Differential Gene Transcription Research Articles

Bidirectional Impact of Varying Severity of Acute Kidney Injury on Calcium Oxalate Stone Formation.

Acute Kidney Injury (AKI) is a prevalent renal disorder. The occurrence of AKI may promote the formation of renal calcium oxalate stones by exerting continuous effects on renal tubular epithelial cells. We aimed to delineate the molecular interplay between AKI and nephrolithiasis. A mild (20 min) and severe (30 min) renal ischemia-reperfusion injury model was established in mice. Seven days after injury, calcium oxalate stones were induced using glyoxylate (Gly) to evaluate the impact of AKI on the formation of kidney stones. Transcriptome sequencing was performed on tubular epithelial cells (TECs) to elucidate the relationship between AKI severity and kidney stones. Key transcription factors (TF) regulating differential gene transcription levels were identified using motif analysis, and pioglitazone, ginkgetin, and fludarabine were used for targeted therapy to validate key transcription factors as potential targets for kidney stone treatment. Severe AKI led to increased deposition of calcium oxalate crystals in renal, impaired kidney function, and upregulation of kidney stone-related gene expression. In contrast, mild AKI was associated with decreased crystal deposition, preserved kidney function, and downregulation of similar gene expression. Transcriptomic analysis revealed that genes associated with inflammation and cell adhesion pathways were significantly upregulated after severe AKI, while genes related to energy metabolism pathways were significantly upregulated after mild AKI. An integrative bioinformatic analysis uncovered a TF regulatory network within TECs, pinpointing that PKNOX1 was involved in the upregulation of inflammation-related genes after severe AKI, and inhibiting PKNOX1 function with Pioglitazone could simultaneously reduce the increase of calcium oxalate crystals after severe AKI in kidney. On the other hand, motif analysis also revealed the protective role of STAT1 in the kidneys after mild AKI, enhancing the function of STAT1 with Ginkgetin could reduce kidney stone formation, while the specific inhibitor of STAT1, Fludarabine, could eliminate the therapeutic effects of mild AKI on kidney stones. Inadequate repair of tubular epithelial cells after severe AKI increases the risk of kidney stone formation, with the upregulation of inflammation-related genes regulated by PKNOX1 playing a role in this process. Inhibiting PKNOX1 function can reduce kidney stone formation. Conversely, after mild AKI, effective cell repair through upregulation of STAT1 expression can protect TEC function, reduce stone formation, and activating STAT1 function can also achieve the goal of treating kidney stones.

Mutations and Differential Transcription of Mating-Type and Pheromone Receptor Genes in Hirsutella sinensis and the Natural Cordyceps sinensis Insect-Fungi Complex.

Sexual reproduction in ascomycetes is controlled by the mating-type (MAT) locus. (Pseudo)homothallic reproduction has been hypothesized on the basis of genetic data from Hirsutella sinensis (Genotype #1 of Ophiocordyceps sinensis). However, the differential occurrence and differential transcription of mating-type genes in the MAT1-1 and MAT1-2 idiomorphs were found in the genome and transcriptome assemblies of H. sinensis, and the introns of the MAT1-2-1 transcript were alternatively spliced with an unspliced intron I that contains stop codons. These findings reveal that O. sinensis reproduction is controlled at the genetic, transcriptional, and coupled transcriptional-translational levels. This study revealed that mutant mating proteins could potentially have various secondary structures. Differential occurrence and transcription of the a-/α-pheromone receptor genes were also found in H. sinensis. The data were inconsistent with self-fertilization under (pseudo)homothallism but suggest the self-sterility of H. sinensis and the requirement of mating partners to achieve O. sinensis sexual outcrossing under heterothallism or hybridization. Although consistent occurrence and transcription of the mating-type genes of both the MAT1-1 and MAT1-2 idiomorphs have been reported in natural and cultivated Cordyceps sinensis insect-fungi complexes, the mutant MAT1-1-1 and α-pheromone receptor transcripts in natural C. sinensis result in N-terminal or middle-truncated proteins with significantly altered overall hydrophobicity and secondary structures of the proteins, suggesting heterogeneous fungal source(s) of the proteins and hybridization reproduction because of the co-occurrence of multiple genomically independent genotypes of O. sinensis and >90 fungal species in natural C. sinensis.

