Publications by authors named "Enrico Petretto"

119 Publications

Implication of sestrin3 in epilepsy and its comorbidities.

Brain Commun 2021 9;3(1):fcaa130. Epub 2020 Oct 9.

Department of Medical Sciences, Section of Pharmacology, University of Ferrara, Italy.

Epilepsy is a serious neurological disorder affecting about 1% of the population worldwide. Epilepsy may arise as a result of acquired brain injury, or as a consequence of genetic predisposition. To date, genome-wide association studies and exome sequencing approaches have provided limited insights into the mechanisms of acquired brain injury. We have previously reported a pro-epileptic gene network, which is conserved across species, encoding inflammatory processes and positively regulated by sestrin3 (SESN3). In this study, we investigated the phenotype of knock-out rats in terms of susceptibility to seizures and observed a significant delay in status epilepticus onset in knock-out compared to control rats. This finding confirms previous and evidence indicating that SESN3 may favour occurrence and/or severity of seizures. We also analysed the phenotype of knock-out rats for common comorbidities of epilepsy, i.e., anxiety, depression and cognitive impairment. knock-out rats proved less anxious compared to control rats in a selection of behavioural tests. Taken together, the present results suggest that SESN3 may regulate mechanisms involved in the pathogenesis of epilepsy and its comorbidities.
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http://dx.doi.org/10.1093/braincomms/fcaa130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966953PMC
October 2020

WNT inhibition creates a BRCA-like state in Wnt-addicted cancer.

EMBO Mol Med 2021 Apr 4;13(4):e13349. Epub 2021 Mar 4.

Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore.

Wnt signaling maintains diverse adult stem cell compartments and is implicated in chemotherapy resistance in cancer. PORCN inhibitors that block Wnt secretion have proven effective in Wnt-addicted preclinical cancer models and are in clinical trials. In a survey for potential combination therapies, we found that Wnt inhibition synergizes with the PARP inhibitor olaparib in Wnt-addicted cancers. Mechanistically, we find that multiple genes in the homologous recombination and Fanconi anemia repair pathways, including BRCA1, FANCD2, and RAD51, are dependent on Wnt/β-catenin signaling in Wnt-high cancers, and treatment with a PORCN inhibitor creates a BRCA-like state. This coherent regulation of DNA repair genes occurs in part via a Wnt/β-catenin/MYBL2 axis. Importantly, this pathway also functions in intestinal crypts, where high expression of BRCA and Fanconi anemia genes is seen in intestinal stem cells, with further upregulation in Wnt-high APC mutant polyps. Our findings suggest a general paradigm that Wnt/β-catenin signaling enhances DNA repair in stem cells and cancers to maintain genomic integrity. Conversely, interventions that block Wnt signaling may sensitize cancers to radiation and other DNA damaging agents.
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http://dx.doi.org/10.15252/emmm.202013349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033517PMC
April 2021

Brugada syndrome genetics is associated with phenotype severity.

Eur Heart J 2021 Mar;42(11):1082-1090

Arrhythmia and Electrophysiology Department, IRCCS Policlinico San Donato, Piazza E. Malan 1, 20097 San Donato Milanese, Milano, Italy.

Aims : Brugada syndrome (BrS) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia/fibrillation (VT/VF) in young, otherwise healthy individuals. Despite SCN5A being the most commonly known mutated gene to date, the genotype-phenotype relationship is poorly understood and remains uncertain. This study aimed to elucidate the genotype-phenotype correlation in BrS.

Methods And Results: Brugada syndrome probands deemed at high risk of future arrhythmic events underwent genetic testing and phenotype characterization by the means of epicardial arrhythmogenic substrate (AS) mapping, and were divided into two groups according to the presence or absence of SCN5A mutation. Two-hundred probands (160 males, 80%; mean age 42.6 ± 12.2 years) were included in this study. Patients harbouring SCN5A mutations exhibited a spontaneous type 1 pattern and experienced aborted cardiac arrest or spontaneous VT/VF more frequently than the other subjects. SCN5A-positive patients exhibited a larger epicardial AS area, more prolonged electrograms and more frequently observed non-invasive late potentials. The presence of an SCN5A mutation explained >26% of the variation in the epicardial AS area and was the strongest predictor of a large epicardial area.

