Rapid Changes In Gene Expression Research Articles

Multi-scale mechanisms driving root regeneration: From regeneration competence to tissue repatterning.

Plants possess an outstanding capacity to regenerate enabling them to repair damages caused by suboptimal environmental conditions, biotic attacks, or mechanical damages impacting the survival of these sessile organisms. Although the extent of regeneration varies greatly between localized cell damage and whole organ recovery, the process of regeneration can be subdivided into a similar sequence of interlinked regulatory processes. That is, competence to regenerate, cell fate reprogramming, and the repatterning of the tissue. Here, using root tip regeneration as a paradigm system to study plant regeneration, we provide a synthesis of the molecular responses that underlie both regeneration competence and the repatterning of the root stump. Regarding regeneration competence, we discuss the role of wound signaling, hormone responses and synthesis, and rapid changes in gene expression observed in the cells close to the cut. Then, we consider how this rapid response is followed by the tissue repatterning phase, where cells experience cell fate changes in a spatial and temporal order to recreate the lost stem cell niche and columella. Lastly, we argue that a multi-scale modeling approach is fundamental to uncovering the mechanisms underlying root regeneration, as it allows to integrate knowledge of cell-level gene expression, cell-to-cell transport of hormones and transcription factors, and tissue-level growth dynamics to reveal how the bi-directional feedbacks between these processes enable self-organized repatterning of the root apex.

Differential Response of Transcription Factors to Activated Kinases in Steroidogenic and Non-Steroidogenic Cells.

Hormone-induced Leydig cell steroidogenesis requires rapid changes in gene expression in response to various hormones, cytokines, and growth factors. These proteins act by binding to their receptors on the surface of Leydig cells leading to activation of multiple intracellular signaling cascades, downstream of which are several kinases, including protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase I (CAMKI), and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). These kinases participate in hormone-induced steroidogenesis by phosphorylating numerous proteins including transcription factors leading to increased steroidogenic gene expression. How these various kinases and transcription factors come together to appropriately induce steroidogenic gene expression in response to specific stimuli remains poorly understood. In the present work, we compared the effect of PKA, CAMKI and ERK1/2 on the transactivation potential of 15 transcription factors belonging to 5 distinct families on the activity of the Star gene promoter. We not only validated known cooperation between kinases and transcription factors, but we also identified novel cooperations that have not yet been before reported. Some transcription factors were found to respond to all three kinases, whereas others were only activated by one specific kinase. Differential responses were also observed within a family of transcription factors. The diverse response to kinases provides flexibility to ensure proper genomic response of steroidogenic cells to different stimuli.

Touch signaling and thigmomorphogenesis are regulated by complementary CAMTA3- and JA-dependent pathways.

Plants respond to mechanical stimuli to direct their growth and counteract environmental threats. Mechanical stimulation triggers rapid gene expression changes and affects plant appearance (thigmomorphogenesis) and flowering. Previous studies reported the importance of jasmonic acid (JA) in touch signaling. Here, we used reverse genetics to further characterize the molecular mechanisms underlying touch signaling. We show that Piezo mechanosensitive ion channels have no major role in touch-induced gene expression and thigmomorphogenesis. In contrast, the receptor-like kinase Feronia acts as a strong negative regulator of the JA-dependent branch of touch signaling. Last, we show that calmodulin-binding transcriptional activators CAMTA1/2/3 are key regulators of JA-independent touch signaling. CAMTA1/2/3 cooperate to directly bind the promoters and activate gene expression of JA-independent touch marker genes like TCH2 and TCH4. In agreement, camta3 mutants show a near complete loss of thigmomorphogenesis and touch-induced delay of flowering. In conclusion, we have now identified key regulators of two independent touch-signaling pathways.

1020 – TARGETED PROTEIN DEGRADATION DEFINES DIRECT TARGETS AND MECHANISMS OF CONTROL BY RUNX1/ETO

Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements. Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements.

Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons.