Lineage-specific patterns of sexually dimorphic antennal transcription in the paper wasp Polistes fuscatus.

Olfaction mediates many behaviors in social Hymenoptera, with sexual dimorphism in antennal transcription associated with different behaviors between sexes. Females display coordinated social behaviors within colonies, while males exhibit limited social behavior but are selected for finding mates. The expanded "9-exon" odorant receptor (OR) gene subfamily is associated with chemical communication and exhibits strongly female biased antennal transcription in ants and honey bees. Polistine wasps represent an independent evolution of sociality and associated expansion of 9-exon ORs, though antennal expression patterns are unknown. Here, we report distinct patterns of sexually dimorphic OR transcription in Polistes fuscatus compared to ants and bees. Most P. fuscatus 9-exon transcripts were detected at similar levels in males and females, and some were male biased. We also report differential antennal transcription of cytochromes P450 and muscle-related genes between sexes. We discuss these patterns in the context of the unique sexual and social behaviors of Polistes wasps, including prolonged male mating aggregations and male antennal tapping and curling during courtship and copulation. These results call attention to the lineage-specific selective pressures shaping sexually dimorphic antennal transcription in social insects.

Transcriptional dynamics orchestrating the development and integration of neurons born in the adult hippocampus.

The adult hippocampus generates new granule cells (aGCs) with functional capabilities that convey unique forms of plasticity to the preexisting circuits. While early differentiation of adult radial glia-like cells (RGLs) has been studied extensively, the molecular mechanisms guiding the maturation of postmitotic neurons remain unknown. Here, we used a precise birthdating strategy to study aGC differentiation using single-nuclei RNA sequencing. Transcriptional profiling revealed a continuous trajectory from RGLs to mature aGCs, with multiple immature stages bearing increasing levels of effector genes supporting growth, excitability, and synaptogenesis. Analysis of differential gene expression, pseudo-time trajectory, and transcription factors (TFs) revealed critical transitions defining four cellular states: quiescent RGLs, proliferative progenitors, immature aGCs, and mature aGCs. Becoming mature aGCs involved a transcriptional switch that shuts down pathways promoting cell growth, such SoxC TFs, to activate programs that likely control neuronal homeostasis. aGCs overexpressing Sox4 or Sox11 remained immature. Our results unveil precise molecular mechanisms driving adult RGLs through the pathway of neuronal differentiation.

Altering CO2 Laser Pulse Energy Settings Did Not Influence Differential Gene Transcription in Normal Skin in a Red DurocPig Model.

Fractional ablative CO2 lasers are used clinically to treat cutaneous burn scars with reported varying degrees of effectiveness. It was hypothesized that different laser pulse energy settings may lead to differential gene transcription in a porcine model. Uninjured skin from red Duroc pigs was treated with a fractional ablative CO2 laser set to 70, 100, or 120 mJ across the abdomen (n = 4 areas per treatment). Punch biopsies of both treated and untreated sites were taken before treatment (baseline), at 30 min, and at each hour for 6 h and stored in All-Protect tissue reagent. The biopsies were then used to isolate RNA, which was subsequently used in qRT-PCR for eight genes associated with wound healing and the extracellular matrix: CCL2, IL6, FGF2, TIMP1, TIMP3, COL1A2, MMP2, andDCN. RPL13a was used as a housekeeping gene to normalize the eightgenes of interest. One-way ANOVA tests were used to assess for differences among laser pulse energies and two-way ANOVA tests were used to assess the differences between treated and untreated areas. While six of the eight genes were upregulated after treatment (p < 0.05), there were no significant differences in gene expression between the different laser pulse energies for any of the eight genes. While laser treatment is correlated with a positive and significant upregulation for six of the eight genes 4 h after intervention, the pulse energy settings of the laser did not lead to a statistically significant difference in gene transcription among the treatment areas. Different laser pulse energies may not be required to induce similar cellular responses in a clinical setting.

