Temporal Patterns Of Gene Expression Research Articles

Nuclear morphogenesis: forming a heterogeneous nucleus during embryogenesis.

ABSTRACTAn embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in Drosophila melanogaster embryos, we observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes. Accordingly, transcription sites driven by different cis-regulatory regions each had their own temporally and spatially varying local histone environments, which could facilitate the finer spatial and temporal regulation of genes to consistently differentiate cells into organs and tissues. Thus, ‘nuclear morphogenesis’ may be a microscopic counterpart of the macroscopic process that shapes the animal body.

Interpretation of exercise-induced changes in human skeletal muscle mRNA expression depends on the timing of the post-exercise biopsies.

BackgroundExercise elicits a range of adaptive responses in skeletal muscle, which include changes in mRNA expression. To better understand the health benefits of exercise training, it is important to investigate the underlying molecular mechanisms of skeletal muscle adaptation to exercise. However, most studies have assessed the molecular events at only a few time-points within a short time frame post-exercise, and the variations of gene expression kinetics have not been addressed systematically.MethodsWe assessed the mRNA expression of 23 gene isoforms implicated in the adaptive response to exercise at six time-points (0, 3, 9, 24, 48, and 72 h post exercise) over a 3-day period following a single session of high-intensity interval exercise.ResultsThe temporal patterns of target gene expression were highly variable and the expression of mRNA transcripts detected was largely dependent on the timing of muscle sampling. The largest fold change in mRNA expression of each tested target gene was observed between 3 and 72 h post-exercise.Discussion and ConclusionsOur findings highlight an important gap in knowledge regarding the molecular response to exercise, where the use of limited time-points within a short period post-exercise has led to an incomplete understanding of the molecular response to exercise. Muscle sampling timing for individual studies needs to be carefully chosen based on existing literature and preliminary analysis of the molecular targets of interest. We propose that a comprehensive time-course analysis on the exercise-induced transcriptional response in humans will significantly benefit the field of exercise molecular biology.

Myocardial Rev-erb-Mediated Diurnal Metabolic Rhythm and Obesity Paradox.

The nuclear receptor Rev-erbα/β, a key component of the circadian clock, emerges as a drug target for heart diseases, but the function of cardiac Rev-erb has not been studied in vivo. Circadian disruption is implicated in heart diseases, but it is unknown whether cardiac molecular clock dysfunction is associated with the progression of any naturally occurring human heart diseases. Obesity paradox refers to the seemingly protective role of obesity for heart failure, but the mechanism is unclear. We generated mouse lines with cardiac-specific Rev-erbα/β knockout (KO), characterized cardiac phenotype, conducted multi-omics (RNA-sequencing, chromatin immunoprecipitation sequencing, proteomics, and metabolomics) analyses, and performed dietary and pharmacological rescue experiments to assess the time-of-the-day effects. We compared the temporal pattern of cardiac clock gene expression with the cardiac dilation severity in failing human hearts. KO mice display progressive dilated cardiomyopathy and lethal heart failure. Inducible ablation of Rev-erbα/β in adult hearts causes similar phenotypes. Impaired fatty acid oxidation in the KO myocardium, in particular, in the light cycle, precedes contractile dysfunctions with a reciprocal overreliance on carbohydrate utilization, in particular, in the dark cycle. Increasing dietary lipid or sugar supply in the dark cycle does not affect cardiac dysfunctions in KO mice. However, obesity coupled with systemic insulin resistance paradoxically ameliorates cardiac dysfunctions in KO mice, associated with rescued expression of lipid oxidation genes only in the light cycle in phase with increased fatty acid availability from adipose lipolysis. Inhibition of glycolysis in the light cycle and lipid oxidation in the dark cycle, but not vice versa, ameliorate cardiac dysfunctions in KO mice. Altered temporal patterns of cardiac Rev-erb gene expression correlate with the cardiac dilation severity in human hearts with dilated cardiomyopathy. The study delineates temporal coordination between clock-mediated anticipation and nutrient-induced response in myocardial metabolism at multi-omics levels. The obesity paradox is attributable to increased cardiac lipid supply from adipose lipolysis in the fasting cycle due to systemic insulin resistance and adiposity. Cardiac molecular chronotypes may be involved in human dilated cardiomyopathy. Myocardial bioenergetics downstream of Rev-erb may be a chronotherapy target in treating heart failure and dilated cardiomyopathy.

