Transcriptome Regulation Research Articles

Long-Term Transcriptomic Changes and Cardiomyocyte Hyperpolyploidy after Lactose Intolerance in Neonatal Rats.

Many cardiovascular diseases originate from growth retardation, inflammation, and malnutrition during early postnatal development. The nature of this phenomenon is not completely understood. Here we aimed to verify the hypothesis that systemic inflammation triggered by neonatal lactose intolerance (NLI) may exert long-term pathologic effects on cardiac developmental programs and cardiomyocyte transcriptome regulation. Using the rat model of NLI triggered by lactase overloading with lactose and the methods of cytophotometry, image analysis, and mRNA-seq, we evaluated cardiomyocyte ploidy, signs of DNA damage, and NLI-associated long-term transcriptomic changes of genes and gene modules that differed qualitatively (i.e., were switched on or switched off) in the experiment vs. the control. Our data indicated that NLI triggers the long-term animal growth retardation, cardiomyocyte hyperpolyploidy, and extensive transcriptomic rearrangements. Many of these rearrangements are known as manifestations of heart pathologies, including DNA and telomere instability, inflammation, fibrosis, and reactivation of fetal gene program. Moreover, bioinformatic analysis identified possible causes of these pathologic traits, including the impaired signaling via thyroid hormone, calcium, and glutathione. We also found transcriptomic manifestations of increased cardiomyocyte polyploidy, such as the induction of gene modules related to open chromatin, e.g., "negative regulation of chromosome organization", "transcription" and "ribosome biogenesis". These findings suggest that ploidy-related epigenetic alterations acquired in the neonatal period permanently rewire gene regulatory networks and alter cardiomyocyte transcriptome. Here we provided first evidence indicating that NLI can be an important trigger of developmental programming of adult cardiovascular disease. The obtained results can help to develop preventive strategies for reducing the NLI-associated adverse effects of inflammation on the developing cardiovascular system.

Spatiotemporally resolved transcriptomics reveals the subcellular RNA kinetic landscape

Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap—a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed ‘kinetic gene clusters’ whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.

Circadian regulation of hippocampal function is disrupted with corticosteroid treatment

Disrupted circadian activity is associated with many neuropsychiatric disorders. A major coordinator of circadian biological systems is adrenal glucocorticoid secretion which exhibits a pronounced preawakening peak that regulates metabolic, immune, and cardiovascular processes, as well as mood and cognitive function. Loss of this circadian rhythm during corticosteroid therapy is often associated with memory impairment. Surprisingly, the mechanisms that underlie this deficit are not understood. In this study, in rats, we report that circadian regulation of the hippocampal transcriptome integrates crucial functional networks that link corticosteroid-inducible gene regulation to synaptic plasticity processes via an intrahippocampal circadian transcriptional clock. Further, these circadian hippocampal functions were significantly impacted by corticosteroid treatment delivered in a 5-d oral dosing treatment protocol. Rhythmic expression of the hippocampal transcriptome, as well as the circadian regulation of synaptic plasticity, was misaligned with the natural light/dark circadian-entraining cues, resulting in memory impairment in hippocampal-dependent behavior. These findings provide mechanistic insights into how the transcriptional clock machinery within the hippocampus is influenced by corticosteroid exposure, leading to adverse effects on critical hippocampal functions, as well as identifying a molecular basis for memory deficits in patients treated with long-acting synthetic corticosteroids.

Single-cell characteristics and malignancy regulation of alpha-fetoprotein-producing gastric cancer.