Hormonal regulation of miRNA during mammary gland development.

The mammary gland is a unique organ as most of its development occurs after birth through stages of proliferation, differentiation and apoptosis that are tightly regulated by circulating hormones and growth factors. Throughout development, hormonal cues induce the regulation of different pathways, ultimately leading to differential transcription and expression of genes involved in this process, but also in the activation or inhibition of post-transcriptional mechanisms of regulation. However, the role of microRNAs (miRNAs) in the different phases of mammary gland remodeling is still poorly understood. The objectives of this study were to analyze the expression of miRNA in key stages of mammary gland development in mice and to determine whether it could be associated with hormonal variation between stages. To do so, miRNAs were isolated from mouse mammary glands at stages of adulthood, pregnancy, lactation and involution, and sequenced. Results showed that 490, 473, 419, and 460 miRNAs are detected in adult, pregnant, lactating and involuting mice, respectively, most of them being common to all four groups, and 58 unique to one stage. Most genes could be divided into six clusters of expression, including two encompassing the highest number of miRNA (clusters 1 and 3) and showing opposite profiles of expression, reaching a peak at adulthood and valley at lactation, or showing the lowest expression at adulthood and peaking at lactation. GO and KEGG analyses suggest that the miRNAs differentially expressed between stages influence the expression of targets associated with mammary gland homeostasis and hormone regulation. To further understand the links between miRNA expression and hormones involved in mammary gland development, miRNAs were then sequenced in breast cells exposed to estradiol, progesterone, prolactin and oxytocin. Four, 38, 24 and 66 miRNAs were associated with progesterone, estradiol, prolactin, and oxytocin exposure, respectively. Finally, when looking at miRNAs modulated by the hormones, differentially expressed during mammary gland development, and having a pattern of expression that could be correlated with the relative levels of hormones known to be found in vivo, 16 miRNAs were identified as likely regulated by circulating hormones. Overall, our study brings a better understanding of the regulation of miRNAs throughout mammary gland development and suggests that there is a relationship between their expression and the main hormones involved in mammary gland development. Future studies will examine this role more in detail.

Renal Endothelial Single-Cell Transcriptomics Reveals Spatiotemporal Regulation and Divergent Roles of Differential Gene Transcription and Alternative Splicing in Murine Diabetic Nephropathy.

Endothelial cell (EC) injury is a crucial contributor to the progression of diabetic kidney disease (DKD), but the specific EC populations and mechanisms involved remain elusive. Kidney ECs (n = 5464) were collected at three timepoints from diabetic BTBRob/ob mice and non-diabetic littermates. Their heterogeneity, transcriptional changes, and alternative splicing during DKD progression were mapped using SmartSeq2 single-cell RNA sequencing (scRNAseq) and elucidated through pathway, network, and gene ontology enrichment analyses. We identified 13 distinct transcriptional EC phenotypes corresponding to different kidney vessel subtypes, confirmed through in situ hybridization and immunofluorescence. EC subtypes along nephrons displayed extensive zonation related to their functions. Differential gene expression analyses in peritubular and glomerular ECs in DKD underlined the regulation of DKD-relevant pathways including EIF2 signaling, oxidative phosphorylation, and IGF1 signaling. Importantly, this revealed the differential alteration of these pathways between the two EC subtypes and changes during disease progression. Furthermore, glomerular and peritubular ECs also displayed aberrant and dynamic alterations in alternative splicing (AS), which is strongly associated with DNA repair. Strikingly, genes displaying differential transcription or alternative splicing participate in divergent biological processes. Our study reveals the spatiotemporal regulation of gene transcription and AS linked to DKD progression, providing insight into pathomechanisms and clues to novel therapeutic targets for DKD treatment.

Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells. Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes. The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified. This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

Bone-derived PDGF-BB drives brain vascular calcification in male mice.

