt is an isoform of RORC, preferentially expressed in Th17 cells, that functions as a critical regulator of type 3 immunity. As murine Th17-driven inflammatory disease models were greatly diminished in RORC knock-out mice, this receptor was prioritised as an attractive therapeutic target for the treatment of several autoimmune diseases. Human genetic studies indicate a significant contributory role for RORC in several human disease conditions. Furthermore, genome-wide association studies (GWAS) report a significant association between inflammatory bowel disease (IBD) and the RORC regulatory variant rs4845604. To investigate if the rs4845604 variant may affect CD4+ T cell differentiation events, naïve CD4+ T cells were isolated from eighteen healthy subjects homozygous for the rs4845604 minor (A) or major (G) allele). Isolated cells from each subject were differentiated into distinct T cell lineages by culturing in either T cell maintenance medium or Th17 driving medium conditions for six days in the presence of an RORC inverse agonist (to prevent constitutive receptor activity) or an inactive diastereomer (control). Our proof of concept study indicated that genotype had no significant effect on the mean number of naïve CD4 T cells isolated, nor the frequency of Th1-like and Th17-like cells following six days of culture in any of the four culture conditions. Analysis of the derived RNA-seq count data identified genotype-driven transcriptional effects in each of the four culture conditions. Subsequent pathway enrichment analysis of these profiles reported perturbation of metabolic signalling networks, with the potential to affect the cellular detoxification response. This investigation reveals that rs4845604 genotype is associated with transcriptional effects in CD4+ T cells that may perturb immune and metabolic pathways. Most significantly, the rs4845604 GG, IBD risk associated, genotype may be associated with a differential detoxification response. This observation justifies further investigation in a larger cohort of both healthy and IBD-affected individuals.

Structural variation of mitochondrial genomes sheds light on evolutionary history of soybeans.

The architecture and genetic diversity of mitogenome (mtDNA) are largely unknown in cultivated soybean (Glycine max), which is domesticated from the wild progenitor, Glycine soja, 5000 years ago. Here, we de novo assembled the mitogenome of the cultivar 'Williams 82' (Wm82_mtDNA) with Illumina PE300 deep sequencing data, and verified it with polymerase chain reaction (PCR) and Southern blot analyses. Wm82_mtDNA maps as two autonomous circular chromosomes (370 871-bp Chr-m1 and 62 661-bp Chr-m2). Its structure is extensively divergent from that of the mono-chromosomal mitogenome reported in the landrace 'Aiganhuang' (AGH_mtDNA). Synteny analysis showed that the structural variations (SVs) between two genomes are mainly attributed to ectopic and illegitimate recombination. Moreover, Wm82_mtDNA and AGH_mtDNA each possess six and four specific regions, which are absent in their counterparts and likely result from differential sequence-loss events. Mitogenome SV was further studied in 39 wild and 182 cultivated soybean accessions distributed world-widely with PCR/Southern analyses or a comparable in silico analysis. The results classified both wild and cultivated soybeans into five cytoplasmic groups, named as GSa-GSe and G1-G5; 'Williams 82' and 'Aiganhuang' belong to G1 and G5, respectively. Notably, except for members in GSe and G5, all accessions carry a bi-chromosomal mitogenome with a common Chr-m2. Phylogenetic analyses based on mtDNA structures and chloroplast gene sequences both inferred that G1-G3, representing >90% of cultigens, likely inherited cytoplasm from the ancestor of domestic soybean, while G4 and G5 likely inherited cytoplasm from wild soybeans carrying GSa- and GSe-like cytoplasm through interspecific hybridization, offering new insights into soybean cultivation history.