Conclusion : In BrS, the genetic background is the main determinant for the extent of the electrophysiological abnormalities. SCN5A mutation carriers exhibit more pronounced epicardial electrical abnormalities and a more aggressive clinical presentation. These results contribute to the understanding of the genetic determinants of the BrS phenotypic expression and provide possible explanations for the varying degrees of disease expression.
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http://dx.doi.org/10.1093/eurheartj/ehaa942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955973PMC
March 2021

Widespread Repression of Gene Expression in Cancer by a Wnt/β-Catenin/MAPK Pathway.

Cancer Res 2021 Jan 17;81(2):464-475. Epub 2020 Nov 17.

Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.

Aberrant Wnt signaling drives a number of cancers through regulation of diverse downstream pathways. Wnt/β-catenin signaling achieves this in part by increasing the expression of proto-oncogenes such as and cyclins. However, global assessment of the Wnt-regulated transcriptome in genetically distinct cancers demonstrates that Wnt signaling suppresses the expression of as many genes as it activates. In this study, we examined the set of genes that are upregulated upon inhibition of Wnt signaling in Wnt-addicted pancreatic and colorectal cancer models. Decreasing Wnt signaling led to a marked increase in gene expression by activating ERK and JNK; these changes in gene expression could be mitigated in part by concurrent inhibition of MEK. These findings demonstrate that increased Wnt signaling in cancer represses MAPK activity, preventing RAS-mediated senescence while allowing cancer cells to proliferate. These results shift the paradigm from Wnt/β-catenin primarily as an activator of transcription to a more nuanced view where Wnt/β-catenin signaling drives both widespread gene repression and activation. SIGNIFICANCE: These findings show that Wnt/β-catenin signaling causes widespread gene repression via inhibition of MAPK signaling, thus fine tuning the RAS-MAPK pathway to optimize proliferation in cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2129DOI Listing
January 2021

Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation.

Genome Med 2020 10 22;12(1):89. Epub 2020 Oct 22.

Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore.

Background: Wnt signaling is an evolutionarily conserved developmental pathway that is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not been systematically characterized.

Methods: We comprehensively mapped Wnt-regulated lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model and examined the response of lncRNAs to Wnt inhibition between in vivo and in vitro cancer models. We further annotated and characterized these Wnt-regulated lncRNAs using existing genomic classifications (using data from FANTOM5) in the context of Wnt signaling and inferred their role in cancer pathogenesis (using GWAS and expression data from the TCGA). To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation both in vivo and in vitro.

Results: We identified 3633 lncRNAs, of which 1503 were regulated by Wnt signaling in an orthotopic Wnt-addicted pancreatic cancer model. These lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. Our analysis suggested that Wnt signaling inhibition could influence the co-expression relationship of Wnt-regulated lncRNAs and their eQTL-linked protein-coding genes. Wnt-regulated lncRNAs were also implicated in specific gene networks involved in distinct biological processes that contribute to the pathogenesis of cancers. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1503) of the Wnt-regulated lncRNAs were found to modify cancer cell growth in vitro. This included CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-regulated lncRNAs (25/1503) that had a functional effect on cancer cell growth.

Conclusions: Our study demonstrates the value of studying lncRNA functions in vivo, provides a valuable resource of lncRNAs regulated by Wnt signaling, and establishes a framework for systematic discovery of functional lncRNAs.
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http://dx.doi.org/10.1186/s13073-020-00788-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580003PMC
October 2020

EpiMogrify Models H3K4me3 Data to Identify Signaling Molecules that Improve Cell Fate Control and Maintenance.

Cell Syst 2020 11 9;11(5):509-522.e10. Epub 2020 Oct 9.

Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore (Duke-NUS) Medical School, 8 College Road, 169857 Singapore, Singapore. Electronic address:

The need to derive and culture diverse cell or tissue types in vitro has prompted investigations on how changes in culture conditions affect cell states. However, the identification of the optimal conditions (e.g., signaling molecules and growth factors) required to maintain cell types or convert between cell types remains a time-consuming task. Here, we developed EpiMogrify, an approach that leverages data from ∼100 human cell/tissue types available from ENCODE and Roadmap Epigenomics consortia to predict signaling molecules and factors that can either maintain cell identity or enhance directed differentiation (or cell conversion). EpiMogrify integrates protein-protein interaction network information with a model of the cell's epigenetic landscape based on H3K4me3 histone modifications. Using EpiMogrify-predicted factors for maintenance conditions, we were able to better potentiate the maintenance of astrocytes and cardiomyocytes in vitro. We report a significant increase in the efficiency of astrocyte and cardiomyocyte differentiation using EpiMogrify-predicted factors for conversion conditions.
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http://dx.doi.org/10.1016/j.cels.2020.09.004DOI Listing
November 2020

A trans-eQTL network regulates osteoclast multinucleation and bone mass.

Elife 2020 06 19;9. Epub 2020 Jun 19.

Centre for Inflammatory Disease, Department of Immunology and Inflammation, Hammersmith Hospital, Imperial College London, London, United Kingdom.

Functional characterisation of cell-type-specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a -regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global (, , , , , ) and myeloid-specific knockout mice displayed abnormal bone phenotypes. We report opposing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast multinucleation and bone mass.
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http://dx.doi.org/10.7554/eLife.55549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351491PMC
June 2020

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Cell Rep 2020 May;31(8):107714

Cardiovascular & Metabolic Disorders Program, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore; Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.celrep.2020.107714DOI Listing
May 2020

Impulsivity is a heritable trait in rodents and associated with a novel quantitative trait locus on chromosome 1.

Sci Rep 2020 04 21;10(1):6684. Epub 2020 Apr 21.

Department of Psychology and Behavioural and Clinical Neuroscience Institute, University of Cambridge, Cambridge, UK.

Impulsivity describes the tendency to act prematurely without appropriate foresight and is symptomatic of a number of neuropsychiatric disorders. Although a number of genes for impulsivity have been identified, no study to date has carried out an unbiased, genome-wide approach to identify genetic markers associated with impulsivity in experimental animals. Herein we report a linkage study of a six-generational pedigree of adult rats phenotyped for one dimension of impulsivity, namely premature responding on the five-choice serial reaction time task, combined with genome wide sequencing and transcriptome analysis to identify candidate genes associated with the expression of the impulsivity trait. Premature responding was found to be heritable (h = 13-16%), with significant linkage (LOD 5.2) identified on chromosome 1. Fine mapping of this locus identified a number of polymorphic candidate genes, however only one, beta haemoglobin, was differentially expressed in both the founder strain and F6 generation. These findings provide novel insights into the genetic substrates and putative neurobiological mechanisms of impulsivity with broader translational relevance for impulsivity-related disorders in humans.
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http://dx.doi.org/10.1038/s41598-020-63646-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174407PMC
April 2020

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly.

Nat Commun 2020 03 11;11(1):1312. Epub 2020 Mar 11.

Cardiovascular Metabolic Disorders Program, Duke-NUS Medical School, Singapore, Singapore.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.
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http://dx.doi.org/10.1038/s41467-020-14999-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066179PMC
March 2020

Genome-wide meta-analysis associates GPSM1 with type 2 diabetes, a plausible gene involved in skeletal muscle function.

J Hum Genet 2020 Apr 21;65(4):411-420. Epub 2020 Jan 21.

National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China, 210016.