Brain vascular calcification is a prevalent age-related condition often accompanying neurodegenerative and neuroinflammatory diseases. The pathogenesis of large-vessel calcifications in peripheral tissue is well studied, but microvascular calcification in the brain remains poorly understood. Here, we report that elevated platelet-derived growth factor BB (PDGF-BB) from bone preosteoclasts contributed to cerebrovascular calcification in male mice. Aged male mice had higher serum PDGF-BB levels and a higher incidence of brain calcification compared with young mice, mainly in the thalamus. Transgenic mice with preosteoclast-specific Pdgfb overexpression exhibited elevated serum PDGF-BB levels and recapitulated age-associated thalamic calcification. Conversely, mice with preosteoclast-specific Pdgfb deletion displayed diminished age-associated thalamic calcification. In an ex vivo cerebral microvascular culture system, PDGF-BB dose-dependently promoted vascular calcification. Analysis of osteogenic gene array and single-cell RNA-Seq (scRNA-Seq) revealed that PDGF-BB upregulated multiple osteogenic differentiation genes and the phosphate transporter Slc20a1 in cerebral microvessels. Mechanistically, PDGF-BB stimulated the phosphorylation of its receptor PDGFRβ (p-PDGFRβ) and ERK (p-ERK), leading to the activation of RUNX2. This activation, in turn, induced the transcription of osteoblast differentiation genes in PCs and upregulated Slc20a1 in astrocytes. Thus, bone-derived PDGF-BB induced brain vascular calcification by activating the p-PDGFRβ/p-ERK/RUNX2 signaling cascade in cerebrovascular cells.

Endotype Characterization Reveals Mechanistic Differences Across Brain Regions in Sporadic Alzheimer's Disease.

While Alzheimer's disease (AD) pathology is associated with altered brain structure, it is not clear whether gene expression changes mirror the onset and evolution of pathology in distinct brain regions. Deciphering the mechanisms which cause the differential manifestation of the disease across different regions has the potential to help early diagnosis. We aimed to identify common and unique endotypes and their regulation in tangle-free neurons in sporadic AD (SAD) across six brain regions: entorhinal cortex (EC), hippocampus (HC), medial temporal gyrus (MTG), posterior cingulate (PC), superior frontal gyrus (SFG), and visual cortex (VCX). To decipher the states of tangle-free neurons across different brain regions in human subjects afflicted with AD, we performed analysis of the neural transcriptome. We explored changes in differential gene expression, functional and transcription factor target enrichment, and co-expression gene module detection analysis to discern disease-state transcriptomic variances and characterize endotypes. Additionally, we compared our results to tangled AD neuron microarray-based study and the Allen Brain Atlas. We identified impaired neuron function in EC, MTG, PC, and VCX resulting from REST activation and reversal of mature neurons to a precursor-like state in EC, MTG, and SFG linked to SOX2 activation. Additionally, decreased neuron function and increased dedifferentiation were linked to the activation of SUZ12. Energetic deficit connected to NRF1 inactivation was found in HC, PC, and VCX. Our findings suggest that SAD manifestation varies in scale and severity in different brain regions. We identify endotypes, such as energetic shortfalls, impaired neuronal function, and dedifferentiation.

Abstract P1141: Molecular Insights Into The Phenotypic Heterogeneity Observed In Two Different Murine Models Of Hypertrophic Cardiomyopathy (HCM)

