Dynamic release of neuronal extracellular vesicles containing miR-21a-5p is induced by hypoxia.

Hypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non-neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)-21-5p, which respond to hypoxia. However, the true EV association of miR-21-5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR-21-5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR-21a-5p secretion in the EVs, which preceded the elevation of hypoxia-induced tissue or cellular miR-21a-5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR-21a-5p from enzymatic degradation but a remarkable fraction of miR-21a-5p remained fragile and non-EV associated. The increase in miR-21a-5p secretion may have biomarker potential, as high blood levels of miR-21-5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke.

Single-cell RNA-seq analysis and cell-cluster deconvolution of the human preovulatory follicular fluid cells provide insights into the pathophysiology of ovarian hyporesponse.

Reduction in responsiveness to gonadotropins or hyporesponsiveness may lead to the failure of in vitro fertilization (IVF), due to a low number of retrieved oocytes. The ovarian sensitivity index (OSI) is used to reflect the ovarian responsiveness to gonadotropin stimulation before IVF. Although introduced to clinical practice already years ago, its usefulness to predict clinical outcomes requires further research. Nevertheless, pathophysiological mechanisms of ovarian hyporesponse, along with advanced maternal age and in younger women, have not been fully elucidated. Follicles consist of multiple cell types responsible for a repertoire of biological processes including responding to pituitary gonadotropins necessary for follicle growth and oocyte maturation as well as ovulation. Encouraging evidence suggests that hyporesponse could be influenced by many contributing factors, therefore, investigating the variability of ovarian follicular cell types and their gene expression in hyporesponders is highly informative for increasing their prognosis for IVF live birth. Due to advancements in single-cell analysis technologies, the role of somatic cell populations in the development of infertility of ovarian etiology can be clarified. Here, somatic cells were collected from the fluid of preovulatory ovarian follicles of patients undergoing IVF, and RNA-seq was performed to study the associations between OSI and gene expression. We identified 12 molecular pathways differentially regulated between hypo- and normoresponder patient groups (FDR<0.05) from which extracellular matrix organization, post-translational protein phosphorylation, and regulation of Insulin-like Growth Factor (IGF) transport and uptake by IGF Binding Proteins were regulated age-independently. We then generated single-cell RNA-seq data from matching follicles revealing 14 distinct cell clusters. Using cell cluster-specific deconvolution from the bulk RNA-seq data of 18 IVF patients we integrated the datasets as a novel approach and discovered that the abundance of three cell clusters significantly varied between hypo- and normoresponder groups suggesting their role in contributing to the deviations from normal ovarian response to gonadotropin stimulation. Our work uncovers new information regarding the differences in the follicular gene expression between hypo- and normoresponders. In addition, the current study fills the gap in understanding the inter-patient variability of cell types in human preovulatory follicles, as revealed by single-cell analysis of follicular fluid cells.

Hsa-mir-548 family expression in human reproductive tissues.

BACKGROUND: Hsa-miR-548ba expressed in ovarian granulosa cells targets PTEN and LIFR, which are essential for ovarian follicle activation and growth. The expression pattern of hsa-miR-548ba correlates with its host gene follicle-stimulating hormone receptor (FSHR), and FSH has a positive influence on hsa-miR-548ba expression. However, hsa-miR-548ba is a member of a large hsa-mir-548 family with potentially overlapping targets. The current study aims to investigate the co-expression of hsa-mir-548 family members in FSHR-positive reproductive tissues and to explore the potential co-regulation of pathways. RESULTS: For the above-described analysis, small RNA sequencing data from public data repositories were used. Sequencing results revealed that hsa-miR-548ba was expressed at the highest level in the ovarian granulosa cells and uterine myometrial samples together with another twelve and one hsa-miR-548 family members, respectively. Pathway enrichment analysis of microRNA targets in the ovarian samples revealed the hsa-miR-548ba and hsa-miR-548b-5p co-regulation of RAB geranylgeranylation in mural granulosa cells. Moreover, other hsa-mir-548 family members co-regulate pathways essential for ovarian functions (PIP3 activates AKT signalling and signalling by ERBB4). In addition to hsa-miR-548ba, hsa-miR-548o-3p is expressed in the myometrium, which separately targets the peroxisome proliferator-activated receptor alpha (PPARA) pathway. CONCLUSION: This study reveals that hsa-mir-548 family members are expressed in variable combinations in the reproductive tract, where they potentially fulfil different regulatory roles. The results provide a reference for further studies of the hsa-mir-548 family role in the reproductive tract.

