Services Publications
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Services Publications
Sigrid Vondra, Anna-Lena Höbler, Andreas Ian Lackner, Johanna Raffetseder, Zala Nikita Mihalic, Leila Saleh, Victoria Kunihs, Peter Haslinger, Markus Wahrmann, Heinrich Husslein, Raimund Oberle, Julia Kargl, Sandra Haider, Paulina Latos, Martin Knöfler, Jan Ernerudh, Jürgen Pollheimer
Abstract
During human pregnancy, placenta-derived extravillous trophoblasts (EVT) invade the decidua and communicate with maternal immune cells. The decidua can be distinguished into basalis (decB) and parietalis (decP), the latter being unaffected by placentation. By defining a novel gating strategy, we report accumulation of myeloid cells in decB. We identified a decidua basalis-associated macrophage (decBAM) population with a differential transcriptome and secretome when compared to decidua parietalis-associated macrophages (decPAMs). decBAMs are CD11chi and efficient inducers of Tregs, proliferate in situ and secrete high levels of CXCL1, CXCL5, M-CSF, and IL-10. In contrast, decPAMs exert a dendritic cell-like, motile phenotype characterized by induced expression of HLA class II molecules, enhanced phagocytosis, and the ability to activate T cells. Strikingly, EVT-conditioned media are able to convert decPAMs into a decBAM phenotype. Cumulatively, these findings assign distinct macrophage phenotypes to decidual areas depending on placentation and further highlight a critical role for EVTs in the induction of pregnancy-tolerant macrophage polarization.
William Garland, Iris Müller, Mengjun Wu, Manfred Schmid, Katsutoshi Imamura, Leonor Rib, Albin Sandelin, Kristian Helin, Torben Heick Jensen
Abstract
Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous “nuclear exosome targeting” (NEXT) and “human silencing hub” (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.
Jennifer S. Mascaro, Amanda Wallace, Brooke Hyman, Carla Haack, Cherie C. Hill, Miranda A. Moore, Maha B. Lund, Eric J. Nehl, Sharon H. Bergquist, Steve W. Cole
Abstract
While much attention has been paid to healthcare provider and trainee burnout, less is known about provider well-being (i.e., flourishing) or about the effects of well-being on immune function. This study examined the demographic and psycho-social correlates of well-being among healthcare trainees (resident physicians and physician assistant (PA) trainees) and evaluated the association of well-being with the “conserved transcriptional response to adversity” (CTRA) characterized by up-regulated expression of pro-inflammatory genes and down-regulated expression of innate antiviral genes. Participants (n = 58) completed self-reported assessments of sleep disturbance, loneliness, depressive symptoms, anxiety, stress, and well-being (flourishing). Blood sample RNA profiles were analyzed by RNA sequencing to assess the CTRA. Slightly over half (n = 32; 55.2%) of healthcare trainees were categorized as flourishing. Flourishing was less prevalent among primary caregivers, and more prevalent among trainees who exercised more frequently and those with fewest days sick. Loneliness (AOR = 0.75; 95% CI = 0.61, 0.91; p = 0.003) and stress (AOR = 0.65; 95% CI = 0.45, 0.94; p = 0.02) were associated with decreased odds of flourishing when controlling for other variables. Flourishing was associated with down-regulated CTRA gene expression, whereas loneliness was associated with up-regulated CTRA gene expression (both p < 0.05). Assessing these relationships in a larger, multi-site study is of critical importance to inform policy, curricula, and interventions to bolster sustainable trainee well-being.
Aleksandra Filipiak-Duliban, Klaudia Brodaczewska, Arkadiusz Kajdasz, Claudine Kieda
Abstract
Hanna Alalam, Jorge Arturo Zepeda-Martinez, Per Sunnerhagen
Abstract
Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives; instead of steady-state RNA level changes. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2 % of our candidates being identified in previous studies. This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.
Anny Devoy, Georgia Price, Francesca De Giorgio, Rosie Bunton-Stasyshyn, David Thompson, Samanta Gasco, Alasdair Allan, Gemma F. Codner, Remya R. Nair, Charlotte Tibbit, Ross McLeod, Zeinab Ali, Judith Noda, Alessandro Marrero-Gagliardi, José M.Brito-Armas, Chloe Williams, Muhammet M. Öztürk, Michelle Simon, Edward O’Neill, Sam Bryce-Smith, Jackie Harrison, Gemma Atkins, Silvia Corrochano, Michelle Stewart, Jonathan D. Gilthorpe, Lydia Teboul, Abraham Acevedo-Arozena, Elizabeth M. C. Fisher, Thomas J.Cunningham
Abstract
Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.