Genome-wide association studies (GWASs) have identified many genetic variations associated with type 2 diabetes mellitus (T2DM) in Asians, but understanding the functional genetic variants that influence traits is often a complex process. In this study, fine mapping and other analytical strategies were performed to investigate the effects of G protein signaling modulator 1 (GPSM1) on insulin resistance in skeletal muscle. A total of 128 single-nucleotide polymorphisms (SNPs) within GPSM1 were analysed in 21,897 T2DM cases and 32,710 healthy controls from seven GWASs. The SNP rs28539249 in intron 9 of GPSM1 showed a nominally significant association with T2DM in Asians (OR = 1.07, 95% CI = 1.04-1.10, P < 10). The GPSM1 mRNA was increased in skeletal muscle and correlated with T2DM traits across obese mice model. An eQTL for the cis-acting regulation of GPSM1 expression in human skeletal muscle was identified for rs28539249, and the increased GPSM1 expression related with T2DM traits within GEO datasets. Another independent Asian cohort showed that rs28539249 is associated with the skeletal muscle expression of CACFD1, GTF3C5, SARDH, and FAM163B genes, which are functionally enriched for endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) pathways. Moreover, rs28539249 locus was predicted to disrupt regulatory regions in human skeletal muscle with enriched epigenetic marks and binding affinity for CTCF. Supershift EMSA assays followed luciferase assays demonstrated the CTCF specifically binding to rs28539249-C allele leading to decreased transcriptional activity. Thus, the post-GWAS annotation confirmed the Asian-specific association of genetic variant in GPSM1 with T2DM, suggesting a role for the variant in the regulation in skeletal muscle.
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http://dx.doi.org/10.1038/s10038-019-0720-3DOI Listing
April 2020

Whole-Genome Sequencing of Finnish Type 1 Diabetic Siblings Discordant for Kidney Disease Reveals DNA Variants associated with Diabetic Nephropathy.

J Am Soc Nephrol 2020 02 9;31(2):309-323. Epub 2020 Jan 9.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;

Background: Several genetic susceptibility loci associated with diabetic nephropathy have been documented, but no causative variants implying novel pathogenetic mechanisms have been elucidated.

Methods: We carried out whole-genome sequencing of a discovery cohort of Finnish siblings with type 1 diabetes who were discordant for the presence (case) or absence (control) of diabetic nephropathy. Controls had diabetes without complications for 15-37 years. We analyzed and annotated variants at genome, gene, and single-nucleotide variant levels. We then replicated the associated variants, genes, and regions in a replication cohort from the Finnish Diabetic Nephropathy study that included 3531 unrelated Finns with type 1 diabetes.

Results: We observed protein-altering variants and an enrichment of variants in regions associated with the presence or absence of diabetic nephropathy. The replication cohort confirmed variants in both regulatory and protein-coding regions. We also observed that diabetic nephropathy-associated variants, when clustered at the gene level, are enriched in a core protein-interaction network representing proteins essential for podocyte function. These genes include protein kinases (protein kinase C isoforms and ) and protein tyrosine kinase 2.

Conclusions: Our comprehensive analysis of a diabetic nephropathy cohort of siblings with type 1 diabetes who were discordant for kidney disease points to variants and genes that are potentially causative or protective for diabetic nephropathy. This includes variants in two isoforms of the protein kinase C family not previously linked to diabetic nephropathy, adding support to previous hypotheses that the protein kinase C family members play a role in diabetic nephropathy and might be attractive therapeutic targets.
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http://dx.doi.org/10.1681/ASN.2019030289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003303PMC
February 2020

A single-cell atlas of entorhinal cortex from individuals with Alzheimer's disease reveals cell-type-specific gene expression regulation.

Nat Neurosci 2019 12;22(12):2087-2097

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia.