Analysis of yellow mutant rainbow trout transcriptomes at different developmental stages reveals dynamic regulation of skin pigmentation genes

Yellow mutant rainbow trout (YR), an economically important aquaculture species, is popular among consumers due to its excellent meat quality and attractive appearance. Skin color is a key economic trait for YR, but little is known about the molecular mechanism of skin color development. In this study, YR skin transcriptomes were analyzed to explore temporal expression patterns of pigmentation-related genes in three different stages of skin color development. In total, 16,590, 16,682, and 5619 genes were differentially expressed between fish at 1 day post-hatching (YR1d) and YR45d, YR1d and YR90d, and YR45d and YR90d. Numerous differentially expressed genes (DEGs) associated with pigmentation were identified, and almost all of them involved in pteridine and carotenoid synthesis were significantly upregulated in YR45d and YR90d compared to YR1d, including GCH1, PTS, QDPR, CSFIR1, SLC2A11, SCARB1, DGAT2, PNPLA2, APOD, and BCO2. Interestingly, many DEGs enriched in melanin synthesis pathways were also significantly upregulated, including melanogenesis (MITF, MC1R, SLC45A2, OCA2, and GPR143), tyrosine metabolism (TYR, TYRP1, and DCT), and MAPK signaling (KITA) pathways. Using short time-series expression miner, we identified eight differential gene expression pattern profiles, and DEGs in profile 7 were associated with skin pigmentation. Protein–protein interaction network analysis showed that two modules were related to xanthophores and melanophores. In addition, 1,812,329 simple sequence repeats and 2,011,334 single-nucleotide polymorphisms were discovered. The results enhance our understanding of the molecular mechanism underlying skin pigmentation in YR, and could accelerate the molecular breeding of fish species with valuable skin color traits and will likely be highly informative for developing new therapeutic approaches to treat pigmentation disorders and melanoma.

Characterization of Squamosa-Promoter Binding Protein-Box Family Genes Reveals the Critical Role of MsSPL20 in Alfalfa Flowering Time Regulation.

SQUAMOSA Promoter-binding protein-Like (SPL) genes affect a broad range of plant biological processes and show potential application in crop improvement by genetic modification. As the most widely planted forage crop in the world, biomass and abiotic stresses tolerance are important breeding targets for alfalfa (Medicago sativa L.). Nevertheless, the systematic analysis of SPL genes in alfalfa genome remains lacking. In the present study, we characterized 22 putative non-redundant SPL genes in alfalfa genome and uncovered the abundant structural variation among MsSPL genes. The phylogenetic analysis of plant SPL proteins separated them into 10 clades and clade J was an alfalfa-specific clade, suggesting SPL genes in alfalfa might have experienced gene duplication and functional differentiation within the genome. Meanwhile, 11 MsSPL genes with perfect matches to miRNA response elements (MREs) could be degraded by miR156, and the cleavage sites were gene specific. In addition, we investigated the temporal and spatial expression patterns of MsSPL genes and their expression patterns in response to multiple treatments, characterizing candidate SPL genes in alfalfa development and abiotic stress tolerant regulation. More importantly, overexpression of the alfalfa-specific SPL gene (MsSPL20) showed stable delayed flowering time, as well as increased biomass. Further studies indicated that MsSPL20 delayed flowering time by regulating the expression of genes involved in floret development, including HD3A, FTIP1, TEM1, and HST1. Together, our findings provide valuable information for future research and utilization of SPL genes in alfalfa and elucidate a possibly alfalfa-specific flowering time regulation, thereby supplying candidate genes for alfalfa molecular-assisted breeding.

Versatile physiological functions of the Nudix hydrolase family in berry development and stress response in grapevine