SUMMARYAlthough axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin. Camptothecin increases ATF3 expression and promotes neurite outgrowth in sensory neurons in vitro and enhances axonal regeneration after sciatic nerve crush in vivo. Given the action of topoisomerases in producing DNA breaks, we determine that they do occur immediately after nerve damage at the ATF3 gene locus in injured sensory neurons and are further increased after camptothecin exposure. Formation of DNA breaks in injured sensory neurons and enhancement of it pharmacologically may contribute to the initiation of those transcriptional changes required for peripheral nerve regeneration.

Integrated Analysis of Transcriptome and Histone Modifications in Granulosa Cells During Ovulation in Female Mice.

The ovulatory luteinizing hormone (LH) surge induces rapid changes of gene expression and cellular functions in granulosa cells (GCs) undergoing luteinization. However, it remains unclear how the changes in genome-wide gene expression are regulated. H3K4me3 histone modifications are involved in the rapid alteration of gene expression. In this study, we investigated genome-wide changes of transcriptome and H3K4me3 status in mouse GCs undergoing luteinization. GCs were obtained from mice treated with equine chorionic gonadotropin (hCG) before, 4 hours, and 12 hours after human chorionic gonadotropin injection. RNA-sequencing identified a number of upregulated and downregulated genes, which could be classified into 8 patterns according to the time-course changes of gene expression. Many genes were transiently upregulated or downregulated at 4 hours after hCG stimulation. Gene Ontology terms associated with these genes included steroidogenesis, ovulation, cumulus-oocyte complex (COC) expansion, angiogenesis, immune system, reactive oxygen species (ROS) metabolism, inflammatory response, metabolism, and autophagy. The cellular functions of DNA repair and cell growth were newly identified as being activated during ovulation. Chromatin immunoprecipitation-sequencing revealed a genome-wide and rapid change in H3K4me3 during ovulation. Integration of transcriptome and H3K4me3 data identified many H3K4me3-associated genes that are involved in steroidogenesis, ovulation, COC expansion, angiogenesis, inflammatory response, immune system, ROS metabolism, lipid and glucose metabolism, autophagy, and regulation of cell size. The present results suggest that genome-wide changes in H3K4me3 after the LH surge are associated with rapid changes in gene expression in GCs, which enables GCs to acquire a lot of cellular functions within a short time that are required for ovulation and luteinization.

Prostaglandin E2 Prevents Apoptosis and Promotes Homeostasis of Hematopoietic Stem Cells after Lethal Irradiation through Alteration of p53 and TNF Signaling