To characterize alpha-fetoprotein (AFP)-producing gastric cancer (AFPGC) at the single-cell level and to identify regulatory factors for AFP expression and malignancy. ScRNA-seq was performed on two tumors collected from patients with AFPGC. InferCNV and sub-clustering were applied to identify typical AFPGC cells, followed by AddModuleScore, pathway enrichment, Pseudo-time, and Scenic analyses. Data from a gastric cancer (GC) cohort were collected for conjoint analysis. The analytical results were verified by cell experiments and immunohistochemistry. AFPGC cells are similar to hepatocytes in transcriptome and transcriptional regulation, with kinetic malignancy-related pathways, compared to the common malignant epithelium. In addition, compared to common GC cells, malignancy-related pathways, such as epithelial-mesenchymal transition (EMT) and angiogenesis, were upregulated in AFPGC. Mechanistically, Dickkopf-1 (DKK1) was found to be associated with AFP expression and malignant phenotype upon combining our scRNA-seq data with a public database, which was further verified by a series of in vitro experiments and immunohistochemistry. We demonstrated the single-cell characteristics of AFPGC and that DKK1 facilitates AFP expression and malignancy.

Abstract 3564: SNAI2 enhances oncogenesis in malignant peripheral nerve sheath tumors

Abstract Background: MPNSTs are highly aggressive soft tissue sarcomas. Recurrent genetic mutations have been linked to its tumorigenesis, including the loss of functional polycomb repressive complex 2 (PRC2). This results in an altered transcription landscape characterized by the global loss of methylation on histone H3 lysine 27 (H3K27me3) and increased super-enhancer (SE) -associated histone acetylation. We have previously identified SNAI2 as a core SE-driven transcription factor (TF) that is essential for the growth of MPNST. Although SNAI2 is known to be critical for oncogenic processes, including epithelial-to-mesenchymal transition (EMT) and dedifferentiation, its role in maintaining MPNST survival through transcriptomic and epigenetic regulation has not been explored. Methods: Using CRISPR mediated knockout (KO), we targeted the SNAI2 DNA binding domain and confirmed KO via western blot, followed by in vitro phenotypic characterization, including 2D growth assays, anchorage-independent colony formation assays, and scratch assays. Additionally, an integrative analysis of SNAI2 genomic distribution and the transcriptomic effect of its KO was performed using chromatin immunoprecipitation coupled with DNA sequencing (ChIPseq) and RNA sequencing (RNAseq). Finally, an inducible re-expression system was employed to examine the effects on the chromatin landscape with SNAI2 rescue. Results: Genetic loss of SNAI2 slowed the growth of MPNST cells by 50% in comparison to the control. In vitro tumorigenic assays revealed that SNAI2 KO diminished the cells’ capacity in invasion but not in anchorage-independent cell growth. Analysis of the RNAseq results revealed that SNAI2 loss upregulated 790 genes and downregulated 1623 genes (log2fold change> 1, p<0.01), indicating a dominant transcriptional activation role. Gene Set Enrichment Analysis of these SNAI2-regulated genes revealed upregulation of hallmark Myc targets (normalized enrichment score (NES)=2.6, FDR=0.0), whilst genes involved in response to alpha interferon proteins were downregulated (NES=-1.87, FDR=0.002). Interestingly, genes defining EMT were upregulated (NES=1.6, FDR=0.006) in cells upon SNAI2 loss, contrasting its known repressive role in this biological process in other systems. Through integrative analysis of the RNAseq and ChIPseq data, we demonstrate SNAI2 binding to promoters of its regulated targets. Downstream targets of interest include CRELD1, a gene involved in cell adhesion, where SNAI2 KO caused loss of promoter binding and gene downregulation (p<0.001). Conclusion: SNAI2 is an essential SE-driven TF in MPNST cells. Transcriptomic and genomic profiling of SNAI2 through loss- and gain-of-function experiments revealed that it is a critical TF that is involved in key biological processes. Future efforts will focus on designing methods to employ targeted degradation of this essential TF and test preclinical efficacy in treating MPNST. Citation Format: Hilda Jafarah, Béga Murray, Jack Shern, Xiyuan Zhang. SNAI2 enhances oncogenesis in malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3564.

Changes and transcriptome regulation of endogenous hormones during somatic embryogenesis in Ormosia henryi Prain.