Background: Phenotypic variability in HCM has long been appreciated to correlate with genotype. However, the molecular drivers underpinning these differences remain poorly understood. We hypothesized that the variable HCM phenotypes observed in 2 different widely used HCM mouse models (R403Q-MyHC, R92W-TnT) result from complex interactions between a variety of cardiac cell types. We performed unbiased assessments of gene expression and chromatin accessibility in mutant and littermate control hearts at an early disease stage, when MyHC-mutants have higher LV-mass and normal LV-function, while TnT-mutants have similar LV-mass, hypercontractility and diastolic dysfunction, compared to littermate-controls. Methods: Nuclei were isolated from hearts of 5-week-old mutant and control animals and subjected to the standard linked ATAC and gene expression profiling pipeline from 10X Genomics w/o enrichment/sorting. Clustering, differential gene expression and transcription factor (TF) motif enrichment was performed using Seurat, ArchR; SCENIC was used to predict TF (Regulon)-activation from gene expression data and Cellchat for cellular crosstalk. Results: Integrated single-nuclei data-based clustering identified all cardiac cell types. Both mutant ventricular(V)-myocytes had similar dysregulation (80-90% agreement) of gene expression and TF enrichment, with matching of 23/30 top TF-motifs, and agreement with gene expression-based TF-prediction. Both mutant V-myocytes had activation of SWI/SNF complex TFs (Jun, Fos, Smarc family), however activation of SREBF-1/2, Atf5, Creb3l2, Egr2 (drivers of fibroblast activation), and Klf2, Klf4, Bcl6b (drivers of shear stress-mediated gene expression in endothelial cells (ECs)) was only present in TnT-mutants. Activation of VCAM signaling from V-myocytes-to-ECs, and PDGF signaling to VSMCs is predicted in both mutants, but TGF-β signaling to fibroblasts, endothelin-1 signaling to V-myocytes, was only predicted in TnT-mutants. Conclusions: Mutant sarcomeric proteins activate the SWI/SNF complex that drives V-myocyte hypertrophy at early disease stage. Mutation-specific activation of TF-programs in non-myocyte cells are important drivers of cardiac phenotypic variability in HCM.

Asynchronous transcription and translation of neurotransmitter-related genes characterize the initial stages of neuronal maturation in Drosophila.

Neuron specification and maturation are essential for proper central nervous system development. However, the precise mechanisms that govern neuronal maturation, essential to shape and maintain neuronal circuitry, remain poorly understood. Here, we analyse early-born secondary neurons in the Drosophila larval brain, revealing that the early maturation of secondary neurons goes through 3 consecutive phases: (1) Immediately after birth, neurons express pan-neuronal markers but do not transcribe terminal differentiation genes; (2) Transcription of terminal differentiation genes, such as neurotransmitter-related genes VGlut, ChAT, or Gad1, starts shortly after neuron birth, but these transcripts are, however, not translated; (3) Translation of neurotransmitter-related genes only begins several hours later in mid-pupa stages in a coordinated manner with animal developmental stage, albeit in an ecdysone-independent manner. These results support a model where temporal regulation of transcription and translation of neurotransmitter-related genes is an important mechanism to coordinate neuron maturation with brain development.

Differential transcription of mating-type genes during sexual reproduction of natural Cordyceps sinensis

Natural Cordyceps sinensis as an insect-fungal complex, which is developed after Ophiocordyceps sinensis infects a larva of Hepialidae family. Seventeen genotypes of O. sinensis have been identified in natural C. sinensis. This paper summarized the literature reports and GenBank database regarding occurrence and transcription of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in natural C. sinensis, in Hirsutella sinensis(GC-biased Genotype #1 of O. sinensis), to infer the mating pattern of O. sinensis in the lifecycle of natural C. sinensis. The mating-type genes and transcripts of MAT1-1 and MAT1-2 idiomorphs were identified in the metagenomes and metatranscriptomes of natural C. sinensis. However, their fungal sources are unclear because of co-colonization of several genotypes of O. sinensis and multiple fungal species in natural C. sinensis. The mating-type genes of MAT1-1 and MAT1-2 idiomorphs were differentially present in 237 H. sinensis strains, constituting the genetic control of the O. sinensis reproduction. Transcriptional control of the O. sinensis reproduction includes: differential transcription or silencing of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs, and the MAT1-2-1 transcript with unspliced intron I that contains 3 stop codons. Research on the H. sinensis transcriptome demonstrated differential and complementary transcriptions of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in Strains L0106 and 1229, which may become mating partners to accomplish physiological heterothallism. The differential occurrence and transcription of the mating-type genes in H. sinensis are inconsistent with the self-fertilization hypothesis under homothallism or pseudohomothallism, but instead indicate the need of mating partners of the same H. sinensis species, either monoecious or dioecious, for physiological heterothallism, or heterospecific species for hybridization. Multiple GC-and AT-biased genotypes of O. sinensis were identified in the stroma, stromal fertile portion(densely covered with numerous ascocarps) and ascospores of natural C. sinensis. It needs to be further explored if the genome-independent O. sinensis genotypes could become mating partners to accomplish sexual reproduction. S. hepiali Strain FENG experienced differential transcription of the mating-type genes with a pattern complementary to that of H. sinensis Strain L0106. Additional evidence is needed to explore a hybridization possibility between S. hepiali and H. sinensis, whether they are able to break the interspecific reproductive isolation. Genotypes #13～14 of O. sinensis feature large DNA segment reciprocal substitutions and genetic material recombination between 2 heterospecific parental fungi, H. sinensis and an AB067719-type fungus, indicating a possibility of hybridization or parasexuality. Our analysis provides important information at the genetic and transcriptional levels regarding the mating-type gene expression and reproduction physiology of O. sinensis in the sexual life of natural C. sinensis and offers crucial reproductive physiology evidence, to assist in the design of the artificial cultivation of C. sinensis to supplement the increasing scarcity of natural resource.

