Identification of selective sweeps in domesticated apple (Malus × domestica Borkh.)

The domesticated apple (Malus × domestica) is one of the most cultivated plant over the world and is one of the most genetically polymorphic agricultural species. Studying the genetic diversity of the apple germplasm could provide important hints about the domestication process as giving a valuable resource for high resolution genetic mapping, QTL analysis and breeding programs. Advances in next generation sequencing technologies have driven the costs of DNA sequencing down to the point that whole genome re-sequencing (WGS) is now feasible for high diversity, large genome species. The aim of this work is to gain information on genome-wide genetic variability patterns in apple and to identify regions of the genome that may have been selected during the process of plant domestication. SNPs were called from Illumina short reads for 63 apple cultivars representative of European germplasm diversity. The identified SNPs (over 15 millions) were filtered for quality and to avoid repeated and paralogous regions. Additional filters (minor allele frequency and Hardy-Weinberg equilibrium) were applied to discard variants derived from genotyping errors resulting in a final number of 426,321 SNPs . The SNPs kept after the quality filters were used to study the population structure and the genetic diversity. A weak stratification of the analyzed population emerged both from the principal component analysis (PCA) and a model based clustering approach performed using fastStructure. This analysis showed the presence of three subpopulations with a high level of admixture. FST between each couple of sub-groups was 0.055, 0.083 and 0.096 that indicate a moderate differentiation. Two different approaches were used to identify selective sweeps. The first is based on allelic frequencies and the site frequency spectrum (SFS) and it is implemented in the software SweeD. The second is based on linkage disequilibrium patterns and the omega statistic and it is implemented in the software OmegaPlus. Regions that were identified by both softwares were merged and used as candidate regions for positive selection resulting in 1,194 sweeps on the whole genome. A total of 153 gene predictions were extracted from these candidate regions and annotated using Gene Ontology terms and mapping on the KEGG pathway database. Similarity searches were also performed against plant databases to find gene orthologs and to better understand the function of candidates. The annotation revealed that genes under positive selection are involved in pathways like photosynthesis, protein ubiquitination, plant hormone signal transduction and starch and sucrose metabolism. In particular for the plant hormone signal transduction, were identified the auxin influx carrier and a SAUR family protein that lead to cell enlargement and plant growth and the ethylene insensitive protein 2 that leads to fruit ripening and senescence. The genes identified in regions under positive selection that were functionally annotated are consistent with the domestication traits for a better fruit: bigger, tastier and sweete

Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influence on MEF2-dependent transcription

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding

CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing

Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in ‘regulation of RNA splicing’ and ‘mRNA catabolic process’. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD

Identification of selective sweeps in domesticated apple (Malus × domestica Borkh.)