André G. Costa-Martins, Karim Mane, Benjamin B. Lindsey, Rodrigo L.T. Ogava, Ícaro Castro, Ya Jankey Jagne, Hadijatou J. Sallah, Edwin P. Armitage, Sheikh Jarju, Bankole Ahadzie, Rebecca Ellis-Watson, John S. Tregoning, Colin D. Bingle, Debby Bogaert, Ed Clarke, Jose Ordovas-Montanes, David Jeffries, Beate Kampmann, Helder I. Nakaya, Thushan I. de Silva
Abstract
In children lacking influenza-specific adaptive immunity, upper respiratory tract innate immune responses may influence viral replication and disease outcome. We use trivalent live attenuated influenza vaccine (LAIV) as a surrogate challenge model in children aged 24–59 months to identify pre-infection mucosal transcriptomic signatures associated with subsequent viral shedding. Upregulation of interferon signaling pathways prior to LAIV is significantly associated with lower strain-specific viral loads (VLs) at days 2 and 7. Several interferon-stimulated genes are differentially expressed in children with pre-LAIV asymptomatic respiratory viral infections and negatively correlated with LAIV VLs. Upregulation of genes enriched in macrophages, neutrophils, and eosinophils is associated with lower VLs and found more commonly in children with asymptomatic viral infections. Variability in pre-infection mucosal interferon gene expression in children may impact the course of subsequent influenza infections. This variability may be due to frequent respiratory viral infections, demonstrating the potential importance of mucosal virus-virus interactions in children.
Monika Chodasiewicz, Olga Kerber, Michal Gorka, Juan C. Moreno, Israel Maruri-Lopez, Romina I. Minen, Arun Sampathkumar, Andrew D. L. Nelson, Aleksandra Skirycz
Abstract
The role of the RNA degradation product 2′,3′-cyclic adenosine monophosphate (2′,3′-cAMP) is poorly understood. Recent studies have identified 2′,3′-cAMP in plant material and determined its role in stress signaling. The level of 2′,3′-cAMP increases upon wounding, in the dark, and under heat, and 2′,3′-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2′,3′-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2′,3′-cAMP treatment. We demonstrated that 2′,3′-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide–adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2′,3′-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2′,3′-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2′,3′-cAMP treatment influences the movement of processing bodies, confirming the role of 2′,3′-cAMP in the formation and motility of membraneless organelles.
Hande Karahan, Daniel C. Smith, Byungwook Kim, Luke C. Dabin, Md Mamun Al-Amin, H. R. Sagara Wijeratne, Taylor Pennington, Gonzalo Viana di Prisco, Brianne McCord, Peter Bor-chian Lin, Yuxin Li, Junmin Peng, Adrian L. Oblak, Shaoyou Chu, Brady K. Atwood, Jungsu Kim
Abstract
Recently, large-scale human genetics studies identified a rare coding variant in the ABI3 gene that is associated with an increased risk of Alzheimer’s disease (AD). However, pathways by which ABI3 contributes to the pathogenesis of AD are unknown. To address this question, we determined whether loss of ABI3 function affects pathological features of AD in the 5XFAD mouse model. We demonstrate that the deletion of Abi3 locus significantly increases amyloid β (Aβ) accumulation and decreases microglia clustering around the plaques. Furthermore, long-term potentiation is impaired in 5XFAD;Abi3 knockout (“Abi3−/−”) mice. Moreover, we identified marked changes in the proportion of microglia subpopulations in Abi3−/− mice using a single-cell RNA sequencing approach. Mechanistic studies demonstrate that Abi3 knockdown in microglia impairs migration and phagocytosis. Together, our study provides the first in vivo functional evidence that loss of ABI3 function may increase the risk of developing AD by affecting Aβ accumulation and neuroinflammation.
Aleksandra Filipiak, Brodaczewska Klaudia, Majewska Aleksandra, Kieda Claudine
Abstract
Background:
Tumor development studies need higher degree of adaptation to the cancer cells specific mechanisms of plasticity in connection with their microenvironment. It appears that standard two-dimensional (2D) cultures and gas composition are not relevant to the real cancer environment. Existing three-dimensional (3D) models are often requiring difficult sophisticated conditions.