Paenibacillus terrae NK3-4 regulates the transcription of growth-related and stress resistance-related genes in rice.

Paenibacillus terrae NK3-4 is a plant growth-promoting rhizobacterium. In this study, the effects of NK3-4 on rice growth and gene transcription were determined. The results indicated that a seed soaking treatment and a pre-germination seed treatment using NK3-4 promoted rice growth, especially spraying rice seedlings with NK3-4 increased the root number and root length by 34.2% and 34.1%, respectively. Moreover, NK3-4 induced the differential transcription of genes annotated with gene ontology (GO) terms; the number of up-regulated genes was 4.38-times higher than the number of down-regulated genes. The NK3-4 treatment induced the differential transcription of genes in 1794 GO functional groups, with 1531 functional groups containing up-regulated genes. Specific growth-related genes up-regulated by NK3-4 are involved in biological processes, including responses to auxin, hormone biosynthesis, cellular component biogenesis, root system development, and other functions. Furthermore, stress resistance-related genes were up-regulated, some of which encode WRKYs, NPK1-related protein kinase, NPR1-like 4, CaM-like proteins, MYBs, ERFs, TIFYs, NACs, EL5s, PR1s, PR2, PR8, PODs, and PAD4. Considered together, these findings imply that NK3-4 may promote plant growth and enhance stress resistance by regulating gene expression, making it a potentially useful microbe for regulating rice growth and stress resistance.

Non-specific and specific DNA binding modes of bacterial histone, HU, separately regulate distinct physiological processes through different mechanisms.

The histone-like protein HU plays a diverse role in bacterial physiology from the maintenance of chromosome structure to the regulation of gene transcription. HU binds DNA in a sequence-non-specific manner via two distinct binding modes: (i) random binding to any DNA through ionic bonds between surface-exposed lysine residues (K3, K18, and K83) and phosphate backbone (non-specific); (ii) preferential binding to contorted DNA of given structures containing a pair of kinks (structure-specific) through conserved proline residues (P63) that induce and/or stabilize the kinks. First, we show here that the P63-mediated structure-specific binding also requires the three lysine residues, which are needed for a non-specific binding. Second, we demonstrate that substituting P63 to alanine in HU had no impact on non-specific binding but caused differential transcription of diverse genes previously shown to be regulated by HU, such as those associated with the organonitrogen compound biosynthetic process, galactose metabolism, ribosome biogenesis, and cell adhesion. The structure-specific binding also helps create DNA supercoiling, which, in turn, may influence directly or indirectly the transcription of other genes. Our previous and current studies show that non-specific and structure-specific HU binding appear to have separate functions- nucleoid architecture and transcription regulation- which may be true in other DNA-binding proteins.