Background: The hematopoietic system is the most sensitive organ to therapeutic or unintended radiation. In a mouse model of the hematopoietic acute radiation syndrome (H-ARS) a single dose of 16,16 dimethyl prostaglandin E2 (dmPGE2) given 15 min to 3 h before LD50/30-90/30 total body irradiation (TBI) provides 100% survival and accelerates hematopoietic recovery. The mechanisms responsible, however, are not known.Methods: C57BL/6 or FGD5-ZsGreen (JAX) mice received 35 µg dmPGE2 or vehicle 30 min before LD50/30 (853 cGy) γ-irradiation, along with non-irradiated dmPGE2 and vehicle controls. Bone marrow (BM) cells were harvested at multiple time points post-TBI and analyzed by flow cytometry for hematopoietic stem cell (HSC) markers as well as cleaved-caspase 3, cleaved-PARP, and p21. HSC were sorted for RNA-seq 1 h post-TBI based on SLAM-LS (Sca-1, CD150, CD48, Lineage-neg) and ZsGreen fluorescence. FGD5-ZsGreen mice express ZsGreen specifically in HSC, and were used to maximize HSC purity as the standard cKit marker is affected by irradiation. RNA-seq data were analyzed using Ingenuity Pathway Analysis.Results: The number of marker-defined HSC in dmPGE2-treated mice was preserved at 24 h post-TBI, but then declined to levels of vehicle-treated mice by day 3, implying that a qualitative rather than quantitative difference in HSC may be facilitating animal survival. To investigate gene expression changes in HSC after dmPGE2 treatment and irradiation, BM cells were harvested 1 h post-TBI and HSC sorted for RNA-seq. We first reasoned that changes observed with dmPGE2 alone may be involved in priming HSC to respond differently to irradiation, perhaps preserving HSC properties and function. The most activated upstream regulator by dmPGE2 alone was TNF, based on the expression patterns of known TNF-regulated genes, with an activation z-score of +5.26 (p = 1.39 x 10-23). Interestingly, TNF was also the upstream regulator most activated by irradiation alone (z-score +8.26, p = 5.88 x 10-37), to a greater degree than dmPGE2 alone, and different sets of TNF regulated genes were increased or decreased in each case. Significantly, with dmPGE2 plus irradiation, TNF-regulated genes in HSC adopted an expression pattern similar to dmPGE2-only, and generally opposite to irradiation alone. The direct comparison of dmPGE2-irradiation with vehicle-irradiation resulted in a significant net inhibition of TNF activation. Approximately one third of these altered genes are also NF-kB-regulated, suggesting that NF-kB-mediated TNF signaling induced by irradiation is prevented by dmPGE2 treatment. TNF mRNA itself was highly upregulated in both irradiated groups, but while TNF receptor 1 expression was not affected, dmPGE2 (with and without irradiation) significantly increased expression of TNF receptor 2, likely influencing TNF downstream effects.Strong changes were also seen in the p53-mediated apoptosis pathway. This pathway was not increased by dmPGE2 alone, but was greatly activated by irradiation (p = 1.47 x 10-27) and was the top inhibited regulator by dmPGE2-irradiation compared to irradiation alone (p = 1.86 x 10-18). At the protein level, a corresponding decrease in HSC production of cleaved caspase-3 and cleaved-PARP was detected from 1 h to 9 d post-TBI in PGE2-treated animals after irradiation, supporting its protection of HSC from irradiation-induced apoptosis. One dramatically affected gene involved in both the TNF and p53 pathways was the cdk inhibitor p21 (Cdkn1a). Confirming the observed gene expression pattern, p21 protein was substantially increased in HSC by 24 h post-TBI, and this was prevented by dmPGE2.Finally, irradiation stimulated a widespread increase in HSC transcriptional activity that was generally prevented by dmPGE2, suggesting preservation of a more quiescent state. In addition, transcription factors implicated in HSC maintenance/function were significantly decreased with irradiation and protected by dmPGE2, including Gata2, Meis1, Hoxa9, Tal1, and Gfi1.Conclusions: DmPGE2 induces rapid gene expression changes in HSC that dampen transcriptional activation induced by high-dose irradiation. This includes inhibiting activation of p53 and NFKB-mediated TNF activity, reducing apoptosis and maintaining essential HSC properties. This work provides insight into the mechanism of how dmPGE2 facilitates survival and hematopoietic recovery from lethal irradiation. DisclosuresNo relevant conflicts of interest to declare.

A stress-responsive miRNA regulates BMP signaling to maintain tissue homeostasis

Adult organisms must sense and adapt to environmental fluctuations. In high-turnover tissues such as the intestine, these adaptive responses require rapid changes in gene expression that, in turn, likely involve posttranscriptional gene control. However, intestinal-tissue-specific microRNA (miRNA)-mediated regulatory pathways remain unexplored. Here, we report the role of an intestinal-specific miRNA, miR-958, that non-cell autonomously regulates stem cell numbers during tissue homeostasis and regeneration in the Drosophila adult midgut. We identify its downstream target cabut, the Drosophila ortholog of mammalian KLF10/11 transcription factors, which mediates this miR-958 function by promoting paracrine enterocyte-to-stem-cell bone morphogenetic protein (BMP) signaling. We also show that mature miR-958 levels transiently decrease in response to stress and that this decrease is required for proper stem cell expansion during tissue regeneration. In summary, we have identified a posttranscriptional mechanism that modulates BMP signaling activity within Drosophila adult intestinal tissue during both normal homeostasis and tissue regeneration to regulate intestinal stem cell numbers.

Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation.

Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injuryand revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.

De novo assembly, annotation and gene expression profiles of gonads of Cytorace-3, a hybrid lineage of Drosophila nasuta nasuta and D. n. albomicans

Cytorace-3 is a laboratory evolved hybrid lineage of Drosophila nasuta nasuta males and Drosophila nasuta albomicans females currently passing ~850 generations. To assess interracial hybridization effects on gene expression in Cytorace-3 we profiled the transcriptomes of mature ovaries and testes by employing Illumina sequencing technology and de novo transcriptome assembling strategies. We found 26% of the ovarian, and 14% of testis genes to be differentially expressed in Cytorace-3 relative to the expressed genes in the parental gonadal transcriptomes. About 5% of genes exhibited additive gene expression pattern in the ovary and 3% in the testis, while the remaining genes were misexpressed in Cytorace-3. Nearly 772 of these misexpressed genes in the ovary and 413 in the testis were either over- or under-dominant. Genes following D. n. nasuta dominance was twice (270 genes) than D. n. albomicans dominance (133 genes) in the ovary. In contrast, only 105 genes showed D. n. nasuta dominance and 207 showed D. n. albomicans dominance in testis transcriptome. Of the six expression inheritance patterns, conserved inheritance pattern was predominant for both ovary (73%) and testis (85%) in Cytorace-3. This study is the first to provide an overview of the expression divergence and inheritance patterns of the transcriptomes in an independently evolving distinct hybrid lineage of Drosophila. This recorded expression divergence in Cytorace-3 surpasses that between parental lineages illustrating the strong impact of hybridization driving rapid gene expression changes.

Toward incorporating epigenetics into regulation of gene expression in the parasite Trichomonas vaginalis.

Trichomonas vaginalis is an extracellular parasite that colonizes the human urogenital tract, causing a highly prevalent sexually transmitted infection. The parasite must change its transcriptional profile in order to establish and maintain infection. However, few core regulatory elements and transcription factors have been identified to date and little is known about other mechanisms that may control these rapid changes in gene expression during parasite infection. In the last years, epigenetic mechanisms involved in the regulation of gene expression have been gaining major attention in this parasite. In this review, we summarize and discuss the major advances of the last few years with regard to epigenetics (DNA methylation, post-translational histone modifications, and histone variants) in the parasite T. vaginalis. These studies can shed light into our current understanding of this parasite's biology with far-reaching implications for the prognosis and treatment of trichomoniasis.

Dynamic chromatin accessibility landscape changes following interleukin-1 stimulation

ABSTRACT Dynamic modifications of chromatin allow rapid access of the gene regulatory machinery to condensed genomic regions facilitating subsequent gene expression. Inflammatory cytokine stimulation of cells can cause rapid gene expression changes through direct signalling pathway-mediated transcription factor activation and regulatory element binding. Here we used the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to assess regions of the genome that are differentially accessible following treatment of cells with interleukin-1 (IL-1). We identified 126,483 open chromatin regions, with 241 regions significantly differentially accessible following stimulation, with 64 and 177 more or less accessible, respectively. These differentially accessible regions predominantly correspond to regions of the genome marked as enhancers. Motif searching identified an overrepresentation of a number of transcription factors, most notably RelA, in the regions becoming more accessible, with analysis of ChIP-seq data confirmed RelA binding to these regions. A significant correlation in differential chromatin accessibility and gene expression was also observed. Functionality in regulating gene expression was confirmed using CRISPR/Cas9 genome-editing to delete regions that became more accessible following stimulation in the genes MMP13, IKBKE and C1QTNF1. These same regions were also accessible for activation using a dCas9-transcriptional activator and showed enhancer activity in a cellular model. Together, these data describe and functionally validate a number of dynamically accessible chromatin regions involved in inflammatory signalling.