Genetic variants in the p53 pathway influence implantation and pregnancy maintenance in IVF treatments using donor oocytes

PurposeSingle-nucleotide polymorphisms (SNPs) in the p53 pathways have shown to play a role in endometrial receptivity and implantation in infertile women undergoing in vitro fertilization (IVF). The present study aimed to assess the influence of these gene variants over pregnancy success through a receptivity model in recipients of egg donation treatments, when factors such as age and quality of the oocytes are standardized.MethodsA nested case–control study was performed on 234 female patients undergoing their first fresh IVF treatment as recipients of donor oocytes. Genotyping of TP53 Arg72Pro (rs1042522), LIF (rs929271), MDM4 (rs1563828), and USP7 (rs1529916) SNPs in the recipients allowed comparison of allele and genotype frequencies and their association with the IVF treatment outcome.ResultsGrouped by genotypes, patients showed differences in IVF outcomes after the embryo transfer. Arg72Pro (rs1042522) gene variant was associated to changes in implantation and clinical pregnancy rates. The polymorphisms USP7 (rs1529916) and MDM4 (rs1563828) were associated to differential ongoing pregnancy rates and variable miscarriage events, respectively.ConclusionsThis study highlights the association between gene polymorphisms related to P53 function and their influence over IVF reproductive outcomes. Arg72Pro variant may influence early events, as lower implantation rates were found in homozygous for Pro72 allele. By contrast, MDM4 (rs1563828) and USP7 (rs1529916) gene variants were associated with the later maintenance of pregnancy.

Physiological Rules of Endocannabinoid Action During Fetal and Neonatal Brain Development.

The endocannabinoid system is chiefly recognized as a homeostatic regulator of synaptic neurotransmission, primarily through the modulation of presynaptic CB1 cannabinoid neurons. Accordingly, the use of plant-derived cannabinoids received significant attention recently given the broad spectrum of physiological and pathobiological processes the endocannabinoid system is involved in. Nevertheless, a parallel line of research from a number of developmental biology groups has uncovered fundamental, evolutionarily conserved, and molecularly unique processes that endocannabinoids drive during development of the central nervous system. This lecture transcript is a concise summary of nearly 20 years of research on endocannabinoid-gated mechanisms of neurogenic specification events, which particularly define the numbers, placement, and connectivity of cortical neurons. A summary of both CB1 and alternative cannabinoid receptor contributions to neural differentiation is also discussed. Besides, insights are given into how phytocannabinoids can bypass physiologically timed and pivoted endocannabinoid action to inflict developmental errors that can significantly compromise the adaptive and computational ability of neurocircuits. By discussing specific subcellular targets of phytocannabinoid action and inferring errant glia versus neuron fate decisions and communication, a cellular basis is outlined for lifelong psychiatric phenotypes in offspring that associate with maternal cannabis seeking during pregnancy.

Population-scale identification of differential adverse events before and during a pandemic

Adverse patient safety events, unintended injuries resulting from medical therapy, were associated with 110,000 deaths in the United States in 2019. A nationwide pandemic (such as COVID-19) further challenges the ability of healthcare systems to ensure safe medication use and the pandemic’s effects on safety events remain poorly understood. Here, we investigate drug safety events across demographic groups before and during a pandemic using a dataset of 1,425,371 reports involving 2,821 drugs and 7,761 adverse events. Among 64 adverse events identified by our analyses, we find 54 increased in frequency during the pandemic, despite a 4.4% decrease in the total number of reports. Out of 53 adverse events with a pre-pandemic gender gap, 33 have seen their gap increase with the pandemic onset. We find that the number of adverse events with an increased reporting ratio is higher in adults (by 16.8%) than in older patients. Our findings have implications for safe medication use and preventable healthcare inequality in public health emergencies.

Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development.

Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. Here we show that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events.

MSphere of Influence: How I Learned to Love Bacteria and their Tangled Evolutionary Tree.

ABSTRACTTera Levin works in the fields of evolution, microbiology, and genetics, studying how adaptation shapes the molecular interactions between eukaryotic hosts and bacterial pathogens. In this mSphere of Influence article, she reflects on how the paper “Population genomics of early events in the ecological differentiation of bacteria” by Shapiro et al. (B. J. Shapiro, J. Friedman, O. X. Cordero, S. P. Preheim, et al., Science 336:48–51, 2012, https://doi.org/10.1126/science.1218198) changed the way she thinks about bacterial gene and genome evolution.

Analysis of Morphogenesis and Flagellar Assembly During Spermatogenesis in Planarian Flatworms.