Profiling of Primary and Mature miRNA Expression in Atherosclerosis-Associated Cell Types.

[Figure: see text].

Peripheral inflammation preceeding ischemia impairs neuronal survival through mechanisms involving miR-127 in aged animals.

Ischemic stroke, the third leading cause of death in the Western world, affects mainly the elderly and is strongly associated with comorbid conditions such as atherosclerosis or diabetes, which are pathologically characterized by increased inflammation and are known to influence the outcome of stroke. Stroke incidence peaks during influenza seasons, and patients suffering from infections such as pneumonia prior to stroke exhibit a worse stroke outcome. Earlier studies have shown that comorbidities aggravate the outcome of stroke, yet the mediators of this phenomenon remain obscure. Here, we show that acute peripheral inflammation aggravates stroke-induced neuronal damage and motor deficits specifically in aged mice. This is associated with increased levels of plasma proinflammatory cytokines, rather than with an increase of inflammatory mediators in the affected brain parenchyma. Nascent transcriptomics data with mature microRNA sequencing were used to identify the neuron-specific miRNome, in order to decipher dysregulated miRNAs in the brains of aged animals with stroke and co-existing inflammation. We pinpoint a previously uninvestigated miRNA in the brain, miR-127, that is highly neuronal, to be associated with increased cell death in the aged, LPS-injected ischemic mice. Target prediction tools indicate that miR-127 interacts with several basally expressed neuronal genes, and of these we verify miR-127 binding to Psmd3. Finally, we report reduced expression of miR-127 in human stroke brains. Our results underline the impact of peripheral inflammation on the outcome of stroke in aged subjects and pinpoint molecular targets for restoring endogenous neuronal capacity to combat ischemic stroke.

NRF2 is a key regulator of endothelial microRNA expression under proatherogenic stimuli.

AIMS: Oxidized phospholipids and microRNAs (miRNAs) are increasingly recognized to play a role in endothelial dysfunction driving atherosclerosis. NRF2 transcription factor is one of the key mediators of the effects of oxidized phospholipids, but the gene regulatory mechanisms underlying the process remain obscure. Here, we investigated the genome-wide effects of oxidized phospholipids on transcriptional gene regulation in human umbilical vein endothelial cells and aortic endothelial cells with a special focus on miRNAs. METHODS AND RESULTS: We integrated data from HiC, ChIP-seq, ATAC-seq, GRO-seq, miRNA-seq, and RNA-seq to provide deeper understanding of the transcriptional mechanisms driven by NRF2 in response to oxidized phospholipids. We demonstrate that presence of NRF2 motif and its binding is more prominent in the vicinity of up-regulated transcripts and transcriptional initiation represents the most likely mechanism of action. We further identified NRF2 as a novel regulator of over 100 endothelial pri-miRNAs. Among these, we characterize two hub miRNAs miR-21-5p and miR-100-5p and demonstrate their opposing roles on mTOR, VEGFA, HIF1A, and MYC expressions. Finally, we provide evidence that the levels of miR-21-5p and miR-100-5p in exosomes are increased upon senescence and exhibit a trend to correlate with the severity of coronary artery disease. CONCLUSION: Altogether, our analysis provides an integrative view into the regulation of transcription and miRNA function that could mediate the proatherogenic effects of oxidized phospholipids in endothelial cells.

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities.

BACKGROUND: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. METHODS: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. RESULTS: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. CONCLUSIONS: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.

Transcriptional Profiling of Hypoxia-Regulated Non-coding RNAs in Human Primary Endothelial Cells.