Maternal Immune Activation Alters Fetal Brain Development and Enhances Proliferation of Neural Precursor Cells in Rats.

Maternal immune activation (MIA) caused by exposure to pathogens or inflammation during critical periods of neurodevelopment is a major risk factor for behavioral deficits and psychiatric illness in offspring. A spectrum of behavioral abnormalities can be recapitulated in rodents by inducing MIA using the viral mimetic, PolyI:C. Many studies have focused on long-term changes in brain structure and behavioral outcomes in offspring following maternal PolyI:C exposure, but acute changes in prenatal development are not well-characterized. Using RNA-Sequencing, we profiled acute transcriptomic changes in rat conceptuses (decidua along with nascent embryo and placenta) after maternal PolyI:C exposure during early gestation, which enabled us to capture gene expression changes provoked by MIA inclusive to the embryonic milieu. We identified a robust increase in expression of genes related to antiviral inflammation following maternal PolyI:C exposure, and a corresponding decrease in transcripts associated with nervous system development. At mid-gestation, regions of the developing cortex were thicker in fetuses prenatally challenged with PolyI:C, with females displaying a thicker ventricular zone and males a thicker cortical mantle. Along these lines, neural precursor cells (NPCs) isolated from fetal brains prenatally challenged with PolyI:C exhibited a higher rate of self-renewal. Expression of Notch1 and the Notch ligand, delta-like ligand 1, which are both highly implicated in maintenance of NPCs and nervous system development, was increased following PolyI:C exposure. These results suggest that MIA elicits rapid gene expression changes within the conceptus, including repression of neurodevelopmental pathways, resulting in profound alterations in fetal brain development.

Negative Elongation Factor (NELF) Regulates Muscle Progenitor Expansion for Efficient Myofiber Repair and Stem Cell Pool Repopulation

Negative Elongation Factor (NELF) is a critical transcriptional regulator that works through stabilizing paused RNA Polymerase to permit rapid gene expression changes in response to environmental cues. Whilst NELF is essential for embryonic development, its role in adult stem cells remains unclear. Here, through a muscle stem cell-specific deletion, we show that NELF is required for efficient muscle regeneration and replenishing the stem cell pool. Mechanistic studies using PRO-seq, single cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of muscle regeneration whereby it mediates massive expansion of muscle progenitors. Strikingly, transplantation experiments indicate that these progenitors are also necessary for stem cell pool repopulation implying they are able to return to quiescence. Thus, we identified a critical role for NELF in expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to stem cell pool repopulation.

Shedding light on the chromatin changes that modulate shade responses.

Perception of vegetation proximity or plant shade informs of potential competition for resources by the neighboring vegetation. As vegetation proximity impacts on both light quantity and quality, perception of this cue by plant photoreceptors reprograms development to result in responses that allow plants to compete with the neighboring vegetation. Developmental reprogramming involves massive and rapid changes in gene expression, with the concerted action of photoreceptors and downstream transcription factors. Changes in gene expression can be modulated by epigenetic processes that alter chromatin compaction, influencing the accessibility and binding of transcription factors to regulatory elements in the DNA. However, little is known about the epigenetic regulation of plant responses to the proximity of other plants. In this manuscript, we review what is known about plant shade effects on chromatin changes at the cytological level, that is, changes in nuclear morphology and high order chromatin density. We address which are the specific histone post-transcriptional modifications that have been associated with changes in shade-regulated gene expression, such as histone acetylation and histone methylation. Furthermore, we explore the possible mechanisms that integrate shade signaling components and chromatin remodelers to settle epigenetic marks at specific loci. This review aims to be a starting point to understand how a specific environmental cue, plant shade, integrates with chromatin dynamics to implement the proper acclimation responses.

Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes.