Ormosia henryi is a rare and endangered plant growing in southern China. Somatic embryo culture is an effective measure for the rapid propagation of O. henryi. It has not been reported how regulatory genes induce somatic embryogenesis by regulating endogenous hormone changes during the process of somatic embryogenesis in O. henryi. In this study, we analysed the endogenous hormone levels and transcriptome data of nonembryogenic callus (NEC), embryogenic callus (EC), globular embryo (GE) and cotyledon embryo (CE) in O. henryi. The results showed that the indole-3-acetic acid (IAA) content was higher and the cytokinins (CKs) content was lower in EC than in NEC, and the gibberellins (GAs) and abscisic acid (ABA) contents were significantly higher in NEC than in EC. The contents of IAA, CKs, GAs and ABA increased significantly with EC development. The expression patterns of differentially expressed genes (DEGs) involved in the biosynthesis and signal transduction of auxin (AUX) (YUCCA and SAUR), CKs (B-ARR), GAs (GA3ox, GA20ox, GID1 and DELLA) and ABA (ZEP, ABA2, AAO3, CYP97A3, PYL and ABF) were consistent with the levels of endogenous hormones during somatic embryogenesis (SE). In this study, 316 different transcription factors (TFs) regulating phytohormones were detected during SE. AUX/IAA were downregulated in the process of EC formation and GE differentiation into CE, but other TFs were upregulated and downregulated. Therefore, we believe that relatively high IAA content and low CKs, GAs and ABA contents contribute to EC formation. The differential expression of AUX, CKs, GAs and ABA biosynthesis and signal transduction genes affected the endogenous hormone levels at different stages of SE in O. henryi. The downregulated expression of AUX/IAA inhibited NEC induction, promoted EC formation and GE differentiation into CE.

Potential Roles of m6A and FTO in Synaptic Connectivity and Major Depressive Disorder

RNA modifications known as epitranscriptomics have emerged as a novel layer of transcriptomic regulation. Like the well-studied epigenetic modifications characterized in DNA and on histone-tails, they have been shown to regulate activity-dependent gene expression and play a vital role in shaping synaptic connections in response to external stimuli. Among the hundreds of known RNA modifications, N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes. Through recognition of its binding proteins, m6A can regulate various aspects of mRNA metabolism and is essential for maintaining higher brain functions. Indeed, m6A is highly enriched in synapses and is involved in neuronal plasticity, learning and memory, and adult neurogenesis. m6A can also respond to environmental stimuli, suggesting an important role in linking molecular and behavioral stress. This review summarizes key findings from fields related to major depressive disorder (MDD) including stress and learning and memory, which suggest that activity-dependent m6A changes may, directly and indirectly, contribute to synaptic connectivity changes underlying MDD. Furthermore, we will highlight the roles of m6A and FTO, a m6A eraser, in the context of depressive-like behaviors. Although we have only begun to explore m6A in the context of MDD and psychiatry, elucidating a link between m6A and MDD presents a novel molecular mechanism underlying MDD pathogenesis.

A comparative gene expression matrix in Apoe-deficient mice identifies unique and atherosclerotic disease stage-specific gene regulation patterns in monocytes and macrophages