There is currently little information available about how individual cell types contribute to Alzheimer's disease. Here we applied single-nucleus RNA sequencing to entorhinal cortex samples from control and Alzheimer's disease brains (n = 6 per group), yielding a total of 13,214 high-quality nuclei. We detail cell-type-specific gene expression patterns, unveiling how transcriptional changes in specific cell subpopulations are associated with Alzheimer's disease. We report that the Alzheimer's disease risk gene APOE is specifically repressed in Alzheimer's disease oligodendrocyte progenitor cells and astrocyte subpopulations and upregulated in an Alzheimer's disease-specific microglial subopulation. Integrating transcription factor regulatory modules with Alzheimer's disease risk loci revealed drivers of cell-type-specific state transitions towards Alzheimer's disease. For example, transcription factor EB, a master regulator of lysosomal function, regulates multiple disease genes in a specific Alzheimer's disease astrocyte subpopulation. These results provide insights into the coordinated control of Alzheimer's disease risk genes and their cell-type-specific contribution to disease susceptibility. These results are available at http://adsn.ddnetbio.com.
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http://dx.doi.org/10.1038/s41593-019-0539-4DOI Listing
December 2019

Decreased microglial Wnt/β-catenin signalling drives microglial pro-inflammatory activation in the developing brain.

Brain 2019 12;142(12):3806-3833

Université de Paris, NeuroDiderot, Inserm, F-75019 Paris, France.

Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/β-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.
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http://dx.doi.org/10.1093/brain/awz319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906599PMC
December 2019

Transcriptional Networks of Microglia in Alzheimer's Disease and Insights into Pathogenesis.

Genes (Basel) 2019 10 12;10(10). Epub 2019 Oct 12.

Programme in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, 8 College Road, 69857 Singapore, Singapore.

Microglia, the main immune cells of the central nervous system, are increasingly implicated in Alzheimer's disease (AD). Manifold transcriptomic studies in the brain have not only highlighted microglia's role in AD pathogenesis, but also mapped crucial pathological processes and identified new therapeutic targets. An important component of many of these transcriptomic studies is the investigation of gene expression networks in AD brain, which has provided important new insights into how coordinated gene regulatory programs in microglia (and other cell types) underlie AD pathogenesis. Given the rapid technological advancements in transcriptional profiling, spanning from microarrays to single-cell RNA sequencing (scRNA-seq), tools used for mapping gene expression networks have evolved to keep pace with the unique features of each transcriptomic platform. In this article, we review the trajectory of transcriptomic network analyses in AD from brain to microglia, highlighting the corresponding methodological developments. Lastly, we discuss examples of how transcriptional network analysis provides new insights into AD mechanisms and pathogenesis.
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http://dx.doi.org/10.3390/genes10100798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826883PMC
October 2019

Broad regulation of gene isoform expression by Wnt signaling in cancer.

RNA 2019 12 10;25(12):1696-1713. Epub 2019 Sep 10.

Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, 169857.

Differential gene isoform expression is a ubiquitous mechanism to enhance proteome diversity and maintain cell homeostasis. Mechanisms such as splicing that drive gene isoform variability are highly dynamic and responsive to changes in cell signaling pathways. Wnt/β-catenin signaling has profound effects on cell activity and cell fate and is known to modify several splicing events by altering the expression of individual splicing factors. However, a global assessment of how extensively Wnt signaling regulates splicing and other mechanisms that determine mRNA isoform composition in cancer is lacking. We used deep time-resolved RNA-seq in two independent in vivo Wnt-addicted tumor models during treatment with the potent Wnt inhibitor ETC-159 and examined Wnt regulated splicing events and splicing regulators. We found 1025 genes that underwent Wnt regulated variable exon usage leading to isoform expression changes. This was accompanied by extensive Wnt regulated changes in the expression of splicing regulators. Many of these Wnt regulated events were conserved in multiple human cancers, and many were linked to previously defined cancer-associated splicing quantitative trait loci. This suggests that the Wnt regulated splicing events are components of fundamental oncogenic processes. These findings demonstrate the wide-ranging effects of Wnt signaling on the isoform composition of the cell and provides an extensive resource of expression changes of splicing regulators and gene isoforms regulated by Wnt signaling.
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http://dx.doi.org/10.1261/rna.071506.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859862PMC
December 2019

Integrated systems-genetic analyses reveal a network target for delaying glioma progression.