Hypoxia occurs in human atherosclerotic lesions and has multiple adverse effects on endothelial cell metabolism. Recently, key roles of long non-coding RNAs (lncRNAs) in the development of atherosclerosis have begun to emerge. In this study, we investigate the lncRNA profiles of human umbilical vein endothelial cells subjected to hypoxia using global run-on sequencing (GRO-Seq). We demonstrate that hypoxia regulates the nascent transcription of ~1800 lncRNAs. Interestingly, we uncover evidence that promoter-associated lncRNAs are more likely to be induced by hypoxia compared to enhancer-associated lncRNAs, which exhibit an equal distribution of up- and downregulated transcripts. We also demonstrate that hypoxia leads to a significant induction in the activity of super-enhancers next to transcription factors and other genes implicated in angiogenesis, cell survival and adhesion, whereas super-enhancers near several negative regulators of angiogenesis were repressed. Despite the majority of lncRNAs exhibiting low detection in RNA-Seq, a subset of lncRNAs were expressed at comparable levels to mRNAs. Among these, MALAT1, HYMAI, LOC730101, KIAA1656, and LOC339803 were found differentially expressed in human atherosclerotic lesions, compared to normal vascular tissue, and may thus serve as potential biomarkers for lesion hypoxia.

HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development.

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616-31. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Analysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture.

Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29a∼29b-1, mir-100∼let-7a-2∼125b-1 and miR-221∼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci.

Transcription-coupled genetic instability marks acute lymphoblastic leukemia structural variation hotspots.

Progression of malignancy to overt disease requires multiple genetic hits. Activation-induced deaminase (AID) can drive lymphomagenesis by generating off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription. The first active transcriptional profiles from acute lymphoblastic leukemia (ALL) patients acquired here reveal striking similarity at structural variation (SV) sites. Specific transcriptional features, namely convergent transcription and Pol2 stalling, were detected at breakpoints. The overlap was most prominent at SV with recognition motifs for the recombination activating genes (RAG). We present signal feature analysis to detect vulnerable regions and quantified from human cells how convergent transcription contributes to R-loop generation and RNA polymerase stalling. Wide stalling regions were characterized by high DNAse hypersensitivity and unusually broad H3K4me3 signal. Based on 1382 pre-B-ALL patients, the ETV6-RUNX1 fusion positive patients had over ten-fold elevation in RAG1 while high expression of AID marked pre-B-ALL lacking common cytogenetic changes.

Combinatorial regulation of lipoprotein lipase by microRNAs during mouse adipogenesis.

MicroRNAs (miRNAs) regulate gene expression directly through base pairing to their targets or indirectly through participating in multi-scale regulatory networks. Often miRNAs take part in feed-forward motifs where a miRNA and a transcription factor act on shared targets to achieve accurate regulation of processes such as cell differentiation. Here we show that the expression levels of miR-27a and miR-29a inversely correlate with the mRNA levels of lipoprotein lipase (Lpl), their predicted combinatorial target, and its key transcriptional regulator peroxisome proliferator-activated receptor gamma (Pparg) during 3T3-L1 adipocyte differentiation. More importantly, we show that Lpl, a key lipogenic enzyme, can be negatively regulated by the two miRNA families in a combinatorial fashion on the mRNA and functional level in maturing adipocytes. This regulation is mediated through the Lpl 3'UTR as confirmed by reporter gene assays. In addition, a small mathematical model captures the dynamics of this feed-forward motif and predicts the changes in Lpl mRNA levels upon network perturbations. The obtained results might offer an explanation to the dysregulation of LPL in diabetic conditions and could be extended to quantitative modeling of regulation of other metabolic genes under similar regulatory network motifs.

Dataset integration identifies transcriptional regulation of microRNA genes by PPARγ in differentiating mouse 3T3-L1 adipocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPARγ binding sites in mouse adipocytes and PPARγ upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPARγ-targets. By integration of four separate datasets of genome-wide PPARγ binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPARγ binding within 50 kb from miRNA transcription start sites. Nineteen mature miRNAs were upregulated ≥2-fold during adipogenesis and for six of these miRNA loci the PPARγ binding sites were confirmed by at least three datasets. The upregulation of five miRNA genes miR-103-1 (host gene Pank3), miR-148b (Copz1), miR-182/96/183, miR-205 and miR-378 (Ppargc1b) followed that of Pparg. The PPARγ-dependence of four of these miRNA loci was demonstrated by PPARγ knock-down and the loci of miR-103-1 (Pank3), miR-205 and miR-378 (Ppargc1b) were also responsive to the PPARγ ligand rosiglitazone. Finally, chromatin immunoprecipitation analysis validated in silico predicted PPARγ binding sites at all three loci and H3K27 acetylation was analyzed to confirm the activity of these enhancers. In conclusion, we identified 22 putative PPARγ target miRNA genes, showed the PPARγ dependence of four of these genes and demonstrated three as direct PPARγ target genes in mouse adipogenesis.