Background and aimsAtherosclerosis is a systemic and chronic inflammatory disease propagated by monocytes and macrophages. Yet, our knowledge on how transcriptome of these cells evolves in time and space is limited. We aimed at characterizing gene expression changes in site-specific macrophages and in circulating monocytes during the course of atherosclerosis. MethodsWe utilized apolipoprotein E-deficient mice undergoing one- and six-month high cholesterol diet to model early and advanced atherosclerosis. Aortic macrophages, peritoneal macrophages, and circulating monocytes from each mouse were subjected to bulk RNA-sequencing (RNA-seq). We constructed a comparative directory that profiles lesion- and disease stage-specific transcriptomic regulation of the three cell types in atherosclerosis. Lastly, the regulation of one gene, Gpnmb, whose expression positively correlated with atheroma growth, was validated using single-cell RNA-seq (scRNA-seq) of atheroma plaque from murine and human. ResultsThe convergence of gene regulation between the three investigated cell types was surprisingly low. Overall 3245 differentially expressed genes were involved in the biological modulation of aortic macrophages, among which less than 1% were commonly regulated by the remote monocytes/macrophages. Aortic macrophages regulated gene expression most actively during atheroma initiation. Through complementary interrogation of murine and human scRNA-seq datasets, we showcased the practicality of our directory, using the selected gene, Gpnmb, whose expression in aortic macrophages, and a subset of foamy macrophages in particular, strongly correlated with disease advancement during atherosclerosis initiation and progression. ConclusionsOur study provides a unique toolset to explore gene regulation of macrophage-related biological processes in and outside the atheromatous plaque at early and advanced disease stages.

Transcriptome Analysis Reveals Differentially Expressed Genes Involved in Cadmium and Arsenic Accumulation in Tea Plant (Camellia sinensis)

Tea (Camellia sinensis) is the second most consumed drink in the world. Rapid industrialization has caused various impacts on nature and increased pollution by heavy metals. However, the molecular mechanisms of cadmium (Cd) and arsenic (As) tolerance and accumulation in tea plants are poorly understood. The present study focused on the effects of heavy metals Cd and As on tea plants. Transcriptomic regulation of tea roots after Cd and As exposure was analyzed to explore the candidate genes involved in Cd and As tolerance and accumulation. In total, 2087, 1029, 1707, and 366 differentially expressed genes (DEGs) were obtained in Cd1 (with Cd treatment for 10 days) vs. CK (without Cd treatment), Cd2 (with Cd treatment for 15 days) vs. CK, As1 (with As treatment for 10 days) vs. CK (without Cd treatment), and As2 (with As treatment for 15 days) vs. CK, respectively. Analysis of DEGs showed that a total of 45 DEGs with the same expression patterns were identified in four pairwise comparison groups. One ERF transcription factor (CSS0000647) and six structural genes (CSS0033791, CSS0050491, CSS0001107, CSS0019367, CSS0006162, and CSS0035212) were only increased at 15 d of Cd and As treatments. Using weighted gene co-expression network analysis (WGCNA) revealed that the transcription factor (CSS0000647) was positively correlated with five structural genes (CSS0001107, CSS0019367, CSS0006162, CSS0033791, and CSS0035212). Moreover, one gene (CSS0004428) was significantly upregulated in both Cd and As treatments, suggesting that these genes might play important roles in enhancing the tolerance to Cd and As stresses. These results provide candidate genes to enhance multi-metal tolerance through the genetic engineering technology.

Characterization of immature ovarian teratomas through single-cell transcriptome.

Immature ovarian teratomas are a type of malignant germ cell tumor composed of complicated cell types and are characterized by pathological features of immature neuroectodermal tubules/rosettes. However, there is a lack of understanding of patient-derived immature ovarian teratomas (PDT) at the single cell level. Moreover, whether stem cell lines derived from immature teratomas (CDT) can be used as models for research on PDT remains to be elucidated. Single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatic analysis was performed on three patient-derived immature ovarian teratomas (PDT) samples to reveal the heterogeneity, evolution trajectory, and cell communication within the tumor microenvironment of PDT. Validations were conducted in additional seven samples through multiplex immunofluorescence. A total of qualified 22,153 cells were obtained and divided into 28 clusters, which can match to the scRNA-seq annotation of CDT as well as human fetal Cell Atlas, but with higher heterogeneity and more prolific cell-cell crosstalk. Radial glia cells (tagged by SOX2) and immature neuron (tagged by DCX) exhibited mutually exclusive expression and differentiated along distinct evolutionary trajectory from cycling neural progenitors. Proportions of these neuroectodermal cell subtypes may play important roles in PDT through contributing to the internal heterogeneity of PDTs. Moreover, the immune cells in PDTs were infiltrated rather than teratoma-derived, with more abundant macrophage in immature neuron than those in radial glia cells, and the infiltrated macrophage subtypes (i.e., M1 and M2) were significantly correlated to clinical grade. Overall, suppressed evolution process and transcriptome regulation in neuroectodermal cells, reduced cell-cell crosstalk, higher M1/M2 proportion ratio, and enhanced T cell effects in tumor microenvironment are enriched in patients with favorable prognosis. This study provides a comprehensive profile of PDT at the single cell level, shedding light on the heterogeneity and evolution of neuroectodermal cells within PDTs and the role of immune cells within the tumor microenvironment. Also, our findings highlight the potential usage of CDTs as a model for research on PDT.

