Dynamic Gene Expression Patterns Research Articles

Heat stress response in Chinese cabbage (Brassica rapa L.) revealed by transcriptome and physiological analysis.

High temperatures have a serious impact on the quality and yield of cold-loving Chinese cabbage, which has evolved to have a unique set of stress mechanisms. To explore the relationship between these mechanisms and the heat-tolerance of Chinese cabbage, the physiological indicators of the heat-tolerant ‘268’ line and heat-sensitive ‘334’ line were measured. Under heat stress, the proline (Pro), soluble sugar (SS), and superoxide dismutase (SOD) indexes of the ‘268’ line increased significantly. When additionally using transcriptome analysis, we found that the identified 3,360 DEGs were abundantly enriched in many metabolic pathways including ‘plant hormone signal transduction’, ‘carbon metabolism’, and ‘glycolysis/gluconeogenesis’. Dynamic gene expression patterns showed that HKL1 in Cluster 15 may be a key factor in the regulation of sugar homeostasis. The interaction network screened four ABA-related genes in Cluster 15, suggesting that high temperatures lead to changes in hormonal signaling, especially an increase in ABA signaling. Compared with the ‘334’ line, the expressions of Prx50, Prx52, Prx54, SOD1, and SOD2 in the ‘268’ line were significantly upregulated, and these genes were actively involved in the reactive oxygen species (ROS) scavenging process. In summary, our results revealed the relationship between plant heat tolerance, physiology, and biochemistry and may also provide ideas for the future development of high-quality and heat-tolerant Chinese cabbage germplasm resources.

The analysis of dynamic gene expression patterns in peripheral blood of multiple sclerosis patients indicates possible diagnostic and prognostic biomarkers

IntroductionAmong numerous invasive procedures for the research of biomarkers, blood-based indicators are regarded as marginally non-invasive procedures in the diagnosis and prognosis of demyelinating disorders, including multiple sclerosis (MS). In this study, we looked into the blood-derived gene expression profiles of patients with multiple sclerosis to investigate their clinical traits and linked them with dysregulated gene expressions to establish diagnostic and prognostic indicators. MethodsWe included 51 patients with relapsing-remitting MS (RRMS, n = 31), clinically isolated syndrome (CIS, n = 12), primary progressive MS (PPMS, n = 8) and a control group (n = 51). Using correlational analysis, the transcriptional patterns of chosen gene panels were examined and subsequently related with disease duration and the expanded disease disability score (EDSS). In addition, principal component analysis, univariate regression, and logistic regression analysis were employed to highlight distinct profiles of genes and prognosticate the excellent biomarkers of this illness. ResultsOur findings demonstrated that neurofilament light (NEFL), tumor necrosis factor α (TNF-α), Tau, and clusterin (CLU) were revealed to be increased in recruited patients, whereas the presenilin-1 (PSEN1) and cell-surface glycoprotein-44 (CD44) were downregulated. Principal Component Analysis revealed distinct patterns between the MS and control groups. Correlation analysis indicated co-dependent dysregulated genes and their differential expression with clinical findings. Furthermore, logistic regression demonstrated that Clusterin (AUC=0.940), NEFL (AUC=0.775), TNF-α (AUC=0.817), Tau (AUC=0.749), PSEN1 (AUC=0.6913), and CD44 (AUC=0.832) had diagnostic relevance. Following the univariate linear regression, a significant regression equation was found between EDSS and IGF-1 (R2 adj = 0.10844; p= 0.0060), APP (R2 adj = 0.1107; p= 0.0098), and PSEN1 (R2 adj = 0.1266; p=0.0102). ConclusionThis study exhibits dynamic gene expression patterns that represent the significance of specified genes that are prospective diagnostic and prognostic biomarkers for multiple sclerosis.

ECO: An Integrated Gene Expression Omnibus for Mouse Endothelial Cells In Vivo.

Endothelial cell (EC) plays critical roles in vascular physiological and pathological processes. With the development of high-throughput technologies, transcriptomics analysis of EC has increased dramatically and a large amount of informative data have been generated. The dynamic patterns of gene expression in ECs under various conditions were revealed. Unfortunately, due to the lack of bioinformatics infrastructures, reuse of these large-scale datasets is challenging for many scientists. Here, by systematic re-analyzing, integrating, and standardizing of 203 RNA sequencing samples from freshly isolated mouse ECs under 71 conditions, we constructed an integrated mouse EC gene expression omnibus (ECO). The ECO database enables one-click retrieval of endothelial expression profiles from different organs under different conditions including disease models, genetic modifications, and clinically relevant treatments in vivo. The EC expression profiles are visualized with user-friendly bar-plots. It also provides a convenient search tool for co-expressed genes. ECO facilitates endothelial research with an integrated tool and resource for transcriptome analysis. The ECO database is freely available at https://heomics.shinyapps.io/ecodb/.