Nudix hydrolases are widely distributed across all classes of organisms and provide the potential capacity to hydrolyze a wide range of organic pyrophosphates. Although Nudix hydrolases are involved in plant detoxification processes in response to abiotic and biotic stresses, the biological functions of Nudix hydrolases remain largely unclear in grapevine. In the present study, a total of 25 putative grapevine Nudix hydrolases (VvNUDXs) were identified by bioinformatics analysis and classified into eight subfamilies based to their preferred substrates. Both tandem and segmental duplications were responsible for the evolution and expansion of the NUDX gene family in grapevine. To investigate the regulatory roles of VvNUDX genes during growth and development, as well as in response to abiotic and biotic stresses in grapevine, the expression patterns were revealed in publicly available microarray data. The spatial and temporal expression patterns of the VvNUDX genes indicated that they might play important roles in multiple developmental processes. Transcriptome and qRT-PCR analyses showed that ten VvNUDX genes were specifically expressed in grapevine berries, suggesting potential roles in grapevine berry development. Expression and phylogenetic analyses demonstrated that VvNUDX1 and VvNUDX3 might be involved in terpenoid biosynthesis in grapevine. Furthermore, most VvNUDX genes active toward the ADP-ribose/NADH showed different patterns in response to various abiotic and biotic stresses, such as salinity and drought, as well as different types of biotic treatments, such as Erysiphe necator, Bois Noir phytoplasma and leaf-roll-associated virus-3 (GLRaV-3). These results indicated that VvNUDX genes were associated with plant detoxification processes in response to abiotic and biotic stresses, and regulate the disease immunity and resistance pathways. The information obtained here may provide good opportunities to explore the physiological functions of VvNUDX genes in berry development and stress response networks in grapevine.

Embryonic expression patterns of TBC1D10 subfamily genes in zebrafish

TBC1D10 subfamily has three members TBC1D10A-C, with the physiological and pathological functions such as melanosome transport, exosome secretion, and T-cell activation. However, the gene expression patterns and functions of this subfamily during embryonic development remain mysterious. In this study, we took advantage of zebrafish model to elucidate the spatial and temporal expression patterns of TBC1D10 subfamily genes including tbc1d10aa, tbc1d10ab, tbc1d10b, and tbc1d10c. Whole-mount in situ hybridization results showed robust tbc1d10aa expression and faint tbc1d10b expression as maternal transcripts except tbc1d10ab and tbc1d10c. In addition to pectoral fins, otic vesicles, and pharyngeal arch tissues, varying degrees of expression of all four subfamily members were observed in brain tissues and eyes (retinal inner nuclear layer). Besides, tbc1d10ab exhibited unique and enriched expression in the developing liver. Despite genetic conservativeness, all four members of zebrafish TBC1D10 subfamily shared several similarities and exhibited some distinctions in the expression patterns, indicating that they might have different and exclusive functions to be further explored.

The age-dependent variations for fatty acid composition and sensory quality of chicken meat and associations between gene expression patterns and meat quality

Chicken meat is accompanied by physicochemical alterations during the process of growth and development, resulting in differences of nutritional value and meat flavor. The primary objective in the present study was to determine the mode of action on intramuscular fat content, serum lipid levels, fatty acid composition, and volatile flavor components of chicken meat. Negative correlations were observed between PUFA and aldehyde compounds and positive correlations were observed between PUFA and heterocyclic compounds. Intramuscular fat was correlated with only three volatile flavor compounds, indicating that the generation of volatile compounds was more dependent on the fatty acid composition rather than total intramuscular fat content. To explore the potential valuable biomarkers for sensorial and nutritional quality of chicken meat, a total of 12 temporal gene expression patterns were identified by transcriptomics. Module eigengenes 2 and 11 were significantly correlated with C14:0, C16:0, C18:1 cis-9, C18:2 cis-6, C18:3 n-3, C20:2, C20:3 n-6, C20:4 n-6, C22:6 n-3, C24:0, C24:0, C24:1, EFA, MUFA, PUFA, PUFA n-6 contents, and MUFA/SFA ratio, while were only significantly correlated with 1-octanol and 2-ethylhexyl glycidyl ether, respectively. Moreover, functional analysis of correlated-trait module eigengenes provided information on the explanation for the age-dependent variations for fatty acid composition and sensory quality of chicken meat.

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo.

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

Neuronal calcium sensor 1 (NCS1) dependent modulation of neuronal morphology and development.

Calcium (Ca2+ ) signaling is critical for neuronal functioning and requires the concerted interplay of numerous Ca2+ -binding proteins, including neuronal calcium sensor 1 (NCS1). Although an important role of NCS1 in neuronal processes and in neurodevelopmental and neurodegenerative diseases has been established, the underlying mechanisms remain enigmatic. Here, we systematically investigated the functions of NCS1 in the brain. Using Golgi-Cox staining, we observed a reduction in dendritic complexity and spine density in the prefrontal cortex and the dorsal hippocampus of Ncs1-/- mice, which may underlie concomitantly observed deficits in memory acquisition. Subsequent RNA sequencing of Ncs1-/- and Ncs1+/+ mouse brain tissues revealed that NCS1 modulates gene expression related to neuronal morphology and development. Investigation of developmental databases further supported a molecular role of NCS1 during brain development by identifying temporal gene expression patterns. Collectively, this study provides insights into NCS1-dependent signaling and lays the foundation for a better understanding of NCS1-associated diseases.