Transcription of MERVL retrotransposons is required for preimplantation embryo development

Zygotic genome activation (ZGA) is a critical postfertilization step that promotes totipotency and allows different cell fates to emerge in the developing embryo. MERVL (murine endogenous retrovirus-L) is transiently upregulated at the two-cell stage during ZGA. Although MERVL expression is widely used as a marker of totipotency, the role of this retrotransposon in mouse embryogenesis remains elusive. Here, we show that full-length MERVL transcripts, but not encoded retroviral proteins, are essential for accurate regulation of the host transcriptome and chromatin state during preimplantation development. Both knockdown and CRISPRi-based repression of MERVL result in embryonic lethality due to defects in differentiation and genomic stability. Furthermore, transcriptome and epigenome analysis revealed that loss of MERVL transcripts led to retention of an accessible chromatin state at, and aberrant expression of, a subset of two-cell-specific genes. Taken together, our results suggest a model in which an endogenous retrovirus plays a key role in regulating host cell fate potential.

Hypoxia induces alterations in tRNA modifications involved in translational control

BackgroundAdaptation to high-altitude hypobaric hypoxia has been shown to require a set of physiological traits enabled by an associated set of genetic modifications, as well as transcriptome regulation. These lead to both lifetime adaptation of individuals to hypoxia at high altitudes and generational evolution of populations as seen for instance in those of Tibet. Additionally, RNA modifications, which are sensitive to environmental exposure, have been shown to play pivotal biological roles in maintaining the physiological functions of organs. However, the dynamic RNA modification landscape and related molecular mechanisms in mouse tissues under hypobaric hypoxia exposure remain to be fully understood. Here, we explore the tissue-specific distribution pattern of multiple RNA modifications across mouse tissues.ResultsBy applying an LC-MS/MS-dependent RNA modification detection platform, we identified the distribution of multiple RNA modifications in total RNA, tRNA-enriched fragments, and 17–50-nt sncRNAs across mouse tissues; these patterns were associated with the expression levels of RNA modification modifiers in different tissues. Moreover, the tissue-specific abundance of RNA modifications was sensitively altered across different RNA groups in a simulated high-altitude (over 5500 m) hypobaric hypoxia mouse model with the activation of the hypoxia response in mouse peripheral blood and multiple tissues. RNase digestion experiments revealed that the alteration of RNA modification abundance under hypoxia exposure impacted the molecular stability of tissue total tRNA-enriched fragments and isolated individual tRNAs, such as tRNAAla, tRNAval, tRNAGlu, and tRNALeu. In vitro transfection experiments showed that the transfection of testis total tRNA-enriched fragments from the hypoxia group into GC-2spd cells attenuated the cell proliferation rate and led to a reduction in overall nascent protein synthesis in cells.ConclusionsOur results reveal that the abundance of RNA modifications for different classes of RNAs under physiological conditions is tissue-specific and responds to hypobaric hypoxia exposure in a tissue-specific manner. Mechanistically, the dysregulation of tRNA modifications under hypobaric hypoxia attenuated the cell proliferation rate, facilitated the sensitivity of tRNA to RNases, and led to a reduction in overall nascent protein synthesis, suggesting an active role of tRNA epitranscriptome alteration in the adaptive response to environmental hypoxia exposure.