Transcriptome Analysis of Developing Wheat Grains at Rapid Expanding Phase Reveals Dynamic Gene Expression Patterns.

Simple SummaryUnderstanding the regulatory mechanism underlying grain development is essential for wheat improvement. The early grain expanding phase boasts critical biological events like embryogenesis and initiation of grain filling. RNA sequencing analysis of this developmental stage revealed dynamic expressions of genes related to cell division, starch biosynthesis, and hormone biosynthesis. An unbalanced expression among triads may play critical roles as shown by multiple enriched metabolic pathways. Our work demonstrated complex regulation mechanisms in early grain development and provided useful information for future wheat improvement.Grain development, as a vital process in the crop’s life cycle, is crucial for determining crop quality and yield. The wheat grain expanding phase is the early process involving the rapid morphological changes and initiation of grain filling. However, little is known about the molecular basis of grain development at this stage. Here, we provide a time-series transcriptome profile of developing wheat grain at 0, 2, 4, 6, 8, and 10 days after pollination of the wheat landrace Chinese Spring. A total of 26,892 differentially expressed genes, including 1468 transcription factors, were found between adjacent time points. Co-expression cluster analysis and Gene Ontology enrichment revealed dynamic expressions of cell division and starch biosynthesis related structural genes and transcription factors. Moreover, diverse, differential and drastically varied expression trends of the key genes related to hormone metabolism were identified. Furthermore, ~30% of triads showed unbalanced expression patterns enriching for genes in multiple pivotal metabolic pathways. Hormone metabolism related genes, such as YUC10 (YUCCA flavin-containing monooxygenase 10), AOS2 (allene oxide synthase 2), CYP90D2 (cytochrome P450 90D2), and CKX1 (cytokinin dehydrogenase 1), were dominantly contributed by A or D homoeologs of the triads. Our study provided a systematic picture of transcriptional regulation of wheat grains at the early grain expanding phase which should deepen our understanding of wheat grain development and help in wheat yield improvement.

Identification of a modular super-enhancer in murine retinal development

Super-enhancers are expansive regions of genomic DNA comprised of multiple putative enhancers that contribute to the dynamic gene expression patterns during development. This is particularly important in neurogenesis because many essential transcription factors have complex developmental stage– and cell–type specific expression patterns across the central nervous system. In the developing retina, Vsx2 is expressed in retinal progenitor cells and is maintained in differentiated bipolar neurons and Müller glia. A single super-enhancer controls this complex and dynamic pattern of expression. Here we show that deletion of one region disrupts retinal progenitor cell proliferation but does not affect cell fate specification. The deletion of another region has no effect on retinal progenitor cell proliferation but instead leads to a complete loss of bipolar neurons. This prototypical super-enhancer may serve as a model for dissecting the complex gene expression patterns for neurogenic transcription factors during development. Moreover, it provides a unique opportunity to alter expression of individual transcription factors in particular cell types at specific stages of development. This provides a deeper understanding of function that cannot be achieved with traditional knockout mouse approaches.

Multiomics landscape of the autosomal dominant osteopetrosis type II disease-specific induced pluripotent stem cells

BackgroundAutosomal dominant osteopetrosis type II (ADO2) is a genetically and phenotypically metabolic bone disease, caused by osteoclast abnormalities. The pathways dysregulated in ADO2 could lead to the defects in osteoclast formation and function. However, the mechanism remains elusive.Materials and methodsTo systematically explore the molecular characterization of ADO2, we performed a multi-omics profiling from the autosomal dominant osteopetrosis type II iPSCs (ADO2-iPSCs) and healthy normal control iPSCs (NC-iPSCs) using whole genome re-sequencing, DNA methylation and N6-methyladenosine (m6A) analysis in this study.ResultsTotally, we detected 7,095,817 single nucleotide polymorphisms (SNPs) and 1,179,573 insertion and deletions (InDels), 1,001,943 differentially methylated regions (DMRs) and 2984 differential m6A peaks, and the comprehensive multi-omics profile was generated from the two cells. Interestingly, the ISG15 m6A level in ADO2-iPSCs is higher than NC-iPSCs by IGV software, and the differentially expressed m6A-modified genes (DEMGs) were highly enriched in the osteoclast differentiation and p53 signaling pathway, which associated with the development of osteopetrosis. In addition, combining our previously published transcriptome and proteome datasets, we found that the change in DNA methylation levels correlates inversely with some gene expression levels.ConclusionOur results indicate that the global multi-omics landscape not only provides a high-quality data resource but also reveals a dynamic pattern of gene expression, and found that the pathogenesis of ADO2 may begin early in life.