Characterization of embryo and protocorm development of Paphiopedilum spicerianum

Paphiopedilum spicerianum (P. spicerianum) is a rare orchid species with high ornamental value. Asymbiotic germination is the most efficient propagation method for conservation and commercial purposes because clonal propagation is very difficult and the separation of native species of Paphiopedilum through aseptic seeding is uncommon owing to their conservatism. However, a high protocorm developmental arresting rate during the asymbiotic germination is the major obstacle for seedling establishment. The fundamental understanding of embryo and protocorm developmental mechanisms will guide the development of an effective propagation method. The morphological and physiological characterization of the key developmental process of embryos and protocorms shows that the mature seeds of P. spicerianum consist of a spherical embryo without an endosperm. Seed coats become heavily lignified once the embryo is mature. Embryo cell size is relatively uniform, and significant structure polarity and cell size gradients occur at the early protocorm stage. The high level of auxin and cytokinin accumulation at the early stage of embryo development and protocorm stage may help to facilitate cell division. The transcriptome profiles of protocorms at three different developmental stages were compared to explore the regulatory mechanism of protocorm development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that differentially expressed genes were implicated in secondary metabolite metabolism, plant hormone signal transduction and photosynthesis. The temporal expression patterns of candidate genes related to embryo and shoot development were analyzed to reveal their roles in protocorm development: in the early stage of protocorm development, embryonic development related genes such as SERKs and BBM1 were active, while in the late stage of protocorm, shoot apical meristem related genes such as WOX8, CLAVATA2, CUC2, and SCR were active.

A transcriptional repressor BrDof2.4 regulates protease genes involved in postharvest leaf senescence in Chinese flowering cabbage

Protein degradation is an integral process of leaf senescence, and this catabolism is strongly associated with proteases in diverse plant species. However, the characterization of proteases as well as their upstream regulators during postharvest senescence, especially in economically important leafy vegetables such as Chinese flowering cabbage, remains to be determined. In this study, temporal and spatial gene expression patterns of eight proteases showed that three of them, namely, BrAPM1, BrASPG2 and BrSAG12, were most up-regulated during postharvest leaf senescence. Their expressions were also significantly induced by senescence-accelerating phytohormones abscisic acid (ABA) and methyl jasmonate (MeJA), but were inhibited by senescence-delaying hormones gibberellin (GA3) and cytokinin (6-BA). Importantly, using yeast one-hybrid screening, we identified a Dof (DNA-binding with one finger) transcription factor, BrDof2.4, as the putative binding protein of BrAPM1, BrASPG2 and BrSAG12 promoters. Nucleus-localized BrDof2.4 was senescence-, ABA- and MeJA-repressible, but was GA3- and 6-BA-inducible. Furthermore, gel mobility shift and transient dual luciferase reporter assays revealed that BrDof2.4 suppressed the transcription of BrAPM1, BrASPG2 and BrSAG12, through binding to their promoters. Taken together, our findings demonstrate that BrDof2.4 acts as a potential repressor of postharvest leaf senescence in Chinese flowering cabbage by the direct suppression of protease expression. Our work contributes towards improving the technology for increasing the shelf-life and commercial value of this important leafy vegetable.

Interpreting machine learning models to investigate circadian regulation and facilitate exploration of clock function

The circadian clock is an important adaptation to life on Earth. Here, we use machine learning to predict complex, temporal, and circadian gene expression patterns in Arabidopsis Most significantly, we classify circadian genes using DNA sequence features generated de novo from public, genomic resources, facilitating downstream application of our methods with no experimental work or prior knowledge needed. We use local model explanation that is transcript specific to rank DNA sequence features, providing a detailed profile of the potential circadian regulatory mechanisms for each transcript. Furthermore, we can discriminate the temporal phase of transcript expression using the local, explanation-derived, and ranked DNA sequence features, revealing hidden subclasses within the circadian class. Model interpretation/explanation provides the backbone of our methodological advances, giving insight into biological processes and experimental design. Next, we use model interpretation to optimize sampling strategies when we predict circadian transcripts using reduced numbers of transcriptomic timepoints. Finally, we predict the circadian time from a single, transcriptomic timepoint, deriving marker transcripts that are most impactful for accurate prediction; this could facilitate the identification of altered clock function from existing datasets.