Severe Sarcoidosis Exhibits Loss of Immunological Regulation In MAPK, WNT, and PI3K Pathways

Sarcoidosis is a complex immunological disorder with varied progression. Disease course can vary greatly between mild and severe cases, with severe cases leading to systemic spread and organ damage. We hypothesized that the transcriptomic regulation of immune function differentiates severe and mild cases.

Post-Transcriptional Modification by Alternative Splicing and Pathogenic Splicing Variants in Cardiovascular Development and Congenital Heart Defects.

Advancements in genomics, bioinformatics, and genome editing have uncovered new dimensions in gene regulation. Post-transcriptional modifications by the alternative splicing of mRNA transcripts are critical regulatory mechanisms of mammalian gene expression. In the heart, there is an expanding interest in elucidating the role of alternative splicing in transcriptome regulation. Substantial efforts were directed toward investigating this process in heart development and failure. However, few studies shed light on alternative splicing products and their dysregulation in congenital heart defects (CHDs). While elegant reports showed the crucial roles of RNA binding proteins (RBPs) in orchestrating splicing transitions during heart development and failure, the impact of RBPs dysregulation or genetic variation on CHDs has not been fully addressed. Herein, we review the current understanding of alternative splicing and RBPs' roles in heart development and CHDs. Wediscuss the impact of perinatal splicing transition and its dysregulation in CHDs. We further summarize the discoveries made of causal splicing variants in key transcription factors that are implicated in CHDs. An improved understanding of the roles of alternative splicing in heart development and CHDs may potentially inform novel preventive and therapeutic advancements for newborn infants with CHDs.

Vascular mechanotransduction.

This review aims to survey the current state of mechanotransduction in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), including their sensing of mechanical stimuli and transduction of mechanical signals that result in the acute functional modulation and longer-term transcriptomic and epigenetic regulation of blood vessels. The mechanosensors discussed include ion channels, plasma membrane-associated structures and receptors, and junction proteins. The mechanosignaling pathways presented include the cytoskeleton, integrins, extracellular matrix, and intracellular signaling molecules. These are followed by discussions on mechanical regulation of transcriptome and epigenetics, relevance of mechanotransduction to health and disease, and interactions between VSMCs and ECs. Throughout this review, we offer suggestions for specific topics that require further understanding. In the closing section on conclusions and perspectives, we summarize what is known and point out the need to treat the vasculature as a system, including not only VSMCs and ECs but also the extracellular matrix and other types of cells such as resident macrophages and pericytes, so that we can fully understand the physiology and pathophysiology of the blood vessel as a whole, thus enhancing the comprehension, diagnosis, treatment, and prevention of vascular diseases.

Phenotypic variation of a new synthetic allotetraploid Arabidopsis kamchatica enhanced in natural environment.

The phenotypic variation of vegetative organs and reproductive organs of newly synthesized and natural Arabidopsis kamchatica genotypes was investigated in both a controlled environment and a natural environment in an experimental garden. When we compared the variation of their leaf shape as a vegetative organ, the synthetic A. kamchatica individuals grown in the garden showed larger variation compared with the individuals incubated in a growth chamber, suggesting enhanced phenotypic variation in a natural fluctuating environment. In contrast, the natural A. kamchatica genotypes did not show significant change in variation by growth condition. The phenotypic variation of floral organs by growth condition was much smaller in both synthetic and natural A. kamchatica genotypes, and the difference in variation width between the growth chamber and the garden was not significant in each genotype as well as among genotypes. The higher phenotypic variation in synthetic leaf may imply flexible transcriptomic regulation of a newly synthesized polyploid compared with a natural polyploid.

Polycomb Repressive Complex 2 in Oncology.