Spermatogenesis is one of the most dramatic cellular differentiation events observed in animals. In particular, spermiogenesis (the final stage of spermatogenesis) involves extensive shedding of cytoplasmic organelles, dramatic nuclear rearrangements, and assembly of long flagellar structures. In planarian flatworms, the spherical nucleus present in round spermatids elongates to produce the filamentous nucleus of mature sperm. Newly formed cortical microtubules participate in cytoskeletal rearrangements observed during spermiogenesis and remain present in sperm. In addition, a pair of flagella assemble at one end of each spermatid in a process that likely involves de novo formation of centrioles. This chapter includes a brief introduction to planarian spermatogenesis and current tools for the analysis of molecular players in this process. Step-by-step protocols for isolating and imaging spermatogenic cells are provided with enough detail to be carried out by newcomers to the field who would like to study this unique organism in the laboratory.

METTL3-dependent MALAT1 delocalization drives c-Myc induction in thymic epithelial tumors

BackgroundThymic epithelial tumors (TETs) are rare neoplasms, originating from epithelial thymic cells. The oncogenic potential of these rare neoplasms is still largely undefined, and a deeper molecular characterization could result in a relevant advance in their management, greatly improving diagnosis, prognosis and treatment choice. Deregulation of N6-methyladenosine (m6A) RNA modification, catalyzed by the METTL3/METTL14 methyltransferase complex, is emerging as a relevant event in cell differentiation and carcinogenesis. Various studies have reported that altered expression of METTL3 is associated with an aggressive malignant phenotype and favors migration and invasiveness, but its role in Thymic Tumors remains unknown.ResultsIn this study, we characterized that METTL3 contributes to Thymic Epithelial Tumor phenotype. We evidenced that METTL3 is overexpressed in tumor tissue compared to normal counterpart. Silencing of METTL3 expression in thymic carcinoma cells results in reduced cell proliferation and overall translation rate. Of note, METTL3 is responsible for the induction of c-MYC expression in TET cells. Specifically, high expression of c-MYC protein is enabled by lncRNA MALAT1, which is methylated and delocalized by METTL3. Interestingly, blocking of c-MYC by using JQ1 inhibitor cooperates with METTL3 depletion in the inhibition of proliferation and induction of cell death.ConclusionThis study highlighted METTL3 as a tumor promoter in Thymic tumors and c-MYC as a promising target to be exploited for the treatment of TET.

Single-cell transcriptomic analysis of bloodstream Trypanosoma brucei reconstructs cell cycle progression and developmental quorum sensing

Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts. Here, using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative ‘slender’ to transmissible ‘stumpy’ bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. We additionally map the cell cycle of proliferating parasites and position stumpy cell-cycle exit at early G1 before progression to a distinct G0 state. A null mutant for one transiently elevated developmental regulator, ZC3H20 is further analysed by scRNA-seq, identifying its point of failure in the developmental atlas. This approach provides a paradigm for the dissection of differentiation events in parasites, relevant to diverse transitions in pathogen biology.

Abstract P501: Comprehensive Analysis Of Alternative Polyadenylation In Human Left Ventricle Failure By Using The Novel Algorithm: Polya-miner

Background: Alternative polyadenylation (APA) is an emerging post-transcriptional mechanism for gene regulation that generates distinct isoforms of mRNA with different 3′ untranslated regions (3’UTR) lengths. APA plays an important role in different biological processes and dysregulation of APA leads to many human diseases. However, the functional consequences of APA events in the left ventricle (LV) failure in humans remain unexplored. Objective: To identify whether the 3′UTR length is modulated by APA in the LV failure in humans compared to healthy LV. Methods and Results: We used Poly(A)-ClickSeq RNA sequencing and PolyA-miner algorithm to measure the global patterns of APA in healthy and failing human LV specimens. We determined shortening versus lengthening of 3′UTRs based on the PolyA index, a metric unit that determines the length of 3′UTR. Based on these scores, we identified 129 genes with a significant shift of cleavage site usage in failing LV compared to healthy LV specimens. By examining polyadenylation events in these hearts, we identified disease-specific APA signatures in many genes. In addition, differential APA events in LV failure regulate many pathways important for the progression of LV failure. Finally, the regulator proteins of APA including cleavage and polyadenylation specificity factor (CPSF) 6 and 7, cleavage factor Im (CFIm) 25 and 59 have been regulated in LV failure compared to healthy LV specimens. Conclusions: Our results provide genome-wide, polyadenylation maps of the human heart and show that APA of mRNA is dynamic in the progression of LV failure in humans. Demonstrating that APA mediated 3’UTR length regulation provides the additional layer of gene expressions in LV failure.