Methods:
Two distinct cancer models were chosen: melanoma (B16F10) and kidney cancer (RenCa) for their different biological reactions in terms of cancer progression. We proposed 3D method which brings simplicity, reproducibility and remarkable mimicry of the in vivo tumor reactions. We characterize and compare the 3D models with standard 2D culture in normoxic and hypoxic condition, depending on presence of hypoxia related genes/proteins and aggressiveness mechanism (EMT- Epithelial Mesenchymal Transition and CSC- Cancer Stem Cells). We validate proposed 3D method by comparing it with in vivo obtained tumors.
Results:
The spheroid formation by both lines indicated a strong potency of melanoma cells to form and grow in 3D structures. These spheroid-growing cells reached a cell cycle arrest at the G0/G1 phase and showed the formation of a hypoxic core while a similar development time produced renal cancer (RCC) 3D cells in growing phase and delayed hypoxia, as shown by hypoxia-related genes/proteins expression. A comparable response to hypoxia was observed in 3D spheroids and in vivo tumors for both lines as opposed to monolayer cultures. RCC model showed induction of CSC characteristic whereas, melanoma spheres contribute to EMT-related mechanism. Such distinction in the use of separate aggressiveness-leading pathways and response to hypoxia was observed in in vivo melanoma vs kidney tumors.
Conclusion:
This work shows that effects of the structural parameters and reaction to hypoxia uncover that these two cell models use distinct mechanisms to reach aggressiveness: epithelial to mesenchymal transition (EMT), cancer stem cells (CSC) by B16F10 and RenCa cells, respectively. These data on aggressiveness pathways and reaction to hypoxia confirm and validate the proposed 3D models for further design of alternative methods.
Anny Devoy, Georgia Price, Francesca De Giorgio, Rosie Bunton-Stasyshyn, David Thompson, Samanta Gasco, Alasdair Allan, Gemma F. Codner, Remya R. Nair, Charlotte Tibbit, Ross McLeod, Zeinab Ali, Judith Noda, Alessandro Marrero-Gagliardi, José M Brito-Armas, Michelle Simon, Edward O’Neill, Jackie Harrison, Gemma Atkins, Silvia Corrochano, Michelle Stewart, Lydia Teboul, Abraham Acevedo-Arozena, Elizabeth M.C Fisher, Thomas J. Cunningham
Abstract
Amyotrophic lateral sclerosis – frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS.
We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan.
These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.
Juan C. Moreno, Silvia Martinez-Jaime, Monika Kosmacz, Ewelina M. Sokolowska, Philipp Schulz, Axel Fischer, Urszula Luzarowska, Michel Havaux and Aleksandra Skirycz
Abstract
Recently, we published a set of tobacco lines expressing the Daucus carota (carrot) DcLCYB1 gene with accelerated development, increased carotenoid content, photosynthetic efficiency, and yield. Because of this development, DcLCYB1 expression might be of general interest in crop species as a strategy to accelerate development and increase biomass production under field conditions. However, to follow this path, a better understanding of the molecular basis of this phenotype is essential. Here, we combine OMICs (RNAseq, proteomics, and metabolomics) approaches to advance our understanding of the broader effect of LCYB expression on the tobacco transcriptome and metabolism. Upon DcLCYB1 expression, the tobacco transcriptome (~2,000 genes), proteome (~700 proteins), and metabolome (26 metabolites) showed a high number of changes in the genes involved in metabolic processes related to cell wall, lipids, glycolysis, and secondary metabolism. Gene and protein networks revealed clusters of interacting genes and proteins mainly involved in ribosome and RNA metabolism and translation. In addition, abiotic stress-related genes and proteins were mainly upregulated in the transgenic lines. This was well in line with an enhanced stress (high light, salt, and H2O2) tolerance response in all the transgenic lines compared with the wild type. Altogether, our results show an extended and coordinated response beyond the chloroplast (nucleus and cytosol) at the transcriptome, proteome, and metabolome levels, supporting enhanced plant growth under normal and stress conditions. This final evidence completes the set of benefits conferred by the expression of the DcLCYB1 gene, making it a very promising bioengineering tool to generate super crops.