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.

Transcriptional signatures underlying dynamic phenotypic switching and novel disease biomarkers in a linear cellular model of melanoma progression

Despite advances in therapeutics, the progression of melanoma to metastasis still confers a poor outcome to patients. Nevertheless, there is a scarcity of biological models to understand cellular and molecular changes taking place along disease progression. Here, we characterized the transcriptome profiles of a multi-stage murine model of melanoma progression comprising a nontumorigenic melanocyte lineage (melan-a), premalignant melanocytes (4C), nonmetastatic (4C11-) and metastasis-prone (4C11+) melanoma cells. Clustering analyses have grouped the 4 cell lines according to their differentiated (melan-a and 4C11+) or undifferentiated/“mesenchymal-like” (4C and 4C11-) morphologies, suggesting dynamic gene expression patterns associated with the transition between these phenotypes. The cell plasticity observed in the murine melanoma progression model was corroborated by molecular markers described during stepwise human melanoma differentiation, as the differentiated cell lines in our model exhibit upregulation of transitory and melanocytic markers, whereas “mesenchymal-like” cells show increased expression of undifferentiated and neural crest-like markers. Sets of differentially expressed genes (DEGs) were detected at each transition step of tumor progression, and transcriptional signatures related to malignancy, metastasis and epithelial-to-mesenchymal transition were identified. Finally, DEGs were mapped to their human orthologs and evaluated in uni- and multivariate survival analyses using gene expression and clinical data of 703 drug-naïve primary melanoma patients, revealing several independent candidate prognostic markers. Altogether, these results provide novel insights into the molecular mechanisms underlying the phenotypic switch taking place during melanoma progression, reveal potential drug targets and prognostic biomarkers, and corroborate the translational relevance of this unique sequential model of melanoma progression.

Integrative pan cancer analysis reveals epigenomic variation in cancer type and cell specific chromatin domains

Epigenetic mechanisms contribute to the initiation and development of cancer, and epigenetic variation promotes dynamic gene expression patterns that facilitate tumor evolution and adaptation. While the NCI-60 panel represents a diverse set of human cancer cell lines that has been used to screen chemical compounds, a comprehensive epigenomic atlas of these cells has been lacking. Here, we report an integrative analysis of 60 human cancer epigenomes, representing a catalog of activating and repressive histone modifications. We identify genome-wide maps of canonical sharp and broad H3K4me3 domains at promoter regions of tumor suppressors, H3K27ac-marked conventional enhancers and super enhancers, and widespread inter-cancer and intra-cancer specific variability in H3K9me3 and H4K20me3-marked heterochromatin domains. Furthermore, we identify features of chromatin states, including chromatin state switching along chromosomes, correlation of histone modification density with genetic mutations, DNA methylation, enrichment of DNA binding motifs in regulatory regions, and gene activity and inactivity. These findings underscore the importance of integrating epigenomic maps with gene expression and genetic variation data to understand the molecular basis of human cancer. Our findings provide a resource for mining epigenomic maps of human cancer cells and for identifying epigenetic therapeutic targets.

Deciphering the dynamic gene expression patterns of pollen abortion in a male sterile line of Avena sativa through transcriptome analysis at different developmental stages

BackgroundMale sterility (MS) has important applications in hybrid seed production, and the abortion of anthers has been observed in many plant species. While most studies have focused on the genetic factors affecting male sterility, the dynamic gene expression patterns of pollen abortion in male sterile lines have not been fully elucidated. In addition, there is still no hybrid oat that is commercially planted due to the lack of a suitable system of male sterility for hybrid breeding.ResultsIn this study, we cultivated a male sterile oat line and a near-isogenic line by crossbreeding to elucidate the expression patterns of genes that may be involved in sterility. The first reported CA male sterile (CAMS) oat line was used for cross-testing and hybridization experiments and was confirmed to exhibit a type of nuclear sterility controlled by recessive genes. Oat stamens of two lines were sampled at four different developmental stages separately. Paired-end RNA sequencing was performed for each sample and generated 252.84 Gb sequences. There were 295,462 unigenes annotated in public databases in all samples, and we compared the histological characteristics and transcriptomes of oat stamens from the two oat lines at different developmental stages. Our results demonstrate that the sterility of the male sterile oat line occurs in the early stage of stamen development and is primarily attributable to abnormal meiosis and the excessive accumulation of superoxide.ConclusionsTo the best of our knowledge, this study is the first to decipher the dynamic expression profiles of pollen abortion CAMS and CA male fertile (CAMF) oat lines, which may represent a valuable resource for further studies attempting to understand pollen abortion and anther development in oats.