UBASH3B knockout Downregulates STAT1 Mediated Interferon Signalling and Megakaryocyte Related Genes in a JAK2 V617F Positive MPN Model

Interferon (IFN) signalling via STAT1 plays an important role in the pathogenesis and treatment of myeloproliferative neoplasms (MPN). Single cell sequencing approaches have implicated megakaryocyte (MKC) biased stem cells with enhanced intrinsic activation of IFN signalling pathways in MPN patients as critical mediators of disease initiation and maintenance. UBASH3B is a histidine phosphatase and has been identified as a potential regulator of malignant haematopoiesis and as a mediator of JAK/STAT1 IFN signalling in lymphoid cells. RNA sequencing (RNA-seq) data in JAK2 V617F positive UKE-1, HEL (both own lab) and SET2 (GSE91062) cells demonstrate that UBASH3B is significantly downregulated following administration of the JAK inhibitor ruxolitinib (RUX). We observed that UBASH3B expression in CD34+ cells in patients with primary myelofibrosis (GSE53482) is significantly enhanced but reduced in patients with essential thrombocythaemia(ET) and polycythaemia (GSE103237) and hypothesized that UBASH3B may contribute to MPN pathogenesis. UKE-1 is a JAK2 V617F positive primitive haematopoietic cell line derived from an ET patient. It shows features of intrinsic IFN signalling and expresses genes related to MKC development and differentiation providing a useful model of MPN stem cell behaviour. CRISPR-Cas9 mediated KO of UBASH3B was performed in UKE-1 cells. A ribonucleoprotein complex of guide RNA and Cas9 protein was nucleofected into cells. Individual colonies were assessed for UBASH3B status using western blotting and Sanger sequencing. Four distinct, healthy KO clones were established for the UKE-1 cell line and compared to the original wildtype cell population. RNA-seq and phospho-proteomic analysis were undertaken in two KO clones. Extracted RNA was sequenced on Illumina platforms with a minimum of 45 million individual paired reads per sample. Differential gene expression was analysed using DESEQ2. Whilst, matched cell lysates underwent tandem mass tagging, high pH fractionation, and phospho-peptide enrichment was undertaken prior to liquid chromatography-mass spectrometry for total and phospho-proteomic analysis. RNA-seq and total proteomic analysis were consistent in demonstrating a significant downregulation of STAT1 (Log2FC-0.79, FDR 9.03E-49) and STAT2 (Log2FC -0.70, FDR 9.05E-20) with over-enrichment analysis identifying downregulation of type 1 IFN signalling pathways and pathways related to MKC and platelet function as shown in Figure 1. The significant downregulation of genes associated with platelet function, MKC development and differentiation included GATA1, GP1BA, ITGA2B, SELP, VWF and genes identified as mediators of fibrosis in MPN as demonstrated in Figure 2. Targeted real time PCR for STAT1, GP1BA and LTPB1 confirmed the same transcriptional patterns were present in both additional knockout clones. Knockout clones show a diminished response to exogenous IFN alpha administration consistent with downregulation of these pathways. Consistently reduced proliferation is observed in the KO clones by a variety of methodologies. Comparison to the transcriptional response in RUX treated UKE-1 cells demonstrates that at 24 hours post RUX treatment significant STAT1 downregulation is observed but not accompanied by the same pattern of down-regulation of MKC and fibrosis associated genes. The same was seen in cells persistently treated with high dose RUX for greater than 5 weeks. These results show a significant downregulation of IFN signalling in UBASH3B KO cells linked to a significant downregulation of MKC associated genes further highlighting the link observed in MPN stem cells. They implicate UBASH3B as a potential mediator of IFN signalling in a JAK2 V617F context and support a hypothesis that downregulating IFN signalling in MPN stem cells may have disease modifying potential by altering differentiation and pro-fibrotic potential. RUX treatment downregulates STAT1 without activating the same transcriptional programme associated with MKC gene downregulation and this may help explain the limited disease modifying effect of RUX observed in patients. This work has generated an important KO cell line model which will permit the molecular characterisation of differential STAT1/IFN pathway activation and allow further study to understand the regulation of MKC differentiation and pro-fibrotic gene transcription observed in MPN stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ubiquitin Proteasome Gene Signatures in Ependymoma Molecular Subtypes.