Seth Jarvis, Nicol Birsa, Maria Secrier, Pietro Fratta and Vincent Plagnol
Abstract
Transcriptomics is a developing field with new methods of analysis being produced which may hold advantages in price, accuracy, or information output. QuantSeq is a form of 3′ sequencing produced by Lexogen which aims to obtain similar gene-expression information to RNA-seq with significantly fewer reads, and therefore at a lower cost. QuantSeq is also able to provide information on differential polyadenylation. We applied both QuantSeq at low read depth and total RNA-seq to the same two sets of mouse spinal cord RNAs, each comprised by four controls and four mutants related to the neurodegenerative disease amyotrophic lateral sclerosis. We found substantial differences in which genes were found to be significantly differentially expressed by the two methods. Some of this difference likely due to the difference in number of reads between our QuantSeq and RNA-seq data. Other sources of difference can be explained by the differences in the way the two methods handle genes with different primary transcript lengths and how likely each method is to find a gene to be differentially expressed at different levels of overall gene expression. This work highlights how different methods aiming to assess expression difference can lead to different results.
Tatiana Lopatina, Enrica Favaro, Ludmila Danilova, Elana J. Fertig, Alexander V. Favorov, Luciane T. Kagohara, Tiziana Martone, Benedetta Bussolati, Renato Romagnoli, Roberto Albera, Giancarlo Pecorari, Maria Felice Brizzi, Giovanni Camussi and Daria A. Gaykalova
Abstract
Head and neck squamous cell carcinoma (HNSCC) has a high recurrence and metastatic rate with an unknown mechanism of cancer spread. Tumor inflammation is the most critical processes of cancer onset, growth, and metastasis. We hypothesize that the release of extracellular vesicles (EVs) by tumor endothelial cells (TECs) induce reprogramming of immune cells as well as stromal cells to create an immunosuppressive microenvironment that favor tumor spread. We call this mechanism as non-metastatic contagious carcinogenesis. Extracellular vesicles were collected from primary HNSCC-derived endothelial cells (TEC-EV) and were used for stimulation of peripheral blood mononuclear cells (PBMCs) and primary adipose mesenchymal stem cells (ASCs). Regulation of ASC gene expression was investigated by RNA sequencing and protein array. PBMC, stimulated with TEC-EV, were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. We validated in vitro the effects of TEC-EV on ASCs or PBMC by measuring invasion, adhesion, and proliferation. We found and confirmed that TEC-EV were able to change ASC inflammatory gene expression signature within 24–48 h. TEC-EV were also able to enhance the secretion of TGF-β1 and IL-10 by PBMC and to increase T regulatory cell (Treg) expansion. TEC-EV carry specific proteins and RNAs that are responsible for Treg differentiation and immune suppression. ASCs and PBMC, treated with TEC-EV, enhanced proliferation, adhesion of tumor cells, and their invasion. These data indicate that TEC-EV exhibit a mechanism of non-metastatic contagious carcinogenesis that regulates tumor microenvironment and reprograms immune cells to sustain tumor growth and progression.
Emma Di Carlo, Giuseppe Cipollone, Felice Mucilli, Carlo Sorrentino
Abstract
The transcriptional profiling of cancer and normal tissues harboring cancer can be a clinical and discovery tool, especially for the study of rare tumors. Invasive mucinous adenocarcinoma (IMA) is a rare lung cancer histotype, which mostly affects the elderly and commonly has a poor prognosis. We investigated the exceptional case of a teenager, exposed to passive smoke and chemical carcinogens, who developed a multifocal IMA with bilateral involvement. The malignancy was asymptomatic and was diagnosed occasionally during hospitalization for acute abdominal pain due to adnexitis. The young patient underwent video-assisted thoracoscopic surgery and lung samples were analysed by RNA-Sequencing. The transcriptome of patient’s normal and neoplastic lung tissues was compared with matched healthy controls and IMA signature cases, using Gene Set Enrichment Analyses, Gene Ontology and Genotype Tissue Expression database. Compared to healthy controls, the patient’s lung tissue lacked the expression of lymphocyte and humoral-mediated immune response genes, whereas genes driving the response to stimulus, chemical and organic substances, primarily, CXCL8, ACKR1, RAB7B, HOXC9, HOXD9, KLF5 and NKX2-8 were overexpressed. Genes driving extracellular structure organization, cell adhesion, cell movement, metabolic and apoptotic processes were down-modulated in patient’s lung tissue. When compared to IMA signature cases, the patient’s IMA revealed a prevalent expression of genes regulating the response to stimulus, myeloid and neutrophil activation and immune system processes, primarily CD1a and CXCL13/BCA1, whereas stemness genes and proto-oncogenes, such as SOX4, HES1, IER3 and SERPINH1 were downmodulated. These transcriptional signature associated with a favorable clinical course, since the patient was healthy five years after initial diagnosis. The transcriptome of the normal tissues bearing tumor provides meaningful information on the gene pathways driving tumor histogenesis, with a prospective impact on early diagnosis. Unlike the tumor histotype-related transcriptional signature, the individual patient’s signature enables tailored treatment and accurate prognosis.