RNA Sequencing Data-Driven Dissection of Human Plasma Cell Differentiation Reveals New Potential Transcription Regulators

Background: Plasma cells play an important role in the adaptive immune system through a continuous production of antibodies. Several hematological disorders, including some autoimmune diseases, multiple myeloma and plasmacytoma result from the malignant transformation of antibody-secreting cells. A clear understanding of the molecular processes underlying human plasma cell differentiation (PCD) and the long-term survival of ASCs will provide greater insight into such pathologies.Methods: We performed RNA-seq analysis to obtain a global transcriptomic map of human PCD including memory B cells, preplasmablasts, plasmablasts and plasma cells. We implemented a DESeq2-based workflow to identify genes with temporal expression pattern during PCD. We used Reactome pathway enrichment analysis to identify key pathways of PCD. We next focused our analysis on the identification of new potential transcription factors and epigenetic enzymes involved in PCD.Results: Our results reveal 8419 genes classified into four temporal gene expression patterns that we refer to one-step-up or one-step-down (mRNA level transitions from low to high or high to low, respectively, in two consecutive differentiation stages) and two-step-up-down (i.e. impulse-up) or two-step-down-up (i.e. impulse-down) (mRNA level transitions from low to high and back down or from high to low and back up, respectively, in a series of differentiation stages).Consistent with known biology, genes upregulated during PCD were mainly involved in protein modification and metabolism. Interestingly, a stringent Reactome pathway enrichment analysis of these expression pattern highlights pathways largely unknown in PCD, including the heme biosynthesis and the glutathione conjugaison pathways.To begin to understand thenature of the regulatory processes during human PCD, we focused on transcription factor (TF) and epigenetic enzyme (EE) genes. Collectively, we identified 617 TF/EE temporally regulated genes during PCD. 123 TF/EE genes fall into the one-step-up groups. 237 TF/EE genes are included in one-step-down groups, 49 in the impulse-up groups, 36 in the impulse-down groups.Among these TFs/EEs we further analyze those exhibiting a one-step-up or impulse-up pattern, because they are highly expressed in one or more PCD stages. Among the one-step-up TFs/EEs are those encoding well-known proteins such as IRF4, BLIMP1 and XBP1. Interestingly we found many other new transcription factors with unknown function and a potential importance in PCD. New TFs with the most consistent and marked overexpression are BATF2, BHLHA15, IRF2, ZSCAN20, MIXL1, MAF and STAT1. Furthermore, our analysis identified EE genes consistently upregulated during PCD including histone methyltransferases (PRDM1, PRDM15, PRMT7, SETDB2, SMYD2 and SMYD4), de-novo DNA methylation enzyme (DNMT3B), DNA methylation readers (MBD1 and ZBTB38), DNA methylation editors/erasers (IDH1, IDH2, TET1, ALKBH1, ALKBH3 and MGMT) and histone phosphorylation editor (EYA2 and EYA3). Additionally, our results reveal several interesting TFs/EEs genes among the genes included in the impulse-up group, including AICDA, MYB, BATF3, FOXM1, ARNTL2, SUV39H2, WHSC1, MYBL2, TP53, EZH2, SUV39H1 and PRMT1.Finally, we experimentally demonstrated that EZH2 inhibition, the catalytic component of polycomb repressive complex 2, affects the survival and the proliferation of plasma cell differentiation cell subpopulations, and significantly accelerates plasma cell differentiation.Conclusion: This analysis thus identifies a discrete set of genes that function together to program plasma cell differentiation. Many of these genes have not yet been implicated in plasma cell biology. Together, the RNA-Seq analysis of temporal stages of PCD helped to identify coexpressed gene sets with associated up- /down- regulated TF/EE genes that could represent regulatory nodes for plasma cell differentiation. DisclosuresNo relevant conflicts of interest to declare.