Zucchini Plants Alter Gene Expression and Emission of (E)-β-Caryophyllene Following Aphis gossypii Infestation.

Zucchini (Cucurbita pepo L.) is widely cultivated in temperate regions. One of the major production challenges is the damage caused by Aphis gossypii (Homoptera: Aphididae), a polyphagous aphid, which can negatively affect its host plant, both directly by feeding and indirectly by vectoring viruses. To gain insights into the transcriptome events that occur during the zucchini–aphid interaction and to understand the early-to-late defense response through gene expression profiles, we performed RNA-sequencing (RNA-Seq) on zucchini leaves challenged by A. gossypii (24, 48, and 96 h post-infestation; hpi). Data analysis indicated a complex and dynamic pattern of gene expression and a transient transcriptional reconfiguration that involved more than 700 differentially expressed genes (DEGs), including a large number of defense-related genes. The down-regulation of key genes of plant immunity, such as leucine-rich repeat (LRR) protein kinases, transcription factors, and genes associated with direct (i.e., protease inhibitors, cysteine peptidases, etc.) and indirect (i.e., terpene synthase) defense responses, suggests the aphid ability to manipulate plant immune responses. We also investigated the emission of volatile organic compounds (VOCs) from infested plants and observed a reduced emission of (E)-β-caryophyllene at 48 hpi, likely the result of aphid effectors, which reflects the down-regulation of two genes involved in the biosynthesis of terpenoids. We showed that (E)-β-caryophyllene emission was modified by the duration of plant infestation and by aphid density and that this molecule highly attracts Aphidius colemani, a parasitic wasp of A. gossypii. With our results we contributed to the identification of genes involved in cucurbit plant interactions with phloem feeders. Our findings may also help pave the way toward developing tolerant zucchini varieties and to identify molecules for sustainable management of harmful insect populations.

Single-Cell Transcriptome Analysis Reveals Dynamic Cell Populations and Differential Gene Expression Patterns in Control and Aneurysmal Human Aortic Tissue.

Ascending thoracic aortic aneurysm (ATAA) is caused by the progressive weakening and dilatation of the aortic wall and can lead to aortic dissection, rupture, and other life-threatening complications. To improve our understanding of ATAA pathogenesis, we aimed to comprehensively characterize the cellular composition of the ascending aortic wall and to identify molecular alterations in each cell population of human ATAA tissues. We performed single-cell RNA sequencing analysis of ascending aortic tissues from 11 study participants, including 8 patients with ATAA (4 women and 4 men) and 3 control subjects (2 women and 1 man). Cells extracted from aortic tissue were analyzed and categorized with single-cell RNA sequencing data to perform cluster identification. ATAA-related changes were then examined by comparing the proportions of each cell type and the gene expression profiles between ATAA and control tissues. We also examined which genes may be critical for ATAA by performing the integrative analysis of our single-cell RNA sequencing data with publicly available data from genome-wide association studies. We identified 11 major cell types in human ascending aortic tissue; the high-resolution reclustering of these cells further divided them into 40 subtypes. Multiple subtypes were observed for smooth muscle cells, macrophages, and T lymphocytes, suggesting that these cells have multiple functional populations in the aortic wall. In general, ATAA tissues had fewer nonimmune cells and more immune cells, especially T lymphocytes, than control tissues did. Differential gene expression data suggested the presence of extensive mitochondrial dysfunction in ATAA tissues. In addition, integrative analysis of our single-cell RNA sequencing data with public genome-wide association study data and promoter capture Hi-C data suggested that the erythroblast transformation-specific related gene(ERG) exerts an important role in maintaining normal aortic wall function. Our study provides a comprehensive evaluation of the cellular composition of the ascending aortic wall and reveals how the gene expression landscape is altered in human ATAA tissue. The information from this study makes important contributions to our understanding of ATAA formation and progression.