Dynamic regulation of the chromatin state by Polycomb Repressive Complex 2 (PRC2) provides an important mean for epigenetic gene control that can profoundly influence normal development and cell lineage specification. PRC2 and PRC2-induced methylation of histone H3 lysine 27 (H3K27) are critically involved in a wide range of DNA-templated processes, which at least include transcriptional repression and gene imprinting, organization of three-dimensional chromatin structure, DNA replication and DNA damage response and repair. PRC2-based genome regulation often goes wrong in diseases, notably cancer. This chapter discusses about different modes-of-action through which PRC2 and EZH2, a catalytic subunit of PRC2, mediate (epi)genomic and transcriptomic regulation. We will also discuss about how alteration or mutation of the PRC2 core or axillary component promotes oncogenesis, how post-translational modification regulates functionality of EZH2 and PRC2, and how PRC2 and other epigenetic pathways crosstalk. Lastly, we will briefly touch on advances in targeting EZH2 and PRC2 dependence as cancer therapeutics.

Nonalcoholic Steatohepatitis Disrupts Diurnal Liver Transcriptome Rhythms in Mice

The liver ensures organismal homeostasis through modulation of physiological functions over the course of the day. How liver diseases such as nonalcoholic steatohepatitis (NASH) affect daily transcriptome rhythms in the liver remains elusive. To start closing this gap, we evaluated the impact of NASH on the diurnal regulation of the liver transcriptome in mice. In addition, we investigated how stringent consideration of circadian rhythmicity affects the outcomes of NASH transcriptome analyses. Comparative rhythm analysis of the liver transcriptome from diet-induced NASH and control mice showed an almost 3-hour phase advance in global gene expression rhythms. Rhythmically expressed genes associated with DNA repair and cell-cycle regulation showed increased overall expression and circadian amplitude. In contrast, lipid and glucose metabolism-associated genes showed loss of circadian amplitude, reduced overall expression, and phase advances in NASH livers. Comparison of NASH-induced liver transcriptome responses between published studies showed little overlap (12%) in differentially expressed genes (DEGs). However, by controlling for sampling time and using circadian analytical tools, a 7-fold increase in DEG detection was achieved compared with methods without time control. NASH had a strong effect on circadian liver transcriptome rhythms with phase- and amplitude-specific effects for key metabolic and cell repair pathways, respectively. Accounting for circadian rhythms in NASH transcriptome studies markedly improves DEG detection and enhances reproducibility.

Transcriptomic Network Regulation of Rat Tooth Germ from Bell Differentiation Stage to Secretory Stage: MAPK Signaling Pathway Is Crucial to Extracellular Matrix Remodeling.

Hard tissues make up the vast majority of teeth and are mineralized from the surrounding matrix. If the development of tooth germ is affected during mineralization, hypoplasia of the tooth tissue can occur. To better understand the mechanisms mediating hypoplasia, we need to first study normal development. Using a rodent model, we highlight the transcriptomic changes that occur from the differentiation to secretion stages of mandibular molar germs. The tooth germ was dissected from rats at postnatal day 1.5 or 3.5 for high-throughput sequencing. Combining transcriptome analysis and DNA methylation, we identified 590 differentially expressed genes (436 upregulated and 154 downregulated) and 551 differentially expressed lncRNAs (long noncoding RNA; 369 upregulated and 182 downregulated) which were linked to the biological processes of odontogenesis, amelogenesis, tooth mineralization, and the alteration of extracellular matrix (ECM), especially matrix metalloproteinases (MMPs) and elastin. We found DNA methylation changes in 32 selected fragments involved in 5 chromosomes, 26 targets, and 2 haplotypes. Finally, three novel genes were identified: MMP20, Tgfb3, and Dusp1. Further analysis revealed that MMP20 has a role in odontogenesis and amelogenesis by influencing Slc24a4 and DSPP; Tgfb3 is involved in epithelial cell proliferation, cellular component disassembly process, ECM cellular component, and decomposition of cell components. But lncRNA expression could affect DNA methylation and mRNA expression. Moreover, the degree of DNA methylation could also affect the transcriptome level. Thus, Tgfb3 had no difference in DNA methylation, and Dusp1 conferred no difference at the transcriptome level. These three genes were all enriched in the MAPK pathway and played an important role in ECM remodeling. These data suggest that during the period of the bell differentiation stage to the secretory stage, along with enamel/dentin matrix secretion and hard tissue occurrence, the ECM is remodeled via MAPK signaling.