Leanna Morinishi, Karl Kochanowski, Ross L. Levine, Lani F. Wu, Steven J. Altschuler
Abstract
A persistent puzzle in cancer biology is how mutations, which neither alter growth signaling pathways nor directly interfere with drug mechanism, can still recur and persist in tumors. One example is the mutation of the DNA demethylase tet methylcytosine dioxygenase 2 (TET2) in acute myeloid leukemias (AMLs) that frequently persists from diagnosis through remission and relapse, but whose fitness advantage in chemotherapy is unclear. Here, we use isogenic human AML cell lines to show that TET2 loss of function alters the dynamics of transitions between differentiated and stem-like states. A conceptual mathematical model and experimental validation suggest that these altered cell-state dynamics can benefit the cell population by slowing population decay during drug treatment and lowering the number of survivor cells needed to re-establish the initial population. These studies shed light on the functional and phenotypic effects of a TET2 mutation in AML and illustrate how a single gene mutation can alter a cells’ phenotypic plasticity. A record of this paper’s transparent peer review process is included in the Supplemental Information.
Audra F Bryan, Jing Wang, Gregory C Howard, Alissa D Guarnaccia, Chase M Woodley, Erin R Aho, Eric J Rellinger, Brittany K Matlock, David K Flaherty, Shelly L Lorey, Dai H Chung, Stephen W Fesik, Qi Liu, April M Weissmiller, William P Tansey
Abstract
WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the ‘WIN’ site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks—if any—that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis.
Yaroslav A. Kainov, Eugene V. Makeyev
Abstract
Eukaryotic gene expression relies on extensive crosstalk between transcription and RNA processing. Changes in this composite regulation network may provide an important means for shaping cell type-specific transcriptomes. Here we show that the RNA-associated protein Srrt/Ars2 sustains embryonic stem cell (ESC) identity by preventing premature termination of numerous transcripts at cryptic cleavage/polyadenylation sites in first introns. Srrt interacts with the nuclear cap-binding complex and facilitates recruitment of the spliceosome component U1 snRNP to cognate intronic positions. At least in some cases, U1 recruited in this manner inhibits downstream cleavage/polyadenylation events through a splicing-independent mechanism called telescripting. We further provide evidence that the naturally high expression of Srrt in ESCs offsets deleterious effects of retrotransposable sequences accumulating in its targets. Our work identifies Srrt as a molecular guardian of the pluripotent cell state.
Carolina Martinelli, Fabio Gabriele, Federico Manai, Roberto Ciccone, Francesca Novara, Elisabetta Sauta, Riccardo Bellazzi, Monica Patane, Isabella Moroni, Rosina Paterra And Sergio Comincini
Abstract
Background/Aim:
We herein presented a case of pediatric spinal cord pilocytic astrocytoma diagnosed on the basis of histopathological and clinical findings. Materials and Methods: Given the paucity of data on genetic features for this tumor, we performed exome, array CGH and RNA sequencing analysis from nucleic acids isolated from a unique and not repeatable very small amount of a formalin-fixed, paraffin-embedded (FFPE) specimen. Results: DNA mutation analysis, comparing tumor and normal lymphocyte peripheral DNA, evidenced few tumor-specific single nucleotide variants in DEFB119, MUC5B, NUDT1, LTBP3 and CPSF3L genes. Differently, tumor DNA was not characterized by for the main pilocytic astrocytoma gene variations, including BRAFV600E. An inframe trinucleotides insertion involving DLX6 or lnc DLX6-AS1 genes was scored in 44.9% of sequenced reads; the temporal profile of this variation on the expression of DLX-AS1 was investigated in patient‘s urine-derived exosomes, reporting no significant variation in the one-year molecular follow-up. Array CGH identified a tumor microdeletion at the 6q25.3 chromosomal region, spanning 1,01 Mb and comprising ZDHHC14, SNX9, TULP4 and SYTL3 genes. The expression of these genes did not change in urine-derived exosomes during the one-year investigation period. Finally, RNAseq did not reveal any of the common pilocytic BRAF-KIAA1549 genes fusion events. Conclusion: To our knowledge, the present report is one of the first described gene-orphan case studies of a pediatric spinal cord pilocytic astrocytoma.