Quantitative gene expression dynamics of key placode signalling factors in the embryonic chicken scleral ossicle system

Development of the scleral ossicles, a ring of bony elements within the sclera, is directed by a series of papillae that arise from placodes in the conjunctival epithelium over a 1.5-day induction period in the chicken embryo. The regular spacing of the papillae around the corneal-scleral limbus suggests that their induction may be regulated by a reaction-diffusion mechanism, similar to other epithelial appendages. Some key placode signalling molecules, including β-catenin, are known to be expressed throughout the induction period. However, others have been studied only at certain stages or have not been successfully detected. Here we use qPCR to study the gene expression patterns of the wingless integration (WNT)/β-catenin, bone morphogenetic protein (BMP), ectodysplasin (EDA), fibroblast growth factor (FGF) and hedgehog (HH) signalling families in discrete regions of the eye throughout the complete conjunctival placode and papillae induction period. This comprehensive analysis revealed a variable level of gene expression within specific eye regions, with some genes exhibiting high, moderate or low changes. Most genes exhibited an initial increase in gene expression, followed by a decrease or plateau as development proceeded, suggesting that some genes are important for a brief initial period whilst the sustained elevated expression level of other genes is needed for developmental progression. The timing or magnitude of these changes, and/or the overall gene expression trend differed in the temporal, nasal and/or dorsal eye regions for some, but not all genes, demonstrating that gene expression may vary across different eye regions. Temporal and nasal EDA receptor (EDAR) had the greatest number of strong correlations (r > 0.700) with other genes and β-catenin had the greatest number of moderate correlations (r = 0.400–0.700), while EDA had the greatest range in correlation strengths. Among the strongly correlated genes, two distinct signalling modules were identified, connected by some intermediate genes. The dynamic gene expression patterns of the five signalling pathways studied here from conjunctival placode formation through to papillae development is consistent with other epithelial appendages and confirms the presence of a conserved induction and patterning signalling network. Two unique gene expression patterns and corresponding gene interaction modules suggest functionally distinct roles throughout placode development. Furthermore, spatial differences in gene expression patterns among the temporal, nasal and dorsal regions of the eye may indicate that the expression of certain genes is influenced by mechanical forces exerted throughout development. Therefore, this study identifies key placode signalling factors and their interactions, as well as some potential region-specific features of gene expression in the scleral ossicle system and provides a basis for further exploration of the spatial expression of these genes and the patterning mechanism(s) active throughout conjunctival placode and papillae formation.

Analysis of expression of candidate genes for polycystic ovary syndrome in adult and fetal human and fetal bovine ovaries†

Polycystic ovary syndrome (PCOS) appears to have a genetic predisposition and a fetal origin. We compared the expression levels of 25 PCOS candidate genes from adult control and PCOS human ovaries (n=16) using microarrays. Only one gene was potentially statistically different. Using qRT-PCR, expression of PCOS candidate genes was examined in bovine fetal ovaries from early stages when they first developed stroma through to completion of development (n=27; 60-270days of gestation). The levels of ERBB3 mRNA negatively correlated with gestational age but positively with HMGA2, FBN3, TOX3, GATA4, and DENND1A.X1,2,3,4, previously identified as correlated with each other and expressed early. PLGRKT and ZBTB16, and less so IRF1, were also correlated with AMH, FSHR, AR, INSR, and TGFB1I1, previously identified as correlated with each other and expressed late. ARL14EP, FDFT1, NEIL2, and MAPRE1 were expressed across gestation and not correlated with gestational age as shown previously for THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX, and KRR1. LHCGR, because of its unusual bimodal expression pattern, had some unusual correlations with other genes. In human ovaries (n=15; <150days of gestation), ERBB3.V1 and ERBB3.VS were expressed and correlated negatively with gestational age and positively with FBN3, HMGA2, DENND1A.V1,3,4, DENND1A.V1-7, GATA4, and FSHR, previously identified as correlated with each other and expressed early. Thus, the general lack of differential expression of candidate genes in adult ovaries contrasting with dynamic patterns of gene expression in fetal ovaries is consistent with a vulnerability to disturbance in the fetal ovary that may underpin development of PCOS.

Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing.

Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.

Independent evolution of transcript abundance and gene regulatory dynamics.

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans. Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.

Epigenetic regulation of cortical neurogenesis; orchestrating fate switches at the right time and place.

Over the last several decades the field has made tremendous progress in understanding the proliferative behavior of cortical progenitors and the lineage relationships of their clonal progeny. The genetic and epigenetic mechanisms that control the dynamic patterns of gene expression during cortical development are only beginning to be characterized. In this review we highlight the most well characterized epigenetic modifications and their influence on progenitor proliferation and cortical neuron cell fate.