Ann Clin Transl Neurol 2019 09 17;6(9):1616-1638. Epub 2019 Aug 17.

Division of Brain Sciences, Imperial College Faculty of Medicine, London, UK.

Objective: To identify a convergent, multitarget proliferation characteristic for astrocytoma transformation that could be targeted for therapy discovery.

Methods: Using an integrated functional genomics approach, we prioritized networks associated with astrocytoma progression using the following criteria: differential co-expression between grade II and grade III IDH1-mutated and 1p/19q euploid astrocytomas, preferential enrichment for genetic risk to cancer, association with patient survival and sample-level genomic features. Drugs targeting the identified multitarget network characteristic for astrocytoma transformation were computationally predicted using drug transcriptional perturbation data and validated using primary human astrocytoma cells.

Results: A single network, M2, consisting of 177 genes, was associated with glioma progression on the basis of the above criteria. Functionally, M2 encoded physically interacting proteins regulating cell cycle processes and analysis of genome-wide gene-regulatory interactions using mutual information and DNA-protein interactions revealed the known regulators of cell cycle processes FoxM1, B-Myb, and E2F2 as key regulators of M2. These results suggest functional disruption of M2 via gene mutation or altered expression as a convergent pathway regulating astrocytoma transformation. By considering M2 as a multitarget drug target regulating astrocytoma transformation, we identified several drugs that are predicted to restore M2 expression in anaplastic astrocytoma toward its low-grade profile and of these, we validated the known antiproliferative drug resveratrol as down-regulating multiple nodes of M2 including at nanomolar concentrations achievable in human cerebrospinal fluid by oral dosing.

Interpretation: Our results identify M2 as a multitarget network characteristic for astrocytoma progression and encourage M2-based drug screening to identify new compounds for preventing glioma transformation.
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http://dx.doi.org/10.1002/acn3.50850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764637PMC
September 2019

WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

Nat Commun 2019 08 9;10(1):3616. Epub 2019 Aug 9.

Programme in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, 169857, Republic of Singapore.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.
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http://dx.doi.org/10.1038/s41467-019-11551-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689010PMC
August 2019

miR-15a/-16 Inhibit Angiogenesis by Targeting the Tie2 Coding Sequence: Therapeutic Potential of a miR-15a/16 Decoy System in Limb Ischemia.

Mol Ther Nucleic Acids 2019 Sep 17;17:49-62. Epub 2019 May 17.

Bristol Heart Institute, University of Bristol, Bristol, UK; National Heart and Lung Institute, Imperial College London, London, UK. Electronic address:

MicroRNA-15a (miR-15a) and miR-16, which are transcribed from the miR-15a/miR-16-1 cluster, inhibit post-ischemic angiogenesis. MicroRNA (miRNA) binding to mRNA coding sequences (CDSs) is a newly emerging mechanism of gene expression regulation. We aimed to (1) identify new mediators of the anti-angiogenic action of miR-15a and -16, (2) develop an adenovirus (Ad)-based miR-15a/16 decoy system carrying a luciferase reporter (Luc) to both sense and inhibit miR-15a/16 activity, and (3) investigate Ad.Luc-Decoy-15a/16 therapeutic potential in a mouse limb ischemia (LI) model. LI increased miR-15a and -16 expression in mouse muscular endothelial cells (ECs). The miRNAs also increased in cultured human umbilical vein ECs (HUVECs) exposed to serum starvation, but not hypoxia. Using bioinformatic tools and luciferase activity assays, we characterized miR-15a and -16 binding to Tie2 CDS. In HUVECs, miR-15a or -16 overexpression reduced Tie2 at the protein, but not the mRNA, level. Conversely, miR-15a or -16 inhibition improved angiogenesis in a Tie2-dependent manner. Local Ad.Luc-Decoy-15a/16 delivery increased Tie2 levels in ischemic skeletal muscle and improved post-LI angiogenesis and perfusion recovery, with reduced toe necrosis. Bioluminescent imaging (in vivo imaging system [IVIS]) provided evidence that the Ad.Luc-Decoy-15a/16 system responds to miR-15a/16 increases. In conclusion, we have provided novel mechanistic evidence of the therapeutic potential of local miR-15a/16 inhibition in LI.
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http://dx.doi.org/10.1016/j.omtn.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586592PMC
September 2019