Human adipose-derived mesenchymal stromal cells (ASC) are showing clinical promise for the treatment of a range of inflammatory and degenerative conditions. These lipoaspirate-derived cells are part of the abundant and accessible source of heterogeneous stromal vascular fraction (SVF). They are typically isolated and expanded from the SVF via adherent cell culture for at least 2 weeks and as such represent a relatively undefined population of cells. We isolated ex vivo ASC directly from lipoaspirate using a cocktail of antibodies combined with immunomagnetic bead sorting. This method allowed for the rapid enrichment of a defined and untouched ex vivo ASC population (referred to as MACS-derived ASC) that were then compared to culture-derived ASC. This comparison found that MACS-derived ASC contain a greater proportion of cells with activity in in vitro differentiation assays. There were also significant differences in the secretion levels of some key paracrine molecules. Moreover, when the MACS-derived ASC were subjected to adherent tissue culture, rapid changes in gene expression were observed. This indicates that culturing cells may alter the clinical utility of these cells. Although MACS-derived ASC are more defined compared to culture-derived ASC, further investigations using a comprehensive multicolor flow cytometry panel revealed that this cell population is more heterogeneous than previously appreciated. Additional studies are therefore required to more precisely delineate phenotypically distinct ASC subsets with the most therapeutic potential. This research highlights the disparity between ex vivo MACS-derived and culture-derived ASC and the need for further characterization.

Comprehensive profiling of alternative splicing landscape during cold acclimation in tea plant

BackgroundAlternative splicing (AS) may generate multiple mRNA splicing isoforms from a single mRNA precursor using different splicing sites, leading to enhanced diversity of transcripts and proteins. AS has been implicated in cold acclimation by affecting gene expression in various ways, yet little information is known about how AS influences cold responses in tea plant (Camellia sinensis).ResultsIn this study, the AS transcriptional landscape was characterized in the tea plant genome using high-throughput RNA-seq during cold acclimation. We found that more than 41% (14,103) of genes underwent AS events. We summarize the possible existence of 11 types of AS events, including the four common types of intron retention (IR), exon skipping (ES), alternative 5′ splice site (A5SS), and alternative 3′ splice site (A3SS); of these, IR was the major type in all samples. The number of AS events increased rapidly during cold treatment, but decreased significantly following de-acclimation (DA). It is notable that the number of differential AS genes gradually increased during cold acclimation, and these genes were enriched in pathways relating to oxidoreductase activity and sugar metabolism during acclimation and de-acclimation. Remarkably, the AS isoforms of bHLH transcription factors showed higher expression levels than their full-length ones during cold acclimation. Interestingly, the expression pattern of some AS transcripts of raffinose and sucrose synthase genes were significantly correlated with sugar contents.ConclusionOur findings demonstrated that changes in AS numbers and transcript expression may contribute to rapid changes in gene expression and metabolite profile during cold acclimation, suggesting that AS events play an important regulatory role in response to cold acclimation in tea plant.

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

LB1057 Ultraviolet B light induces rapid changes in gene expression as detected by non-invasive, adhesive skin biopsies

LB1057 Ultraviolet B light induces rapid changes in gene expression as detected by non-invasive, adhesive skin biopsies

Auxin Induces Widespread Proteome Remodeling in Arabidopsis Seedlings.

It is known that auxin induces rapid gene expression changes throughout plant development, but how these transcriptional responses relate to changes in protein abundance is not well characterized. This report identifies early auxin responsive proteins in whole Arabidopsis seedlings using an isobaric tags for relative and absolute quantification-based quantitative proteomics approach. Approximately 25% of the detected proteins (1045 out of 4257 proteins) are auxin responsive, which is in line with the central role of auxin in the regulation of plant growth and development. Several well-known auxin pathway proteins are identified as differentially expressed, validating this quantitative proteomics approach. Additionally, functional categorization of these auxin responsive proteins indicates that rapid and complex metabolic changes occur in seedlings in response to auxin, including lipid metabolism. Altogether, these data describe novel auxin-regulated proteins and are an excellent resource for identifying new downstream signaling components related to auxin-mediated plant growth and development.