The domesticated apple (Malus × domestica) is one of the most cultivated plant over the world and is one of the most genetically polymorphic agricultural species. Studying the genetic diversity of the apple germplasm could provide important hints about the domestication process as giving a valuable resource for high resolution genetic mapping, QTL analysis and breeding programs. Advances in next generation sequencing technologies have driven the costs of DNA sequencing down to the point that whole genome re-sequencing (WGS) is now feasible for high diversity, large genome species. The aim of this work is to gain information on genome-wide genetic variability patterns in apple and to identify regions of the genome that may have been selected during the process of plant domestication. SNPs were called from Illumina short reads for 63 apple cultivars representative of European germplasm diversity. The identified SNPs (over 15 millions) were filtered for quality and to avoid repeated and paralogous regions. Additional filters (minor allele frequency and Hardy-Weinberg equilibrium) were applied to discard variants derived from genotyping errors resulting in a final number of 426,321 SNPs . The SNPs kept after the quality filters were used to study the population structure and the genetic diversity. A weak stratification of the analyzed population emerged both from the principal component analysis (PCA) and a model based clustering approach performed using fastStructure. This analysis showed the presence of three subpopulations with a high level of admixture. FST between each couple of sub-groups was 0.055, 0.083 and 0.096 that indicate a moderate differentiation. Two different approaches were used to identify selective sweeps. The first is based on allelic frequencies and the site frequency spectrum (SFS) and it is implemented in the software SweeD. The second is based on linkage disequilibrium patterns and the omega statistic and it is implemented in the software OmegaPlus. Regions that were identified by both softwares were merged and used as candidate regions for positive selection resulting in 1,194 sweeps on the whole genome. A total of 153 gene predictions were extracted from these candidate regions and annotated using Gene Ontology terms and mapping on the KEGG pathway database. Similarity searches were also performed against plant databases to find gene orthologs and to better understand the function of candidates. The annotation revealed that genes under positive selection are involved in pathways like photosynthesis, protein ubiquitination, plant hormone signal transduction and starch and sucrose metabolism. In particular for the plant hormone signal transduction, were identified the auxin influx carrier and a SAUR family protein that lead to cell enlargement and plant growth and the ethylene insensitive protein 2 that leads to fruit ripening and senescence. The genes identified in regions under positive selection that were functionally annotated are consistent with the domestication traits for a better fruit: bigger, tastier and sweeterIl melo domestico (Malus × domestica) è una delle piante più coltivate al mondo ed è tra le specie agricole geneticamente più polimorfiche. Studiare la diversità genetica in melo può dare importanti suggerimenti sul processo di domesticazione e valide risorse per creare mappe genetiche ad alta risoluzione, per analisi di QTL e nei programmi di breeding. I miglioramenti nelle tecnologie di sequenziamento del DNA, dette NGS, hanno ridotto di molto i costi del sequenziamento al punto che i risequenziamenti completi di genomi sono ora fattibili anche per specie ad alta diversità genetica e dal genoma molto grande. Lo scopo di questo lavoro è l'analisi della variabilità genetica dell’intero genoma di melo e l'identificazione di regioni genomiche sottoposte a selezione durante il processo di domesticazione. A tale scopo 63 cultivar di melo, rappresentanti l’intera diversità del germoplasma europeo, sono state sequenziate con teconolgia Illumina. Dalle sequenze sono stati predetti oltre 15 milioni di SNP che sono stati filtrati eliminare le predizioni scadenti o legate a regioni ripetute e paraloghe. Ulteriori filtri (minor allele frequency e Hardy-Weinberg equilibrium) sono stati applicati per eliminare gli SNP derivati da errori di genotipizzazione. Il numero finale degli SNP filtrati è risultato di 426'321. Gli SNP rimasti dopo i filtri di qualità sono stati usati per studiare la struttura di popolazione e la diversità genetica. Dall'analisi delle componenti principali e da un metodo di clusterizzazione implementato in fastStructure, è emersa una debole stratificazione della popolazione analizzata. Questa analisi ha mostrato la presenza di tre sottopopolazioni con un alto livello di admixture. L’FST tra ogni coppia di sottopopolazioni è risultato di 0,055, 0,083 and 0,096 indicando un livello di differenziazione moderato. Due diversi approcci sono stati usati per identificare 'selective sweep'. Il primo è basato sulle frequenze alleliche e sul 'site frequency spectrum' (SFS) ed è implementato nel software SweeD. Il secondo è basato sui pattern di 'linkage disequilibrium' e la statistica ω ed è implementato nel software OmegaPlus. Le regioni del genoma che sono state identificate da entrambi i software sono state usate come regioni candidate sotto selezione positiva. In tutto il genoma le regioni sotto selezione sono risultate 1'194. In totale 153 predizioni geniche sono state estratte dalle regioni candidate e annotate usando i termini della Gene Ontology e con i pathway metabolici descritti nel database KEGG. Ricerche di similarità in database di piante sono state fatte per trovare geni ortologhi e per capire meglio la funzione dei geni candidati. L'annotazione ha rivelato che i geni sotto selezione positiva sono coinvolti in vari processi quali la fotosintesi, l'ubiquitinazione di proteine, la trasduzione del segnale ormonale delle piante o il metobolismo di amidi e zuccheri. In particolare, per la trasduzione del segnale, sono stati identificati l'importatore dell'auxina e una proteina della famiglia SAUR che agiscono sull'aumento della dimensione cellulare e sulla crescita della pianta e la proteina 2 insensibile all'etilene che porta alla maturazione del frutto e alla senescenza. Le annotazioni funzionali disponibili ascrivono i geni identificati a ruoli fisiologici coerenti con i tratti fenotipici attesi per un processo di domesticazione. Per esempio i tratti legati al miglioramento delle caratterisitche del frutto come la dimensione, il gusto e la dolcezz

Zebrafish Melanoma-Derived Interstitial EVs Are Carriers of ncRNAs That Induce Inflammation

International audienceExtracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRASV12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system

Hypomorphic mutation of the mouse Huntington's disease gene orthologue.

Rare individuals with inactivating mutations in the Huntington's disease gene (HTT) exhibit variable abnormalities that imply essential HTT roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in the murine HTT orthologue Htt, (HdhneoQ20, HdhneoQ50, HdhneoQ111), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate, though still severe, alleles yielded recessive perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development that nominate polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that Htt is required both pre- and post-gastrulation to support normal development

CAG repeat expansion in the Huntington's disease gene shapes linear and circular RNAs biogenesis.

Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis

The Class IIA Histone Deacetylase (HDAC) Inhibitor TMP269 Downregulates Ribosomal Proteins and Has Anti-Proliferative and Pro-Apoptotic Effects on AML Cells

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML

Kinome-wide synthetic lethal screen identifies PANK4 as a modulator of temozolomide resistance in glioblastoma

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterized. Here, following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ-resistant GBM cell models, patient-derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)-based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.</p

Kinome-wide synthetic lethal screen identifies PANK4 as a modulator of temozolomide resistance in glioblastoma

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterized. Here, following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ-resistant GBM cell models, patient-derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)-based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.</p