BCR-ABL1-driven exosome-miR130b-3p-mediated gap-junction Cx43 MSC intercellular communications imply therapies of leukemic subclonal evolution.

Rationale: In the bone marrow microenvironment (BMME), mesenchymal stem/stromal cells (MSCs) control the self-renewal of both healthy and cancerous hematopoietic stem/progenitor cells (HSPCs). We previously showed that in vivo leukemia-derived MSCs change neighbor MSCs into leukemia-permissive states and boost leukemia cell proliferation, survival, and chemotherapy resistance. But the mechanisms behind how the state changes are still not fully understood. Methods: Here, we took a reverse engineering approach to determine BCR-ABL1+ leukemia cells activated transcriptional factor C/EBPβ, resulting in miR130a/b-3p production. Then, we back-tracked from clinical specimen transcriptome sequencing to cell co-culture, molecular and cellular assays, flow cytometry, single-cell transcriptome, and transcriptional regulation to determine the molecular mechanisms of BCR-ABL1-driven exosome-miR130b-3p-mediated gap-junction Cx43 MSC intercellular communications. Results: BCR-ABL1-driven exosome-miR130a/b-3p mediated gap-junction Cx43 (a.k.a., GJA1) BMSC intercellular communications for subclonal evolution in leukemic microenvironment by targeting BMSCs-expressed HLAs, thereby potentially maintaining BMSCs with self-renewal properties and reduced BMSC immunogenicity. The Cx43low and miR-130a/bhigh subclonal MSCs subsets of differentiation state could be reversed to Cx43high and miR-130a/blow subclones of the higher stemness state in Cx43-overexpressed subclonal MSCs. Both miR-130a and miR-130b might only inhibit Cx43 translation or degrade Cx43 proteins and did not affect Cx43 mRNA stability. The subclonal evolution was further confirmed by single-cell transcriptome profiling of MSCs, which suggested that Cx43 regulated their stemness and played normal roles in immunomodulation antigen processing. Thus, upregulated miR-130a/b promoted osteogenesis and adipogenesis from BMSCs, thereby decreasing cancer progression. Our clinical data validated that the expression of many genes in human major histocompatibility was negatively associated with the stemness of MSCs, and several immune checkpoint proteins contributing to immune escape in tumors were overexpressed after either miR-130a or miR-130b overexpression, such as CD274, LAG3, PDCD1, and TNFRSF4. Not only did immune response-related cytokine-cytokine receptor interactions and PI3K-AKT pathways, including EGR3, TNFRSF1B, but also NDRG2 leukemic-associated inflammatory factors, such as IFNB1, CXCL1, CXCL10, and CCL7 manifest upon miR-130a/b overexpression. Either BCR siRNAs or ABL1 siRNAs assay showed significantly decreased miR-130a and miR-130b expression, and chromatin immunoprecipitation sequencing confirmed that the regulation of miR-130a and miR-130b expression is BCR-ABL1-dependent. BCR-ABL1 induces miR-130a/b expression through the upregulation of transcriptional factor C/EBPβ. C/EBPβ could bind directly to the promoter region of miR-130b-3p, not miR-130a-3p. BCR-ABL1-driven exosome-miR130a-3p could interact with Cx43, and further impact GJIC in TME. Conclusion: Our findings shed light on how leukemia BCR-ABL1-driven exosome-miR130b-3p could interact with gap-junction Cx43, and further impact GJIC in TME, implications for leukemic therapies of subclonal evolution.