Disregulation of CTCF Boundary at the HOX Locus Enhances Sensitity to Multiple Drug Treatment in Myeloid Malignancies

Dysregulation of HOX genes is a dominant mechanism of leukemic transformation by altering self-renewal and differentiation properties of HS/PCs leading to leukemic transformation. The overexpression of posterior HOXA9 gene is also a poor prognostic marker in leukemia patients. Recent studies revealed that CTCF insulator constrains temporal HOX gene expression patterns within confined chromatin domains suggesting that CTCF mediated chromatin boundary activity may involve in regulating HOX gene patterns during AML prognosis.To investigate the role of CTCF in aberrant HOX gene expression and AML prognosis, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the core CTCF motif located between HOXA7 and HOXA9 genes (CBS7/9) in different subtype of AML cells with either MLL-rearrangement or NPM1 mutation. RNA-seq analysis revealed that transcription factors, HOXA9 , RUNX1 , and ZEB1 , as well as MAPK signaling pathway, all of them are involved in AML survival, prognosis and chemo-resistance, are significantly dowregulated in response to the CBS7/9 boundary deletion. Given that these genes and pathways are involved in AML prognosis and chemo-resistance, we then examined whether perturbation of CBS7/9 chromatin boundary resulted in alteration of drug resposes of AML cells. Both WT AML cells and the CBS7/9 defected AML cells derived from MLL-AF9 rearranged MOLM-13 and NPM mutated OCI-AML3 cells were treated with 1 mM of Ara-C or 100 nM of bromodomain (BET) inhibitors. Compared to the WT controls, the defected CBS7/9 boundary resulted in a significantly increase in AML sensitivity to both Ara-C and BET inhibitor treatment in both MOLM-13 and OCI-AML3 cells as evidence by significantly reducing percentage of proliferating cells.To further examine if treatment related drug sensitivity in AML cells is depended on the levels of HOXA9 expression, we overexpressed HOXA9 cDNA in the CBS7/9 deleted MOLM-13 and OCI-AML3 cells. Gain-of-function (GOF) expression of HOXA9 in the CBS7/9 defected AML cells rescued and restored the resistance to the Ara-C treatment of the CBS7/9 KO cells to the levels compatible to that of the WT control MOLM-13 and OCI-AML3 cells, but HOXA9 overexpression did not rescued the resistance of the CBS7/9 KO AML cells to the BET inhibitor treatment. The data suggest that HOXA9 is directly associated with cytotoxic drug Ara-C resistance, but not BET inhibitor mediated drug responses. KO of CBS7/9 specifically decreased enrichment of H3K9ac and H4K5ac, as well as recruitment of BRD4 to the posterior HOXA domain. Furthermore, overexpression of HOXA9 gene in the CBS7/9 KO AML cells indeed rescued the mRNA transcripts affected by CBS7/9 deletion to the levels compatible to that of WT control MOLM-13 or OCI-AML3 cells. Among rescued genes, ZEB1, STAT5A, and RUNX1 play essential roles in pathogenesis of myeloid malignancies and chemo-drug sensitivity.Finally, in order to further explore biological role of CTCF boundaries in AML pathogenesis, we analyze the whole genome sequencing data of 183 primary AML patients obtained from public available Cosmic Database for somatic mutations occurred in the CTCF binding sites. Several core CTCF motifs in the HOXA locus, including CBS5/6 and CBS7/9 boundaries, are highly mutated in AML patients. Thus, disruption of CTCF boundary located between HOXA7 and HOXA9 genes perturbs HOXA9 oncogene transcription and regulates chemo- or epigenetic drug sensitivity of acute myeloid leukemia. Alteration of CTCF boundaries in the oncogene loci may provide a novel therapeutic target for treatment of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Temporal transcriptomic landscape of postnatal mouse ovaries reveals dynamic gene signatures associated with ovarian aging

The ovary is the most important organ for maintaining female reproductive health, but it fails before most other organs. Aging-associated alterations in gene expression patterns in mammalian ovaries remain largely unknown. In this study, the transcriptomic landscape of postnatal mouse ovaries over the reproductive lifespan was investigated using bulk RNA sequencing in C57BL/6 mice. Gene expression dynamics revealed that the lifespan of postnatal mouse ovaries comprised four sequential stages, during which 2517 genes were identified as differentially enriched. Notably, the DNA repair pathway was found to make a considerable and specific contribution to the process of ovarian aging. Temporal gene expression patterns were dissected to identify differences in gene expression trajectories over the lifespan. In addition to DNA repair, distinct biological functions (including hypoxia response, epigenetic modification, fertilization, mitochondrial function, etc.) were overrepresented in particular clusters. Association studies were further performed to explore the relationships between known genes responsible for ovarian function and differentially expressed genes identified in this work. We found that the causative genes of human premature ovarian insufficiency were specifically enriched in distinct gene clusters. Taken together, our findings reveal a comprehensive transcriptomic landscape of the mouse ovary over the lifespan, providing insights into the molecular mechanisms underlying mammalian ovarian aging and supporting future etiological studies of aging-associated ovarian disorders.