Marianna N. Rossi, Antonia Pascarella, Valerio Licursi, Ivan Caiello, Anna Taranta, Laura R. Rega, Elena Levtchenko, Francesco Emma, Fabrizio De Benedetti and Giusi Prencipe
Abstract
Nod-like Receptor Pyrin domain containing proteins (NLRPs) expressed by resident renal cells may contribute to the pathogenesis of multiple renal diseases. Cystinosis is a genetic disorder that affects kidney and particularly proximal tubular epithelial cells (PTEC). Here, we investigated the expression of NLRP family members in human control and cystinotic conditionally immortalized PTEC. Among all the NLRPs tested, we found that NLRP2 is highly expressed in cystinostic PTEC, but not in PTEC from healthy subjects. The NLRP2 overexpression was confirmed in primary PTEC and in kidney biopsies from cystinotic patients. In order to elucidate the role of NLRP2 in PTEC, we stably transfected control PTEC with an NLRP2-containing plasmid. We showed that NLRP2 markedly increases the production of several NF-κB regulated cytokines and chemokines. Accordingly, we demonstrated that NLRP2 interacts with IKKa and positively regulates the DNA-binding activity of p50 and p65 NF-κB, by modulating the p65 NF-κB phosphorylation status in Serine 536. Transcriptome analysis revealed that NLRP2 also upregulates the expression of profibrotic mediators and reduces that of several interferon-inducible genes. Finally, NLRP2 overexpression decreased the apoptotic cell rate. Consistently, silencing of NLRP2 by small-interfering RNA in cystinotic PTEC resulted in a significant decrease in cytokine and chemokine production as well as in an increase in the apoptosis rate. Altogether, our data reveals a previously unrecognized role for NLRP2 in regulating proinflammatory, profibrotic and antiapoptotic responses in PTEC, through NF-κB activation. Moreover, our findings unveil a novel potential mechanism involving NLRP2 overexpression in the pathogenesis of cystinosis.
K. Arnauts, B. Verstockt, M. Vancamelbeke, S. Vermeire, C. Verfaillie, M. Ferrante
Abstract
Background
Patient-derived intestinal organoids provide an excellent tool to unravel the multi-factorial mechanisms underlying ulcerative colitis (UC). Organoids develop from stem cell-containing intestinal crypts and recapitulate many features of the source tissue. However, it remains unclear whether ex vivo organoids retain the inflammatory character of their origin. To address this, we isolated crypts from both inflamed and non-inflamed regions of the colon, created organoids, and compared the transcriptome of whole biopsies, crypts and ex vivo cultured organoids.
Methods
Fresh biopsies from both inflamed and non-inflamed segments were obtained during endoscopy from eight patients with active UC (endoscopic Mayo sub-score of ≥2) and an accessible border of inflammation. Crypts were isolated and cultured as organoids for 4 weeks with weekly mechanical splitting. RNA was extracted from biopsies, crypts, and 1- and 4-week-old organoids. RNA sequencing was performed by Lexogen QuantSeq for Illumina. Differential gene expression and pathways were studied through DESeq2 and Ingenuity Pathway Analysis (FDR < 0.05).
Results
Biopsies and crypts from inflamed regions showed separate clustering on principal component analysis (PCA, Figure) and significantly higher activation of inflammatory pathways, including antigen presentation (p < 0.01 and p < 0.001), interferon signalling (p < 0.05 and p < 0.001) and granulocyte adhesion (both p < 0.001) compared with non-inflamed biopsies and crypts. However, organoids derived from inflamed crypts lost part of their inflammatory character after 1 week in culture. Several inflammatory markers (IFN-γ [p = 0.01], IL-1β [p < 0.001], JAK1 [p < 0.001]), and pathways involved in antigen presentation (p < 0.005) and interferon signalling (p < 0.001) were significantly decreased after 1 week ex vivo culture compared with inflamed crypts. After 4 weeks in culture, organoids derived from inflamed and non-inflamed regions were indistinguishable in PCA clustering, and expression levels of inflammatory signalling pathways were not significant.
Conclusions
We conclude that ex vivo organoids lose their inflammatory transcriptional signature in culture. After 4 weeks in culture, organoids derived from inflamed and non-inflamed biopsies were no longer distinguishable. Therefore, it is not essential to obtain biopsies from inflamed regions to culture organoids from UC patients. We hypothesise that to mimic the inflammatory phenotype and create a physiological representative model, inflammatory components, and/or immune cells should be added to the ex vivo culture system.