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Temporal transcriptome change of Oncomelania hupensis revealed by Schistosoma japonicum invasion

BackgroundThe freshwater snail Oncomelania hupensis is the obligate intermediate host for Schistosoma japonicum in China. Transcriptomic examination of snail–schistosome interactions can provide valuable information of host response at physiological and immune levels.MethodsTo investigate S. japonicum-induced changes in O. hupensis gene expression, we utilized high-throughput sequencing to identify transcripts that were differentially expressed between infected snails and their uninfected controls at two key time-point, Day 7 and Day 30 after challenge. Time-series transcriptomic profiles were analyzed using R package DESeq 2, followed by GO, KEGG and (weighted gene correlation network analysis) WGCNA analysis to elucidate and identify important molecular mechanism, and subsequently understand host–parasite relationship. The identified unigenes was verified by bioinformatics and real-time PCR. Possible adaptation molecular mechanisms of O. hupensis to S. japonicum challenge were proposed.ResultsTranscriptomic analyses of O. hupensis by S. japonicum invasion yielded billion reads including 92,144 annotated transcripts. Over 5000 differentially expressed genes (DEGs) were identified by pairwise comparisons of infected libraries from two time points to uninfected libraries in O. hupensis. In total, 6032 gene ontology terms and 149 KEGG pathways were enriched. After the snails were infected with S. japonicum on Day 7 and Day 30, DEGs were shown to be involved in many key processes associated with biological regulation and innate immunity pathways. Gene expression patterns differed after exposure to S. japonicum. Using WGCNA, 16 modules were identified. Module-trait analysis identified that a module involved in RNA binding, ribosome, translation, mRNA processing, and structural constituent of ribosome were strongly associated with S. japonicum invasion. Many of the genes from enriched KEGG pathways were involved in lysosome, spliceosome and ribosome, indicating that S. japonicum invasion may activate the regulation of ribosomes and immune response to infection in O. hupensis.ConclusionsOur analysis provided a temporally dynamic gene expression pattern of O. hupensis by S. japonicum invasion. The identification of gene candidates serves as a foundation for future investigations of S. japonicum infection. Additionally, major DEGs expression patterns and putative key regulatory pathways would provide useful information to construct gene regulatory networks between host-parasite crosstalk.

Time-Course Transcriptome Analysis of Gingiva-Derived Mesenchymal Stem Cells Reveals That Fusobacterium nucleatum Triggers Oncogene Expression in the Process of Cell Differentiation.

Fusobacterium nucleatum has pathogenic effects on oral squamous cell carcinoma and colon cancer, while the effects of continuously altered gene expression in normal human cells, as induced by persistent exposure to F. nucleatum, remain unclear. In this study, a microarray Significant Profiles (maSigPro) analysis was used to obtain the transcriptome profile of gingiva-derived mesenchymal stem cells (GMSCs) stimulated by F. nucleatum for 3, 7, 14, and 21 day, and the results revealed 790 (nine clusters) differentially expressed genes (DEGs), which were significantly enriched in cell adherens junctions and cancer-related pathways. On the basis of a short time-series expression miner (STEM) analysis, all the expressed genes in the GMSCs were grouped into 50 clusters according to dynamic gene expression patterns, and the expression levels of three gene clusters in the F. nucleatum-treated GMSCs were significantly different than the predicted values. Among the 790 DEGs, 50 tumor-associated genes (TAGs; such as L3MBTL4, CD163, CCCND2, CADM1, BCL7A, and IGF1) and five core dynamic DEGs (PLCG2, CHI3L2, L3MBTL4, SH2D2A, and NLRP3) were identified during F. nucleatum stimulation. Results from a GeneMANIA database analysis showed that PLCG2, CHI3L2, SH2D2A, and NLRP3 and 20 other proteins formed a complex network of which 12 genes were enriched in cancer-related pathways. Based on the five core dynamic DEGs, the related microRNAs (miRNAs) and transcription factors (TFs) were obtained from public resources, and an integrated network composed of the related TFs, miRNAs, and mRNAs was constructed. The results indicated that these genes were regulated by several miRNAs, such as miR-372-3p, miR-603, and miR-495-3p, and several TFs, including CREB3, GATA2, and SOX4. Our study suggests that long-term stimulation by F. nucleatum may trigger the expression of cancer-related genes in normal gingiva-derived stem cells.