Axial elongation of caudalized human organoids mimics aspects of neural tube development.

Axial elongation of the neural tube is crucial during mammalian embryogenesis for anterior-posterior body axis establishment and subsequent spinal cord development, but these processes cannot be interrogated directly in humans as they occur post-implantation. Here, we report an organoid model of neural tube extension derived from human pluripotent stem cell (hPSC) aggregates that have been caudalized with Wnt agonism, enabling them to recapitulate aspects of the morphological and temporal gene expression patterns of neural tube development. Elongating organoids consist largely of neuroepithelial compartments and contain TBXT+SOX2+ neuro-mesodermal progenitors in addition to PAX6+NES+ neural progenitors. A critical threshold of Wnt agonism stimulated singular axial extensions while maintaining multiple cell lineages, such that organoids displayed regionalized anterior-to-posterior HOX gene expression with hindbrain (HOXB1) regions spatially distinct from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR interference-mediated silencing of TBXT, a Wnt pathway target, increased neuroepithelial compartmentalization, abrogated HOX expression and disrupted uniaxial elongation. Together, these results demonstrate the potent capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early human nervous system development.

Transcriptome analysis associated with polysaccharide synthesis and their antioxidant activity in Cyclocarya paliurus leaves of different developmental stages.

BackgroundCyclocarya paliurus (Batal.) Iljinskaja is a common endemic tree species and used as a Chinese medicine. The main active components in the leaves of this plant are polysaccharides. However, the temporal patterns of gene expression underlying the synthesis of polysaccharides in C. paliurus at different leaf developmental stages and its relationship with the polysaccharide content and antioxidant activities has not been reported to date.MethodsRNA-seq was used to investigate the biosynthesis pathway of polysaccharides at the four developmental stages of C. paliurus leaves. The content and the antioxidant activities of polysaccharides were measured with typical biochemical methods and the identified correlations were statistically evaluated.ResultsSixty-nine differentially expressed genes were found in the leaves during different developmental stages of C. paliurus. These are associated with glycosyltransferases and belong to 18 families. During different developmental stages of C. paliurus, the polysaccharide content first increased and then decreased, and the UDP-glucose 4-epimerase gene was found to be significantly positively correlated with the polysaccharide content. The clearance rates of DPPH radicals, superoxide anion radicals, hydroxyl radicals, and the reducing power of polysaccharides in the leaves of C. paliurus at different developmental stages showed a dose-dependent relationship with the concentration of polysaccharides.ConclusionsThe smallest fully expanded leaves are suitable for high-quality tea, and leaves with sizes below the largest fully expanded leaves are suitable for industrial production of polysaccharides.

Enhancing Gonadotrope Gene Expression Through Regulatory lncRNAs.

The world of long non-coding RNAs (lncRNAs) has opened up massive new prospects in understanding the regulation of gene expression. Not only are there seemingly almost infinite numbers of lncRNAs in the mammalian cell, but they have highly diverse mechanisms of action. In the nucleus, some are chromatin-associated, transcribed from transcriptional enhancers (eRNAs) and/or direct changes in the epigenetic landscape with profound effects on gene expression. The pituitary gonadotrope is responsible for activation of reproduction through production and secretion of appropriate levels of the gonadotropic hormones. As such, it exemplifies a cell whose function is defined through changes in developmental and temporal patterns of gene expression, including those that are hormonally induced. Roles for diverse distal regulatory elements and eRNAs in gonadotrope biology have only just begun to emerge. Here, we will present an overview of the different kinds of lncRNAs that alter gene expression, and what is known about their roles in regulating some of the key gonadotrope genes. We will also review various screens that have detected differentially expressed pituitary lncRNAs associated with changes in reproductive state and those whose expression is found to play a role in gonadotrope-derived nonfunctioning pituitary adenomas. We hope to shed light on this exciting new field, emphasize the open questions, and encourage research to illuminate the roles of lncRNAs in various endocrine systems.