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

Envelliment; Inflamació; MicroARNEnvejecimiento; Inflamación; MicroARNAging; Inflammation; MicroRNAIschemic 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.This study was supported by Emil Aaltonen Foundation, Academy of Finland and Finnish Cultural Foundation

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

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

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

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

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

© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.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.This work was supported by the University of Eastern Finland, Emil Aaltonen Foundation, Paavo Nurmi Foundation, Saastamoinen Foundation, Instrumentarium Science Foundation and Business Finland (Grant number 4399/31/2019). Work with clinical samples was supported by the European Regional Development Fund - Project INBIO (No. CZ.02.1.01/0.0/0.0/16_026/0008451). Work with EVQuant was supported by the IMMPROVE Alpe d'HuZes grant of the Dutch Cancer Society (EMCR2015-8022) and the Daniel den Hoed Foundation grant for Erasmus MC Cancer Treatment Screening Facility. L.R. is supported by a predoctoral fellowship grant (IFI17/00012) and J.M. is the principal investigator of the grant PI18/804 ‘MULTI-BIO-TARGETS: a new strategy for stroke management combining outcome biomarkers and neuroprotection’, both from the Instituto de Salud Carlos III.Peer reviewe

Modelling and global analysis of transcript profiles reveals dynamic roles for microRNAs in transcriptional networks controlling lineage commitment.

Controlled maintenance of multipotent stem cells is a key component for the development and sustainment of complex multicellular organisms. Various signalling pathways contribute to these processes being either lineage specific or more ubiquitously distributed over different tissue types. Transcription factors are considered as the primary propagators of signals that induce multipotent precursor cells to differentiate into specified cell types. These processes are required to revolve in a constrained and timely manner, with different cell types using variable sets of transcription factors and time scales. microRNA molecules represent an efficient and specific class of regulatory non-coding RNA molecules that efficiently constrain and specify differentiation cascades. New findings suggest that various endogenous non-coding RNA species, whose expression is governed through elaborate transcription factor networks, contribute to the regulation of genomewide transcriptional output. Here, evidence is presented of microRNA and transcription factor connectivity during differentiation cascades. First, these two classes of RNA regulatory molecules are shown to share a common target, lipoprotein lipase, and exert dynamical regulation over its expression during adipogenic differentiation. Second, investigating the genome-wide initial events of adipogenic and osteoblastic lineage commitment cascades reveals extensive transcription in non-protein-coding genomic regions. Further analysis of a select cohort of these non-coding transcripts allows for inferring transcription factor binding dynamics through enhancer-related RNA sequences as well as suggests a more wide-spread role for long non-coding RNA species in regulating transcriptional output. These findings contribute to unravelling basic transcriptional circuitry during cellular transitions

Keskkonna kui terviksüsteemi areng

https://www.ester.ee/record=b5466502*es

Hedersmord, baserad på en verklig händelse

Större delen av vårt arbete baseras på en vecka då vår lägenhet blev ett gömställe för Sara, efter att släktingar som hotat henne till livet, ännu en gång hittat henne. Idag är begreppet hedersmord något de flesta känner till. Dock har vi svårt att i den svenska kulturen sammankoppla hedern med mord. I vad grundar sig hedersmord, beror det på religion, kultur eller gamla seder? Vad är bakgrunden till att män dödar kvinnor för att upprätthålla en familjs heder? Vi vill belysa detta ämne och påvisa konsekvenser som drabbar de utsatta. Med våra egna erfarenheter har vi också stått öga mot öga med den problematik som uppstår när man försöker finna hjälp för någon som är utsatt. Vi vill reda ut vilken hjälp som faktiskt finns att få, och jämföra den med den hjälp som i vårat fall Sara har fått. Motivet bakom hedersmord är att återupprätta en familjs eller en hel släkts förlorade heder, männens heder. Männens heder har ett tydligt samband med kvinnornas frigörelse, beteende och sexualitet. Detta handlar om tusenåriga traditioner och normer som är kulturellt betingade

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

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPARgamma binding sites in mouse adipocytes and PPARgamma upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPARgamma-targets. By integration of four separate datasets of genome-wide PPARgamma binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPARgamma 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 PPARgamma 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 PPARgamma-dependence of four of these miRNA loci was demonstrated by PPARgamma knock-down and the loci of miR-103-1 (Pank3), miR-205 and miR-378 (Ppargc1b) were also responsive to the PPARgamma ligand rosiglitazone. Finally, chromatin immunoprecipitation analysis validated in silico predicted PPARgamma binding sites at all three loci and H3K27 acetylation was analyzed to confirm the activity of these enhancers. In conclusion, we identified 22 putative PPARgamma target miRNA genes, showed the PPARgamma dependence of four of these genes and demonstrated three as direct PPARgamma target genes in mouse adipogenesis