Abstract P507: Comprehensive Characterization Of Poly(a)-clickseq Data Sets Reveal Novel Polyadenylation Signals In Human Right Ventricle Failure

Background: Many human genes hold more than 1 poly(A) site, and polyadenylation gives rise to transcripts with different 3′ untranslated regions (3’UTR) lengths, and this process known as alternative polyadenylation (APA). It plays a significant role in many cellular processes and dysregulation of APA leads to many human diseases. However, the functional consequences of APA events in the right ventricle (RV) failure in humans remain unexplored. Objective: To understand whether a global APA event is presented in the progression of RV failure by using the novel algorithm PolyA miner in the RV of healthy donors and patients with RV failure. Methods and Results: RV tissue samples were obtained from failing human hearts at the time of cardiac transplantation at the Houston Methodist DeBakey Heart & Vascular Center, Houston Methodist Hospital. The RV tissues were immediately frozen in liquid nitrogen and stored at –80°C until use. Normal tissue samples were obtained from donor hearts that were not used for transplantation and were collected and stored in the same manner. We characterized the APA profiles of RV failing patients and compared them to healthy RV specimens by using Poly(A)-ClickSeq (PAC-seq) RNA sequencing and the PolyA-miner algorithm. We determined shortening versus lengthening of 3′UTRs based on the PolyA index, a metric unit that determines the length of 3′UTR. Based on these scores, we identified 435 transcripts with a significant shift in cleavage site usage. Further, we discovered 3'UTR shortening and lengthening of many genes in failing RV compared to healthy RV specimens. By examining polyadenylation events in these hearts, we identified disease-specific APA signatures in many genes. In addition, differential APA events in RV failure regulate many pathways important for the progression of the RV failure. Conclusions: Our study highlights the important roles for APA in human RV failure, including reforming multiple pathways and regulating specific gene expression, representing the complex interplay between APA and other biological processes in the RV failure.

CD8+ TILs in NSCLC differentiate into TEMRA via a bifurcated trajectory: deciphering immunogenicity of tumor antigens

BackgroundCD8+ tumor-infiltrating lymphocytes (TILs) comprise phenotypically and functionally heterogeneous subpopulations. Of these, effector memory CD45RA re-expressing CD8+ T cells (Temra) have been discovered and characterized as the most terminally differentiated...

932Using marginal structural models to account for selection bias in the analysis of legacy effect

Abstract Background Analyses of legacy effects are mainly based on post-trial follow-up study after initial randomized controlled trials (RCT). However, the differential event rates between arms may cause a violation of randomization balance and induce selection bias in the post-trial analysis. We conducted a simulation to illustrate the bias and explore if marginal structural model (MSM) can address it. Methods Our simulation combined an RCT and an extended follow-up study. The scenarios investigated include different settings of direct treatment effect, legacy effect and underlying event rate. To fit the MSM, we used the inverse probability weighting method. The performance of MSM was compared to the standard model with and without adjustment of baseline covariate. Results Post-trial analysis without making adjustment resulted in biased estimates, and the bias increased with the underlying event rate and treatment effect. Both MSM and standard baseline covariate adjustment equally corrected for the bias if no patients took treatment after the trial. We are currently undertaking analysis for scenarios where some people continue treatment post-trial, and there is treatment confounder feedback; these results will be presented at the Congress. Conclusions Estimation of legacy effects using post-trial data without adjusting for differential survival between randomised treatment arms results in biased estimates. Although both standard covariate adjustment and MSM correct the bias if no patients take treatment in the post-trial period, MSM is expected to be the best method in the more realistic scenario where some patients continue to take treatment, and there is treatment confounder feedback. Key messages Post-trial analysis without making adjustment results in biased estimation of legacy effect. Marginal structural models may be used to address the selection bias.