The ubiquitin proteasome system (UPS) is critically important for cellular homeostasis and affects virtually all key functions in normal and neoplastic cells. Currently, a comprehensive review of the role of the UPS in ependymoma (EPN) brain tumors is lacking but may provide valuable new information on cellular networks specific to different EPN subtypes and reveal future therapeutic targets. We have reviewed publicly available EPN gene transcription datasets encoding components of the UPS pathway. Reactome analysis of these data revealed genes and pathways that were able to distinguish different EPN subtypes with high significance. We identified differential transcription of several genes encoding ubiquitin E2 conjugases associated with EPN subtypes. The expression of the E2 conjugase genes UBE2C, UBE2S, and UBE2I was elevated in the ST_EPN_RELA subtype. The UBE2C and UBE2S enzymes are associated with the ubiquitin ligase anaphase promoting complex (APC/c), which regulates the degradation of substrates associated with cell cycle progression, whereas UBE2I is a Sumo-conjugating enzyme. Additionally, elevated in ST_EPN_RELA were genes for the E3 ligase and histone deacetylase HDAC4 and the F-box cullin ring ligase adaptor FBX031. Cluster analysis demonstrated several genes encoding E3 ligases and their substrate adaptors as EPN subtype specific genetic markers. The most significant Reactome Pathways associated with differentially expressed genes for E3 ligases and their adaptors included antigen presentation, neddylation, sumoylation, and the APC/c complex. Our analysis provides several UPS associated factors that may be attractive markers and future therapeutic targets for the subtype-specific treatment of EPN patients.

Differential cofactor dependencies define distinct types of human enhancers.

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3-5, yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq6 in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator7 seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity.

Development of fluorescence-based nucleic acid blot hybridization method using Cy5.5 labeled DNA probes

Near-infrared (NIR) fluorophores are widely used as fluorescent probes for bioimaging because of their minimal photodamage to biological samples, deep penetration, and low interference from background autofluorescence. Here, we employed a NIR fluorescent cyanine dye Cy5.5 to label DNA probes for nucleic acid blot hybridization. The specificity and sensitivity of fluorescent DNA probes were proven by both Southern blot and Northern blot using cellulolytic bacterium Ruminiclostridium cellulolyticum as a model. Furthermore, employing the method, we successfully identified the gene disruption of ClosTron to rule out off-target, analyzed the differential transcription of genes under different conditions, and confirmed RNA cleavage. Compared to other nonradioactive probes, the preparation and detection of Cy5.5-labeled probes are more simple, more economical, and versatile, suggesting that the Cy5.5-labeled probes are suitable for nucleic acid blot hybridization in addition to bioimaging.

Concentration-dependent change in hypothalamic neuronal transcriptome by the dietary fatty acids: oleic and palmitic acids

Prenatal high-fat diet exposure increases hypothalamic neurogenesis events in embryos and programs offspring to be obesity-prone. The molecular mechanism involved in these dietary effects of neurogenesis is unknown. This study investigated the effects of oleic and palmitic acids, which are abundant in a high-fat diet, on the hypothalamic neuronal transcriptome and how these changes impact neurogenesis events. The results show the differential effects of low and high concentrations of oleic or palmitic acid treatment on differential gene transcription. Gene ontology analysis uncovered significant gene enrichment in several cellular pathways involved in gene regulation and protein production, particularly with proliferation, migration, and cell survival. The enriched signaling pathways include Wnt, integrin, PDGF, and apoptosis, in addition to endocrine function signaling pathways CCKR and GnRH. Further examination of proliferation and migration shows low concentrations of oleic acid to stimulate proliferation and high concentrations of both oleic and palmitic acid to stimulate apoptosis. Oleic acid also reduces hypothalamic neuronal migration, with little effect from palmitic acid. The results show the two most abundant fatty acids in a high-fat diet impact hypothalamic neuronal proliferation and migration. The results also uncovered potential signaling pathways affected by oleic and palmitic acid and suggest one mechanism of prenatal high-fat diet-induced neurogenesis events may be through these two abundant fatty acids.