Intrinsic mutant HTT-mediated defects in oligodendroglia cause myelination deficits and behavioral abnormalities in Huntington disease.

Proc Natl Acad Sci U S A 2019 05 23;116(19):9622-9627. Epub 2019 Apr 23.

Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research (A*STAR), Immunos, 138648 Singapore;

White matter abnormalities are a nearly universal pathological feature of neurodegenerative disorders including Huntington disease (HD). A long-held assumption is that this white matter pathology is simply a secondary outcome of the progressive neuronal loss that manifests with advancing disease. Using a mouse model of HD, here we show that white matter and myelination abnormalities are an early disease feature appearing before the manifestation of any behavioral abnormalities or neuronal loss. We further show that selective inactivation of mutant huntingtin (mHTT) in the NG2+ oligodendrocyte progenitor cell population prevented myelin abnormalities and certain behavioral deficits in HD mice. Strikingly, the improvements in behavioral outcomes were seen despite the continued expression of mHTT in nonoligodendroglial cells including neurons, astrocytes, and microglia. Using RNA-seq and ChIP-seq analyses, we implicate a pathogenic mechanism that involves enhancement of polycomb repressive complex 2 (PRC2) activity by mHTT in the intrinsic oligodendroglial dysfunction and myelination deficits observed in HD. Our findings challenge the long-held dogma regarding the etiology of white matter pathology in HD and highlight the contribution of epigenetic mechanisms to the observed intrinsic oligodendroglial dysfunction. Our results further suggest that ameliorating white matter pathology and oligodendroglial dysfunction may be beneficial for HD.
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http://dx.doi.org/10.1073/pnas.1818042116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511031PMC
May 2019

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Cell Rep 2019 03;26(12):3231-3245.e9

Cardiovascular & Metabolic Disorders Program, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore; Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden. Electronic address:

Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical-quality stem cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein and showed that LN-221 promotes differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulates pluripotency and teratoma-associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. We show high reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from different hESC lines. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical-quality cells for use in regenerative cardiology.
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http://dx.doi.org/10.1016/j.celrep.2019.02.083DOI Listing
March 2019

Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance.

PLoS One 2019 14;14(3):e0213685. Epub 2019 Mar 14.

National Heart and Lung Institute, ICTEM, The Hammersmith Hospital, Imperial College London, London, United Kingdom.

This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213685PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417664PMC
December 2019

Unbiased Profiling of Isogenic Huntington Disease hPSC-Derived CNS and Peripheral Cells Reveals Strong Cell-Type Specificity of CAG Length Effects.

Cell Rep 2019 02;26(9):2494-2508.e7

Translational Laboratory in Genetic Medicine (TLGM), Agency for Science, Technology and Research (A(∗)STAR), Singapore 138648, Singapore; Department of Medicine, National University of Singapore (NUS), Singapore 117597, Singapore; Department of Physiology, National University of Singapore (NUS), Singapore 117597, Singapore. Electronic address:

In Huntington disease (HD), the analysis of tissue-specific CAG repeat length effects has been challenging, given the difficulty in obtaining relevant patient tissues with a broad range of CAG repeat lengths. We used genome editing to generate an allelic panel of isogenic HD (IsoHD) human embryonic stem cell (hESC) lines carrying varying CAG repeat lengths in the first exon of HTT. Functional analyses in differentiated neural cells revealed CAG repeat length-related abnormalities in mitochondrial respiration and oxidative stress and enhanced susceptibility to DNA damage. To explore tissue-specific effects in HD, we differentiated the IsoHD panel into neural progenitor cells, neurons, hepatocytes, and muscle cells. Transcriptomic and proteomic analyses of the resultant cell types identified CAG repeat length-dependent and cell-type-specific molecular phenotypes. We anticipate that the IsoHD panel and transcriptomic and proteomic data will serve as a versatile, open-access platform to dissect the molecular factors contributing to HD pathogenesis.
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http://dx.doi.org/10.1016/j.celrep.2019.02.008DOI Listing
February 2019

Sleeve gastrectomy rapidly enhances islet function independently of body weight.

JCI Insight 2019 03 21;4(6). Epub 2019 Mar 21.

Division of Endocrinology, Duke Molecular Physiology Institute, Duke University, Durham, North Carolina, USA.

Bariatric surgeries including vertical sleeve gastrectomy (VSG) ameliorate obesity and diabetes. Weight loss and accompanying increases to insulin sensitivity contribute to improved glycemia after surgery; however, studies in humans also suggest weight-independent actions of bariatric procedures to lower blood glucose, possibly by improving insulin secretion. To evaluate this hypothesis, we compared VSG-operated mice with pair-fed, sham-surgical controls (PF-Sham) 2 weeks after surgery. This paradigm yielded similar postoperative body weight and insulin sensitivity between VSG and calorically restricted PF-Sham animals. However, VSG improved glucose tolerance and markedly enhanced insulin secretion during oral nutrient and i.p. glucose challenges compared with controls. Islets from VSG mice displayed a unique transcriptional signature enriched for genes involved in Ca2+ signaling and insulin secretion pathways. This finding suggests that bariatric surgery leads to intrinsic changes within the islet that alter function. Indeed, islets isolated from VSG mice had increased glucose-stimulated insulin secretion and a left-shifted glucose sensitivity curve compared with islets from PF-Sham mice. Isolated islets from VSG animals showed corresponding increases in the pulse duration of glucose-stimulated Ca2+ oscillations. Together, these findings demonstrate a weight-independent improvement in glycemic control following VSG, which is, in part, driven by improved insulin secretion and associated with substantial changes in islet gene expression. These results support a model in which β cells play a key role in the adaptation to bariatric surgery and the improved glucose tolerance that is typical of these procedures.
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http://dx.doi.org/10.1172/jci.insight.126688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483064PMC
March 2019

Systems genetics identifies a macrophage cholesterol network associated with physiological wound healing.

JCI Insight 2019 Jan 24;4(2). Epub 2019 Jan 24.

Centre for Inflammatory Disease, Imperial College London, Hammersmith Hospital, London, United Kingdom (UK).

Among other cells, macrophages regulate the inflammatory and reparative phases during wound healing but genetic determinants and detailed molecular pathways that modulate these processes are not fully elucidated. Here, we took advantage of normal variation in wound healing in 1,378 genetically outbred mice, and carried out macrophage RNA-sequencing profiling of mice with extreme wound healing phenotypes (i.e., slow and fast healers, n = 146 in total). The resulting macrophage coexpression networks were genetically mapped and led to the identification of a unique module under strong trans-acting genetic control by the Runx2 locus. This macrophage-mediated healing network was specifically enriched for cholesterol and fatty acid biosynthetic processes. Pharmacological blockage of fatty acid synthesis with cerulenin resulted in delayed wound healing in vivo, and increased macrophage infiltration in the wounded skin, suggesting the persistence of an unresolved inflammation. We show how naturally occurring sequence variation controls transcriptional networks in macrophages, which in turn regulate specific metabolic pathways that could be targeted in wound healing.
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http://dx.doi.org/10.1172/jci.insight.125736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413785PMC
January 2019

Systems Genetics Approaches in Rat Identify Novel Genes and Gene Networks Associated With Cardiac Conduction.

J Am Heart Assoc 2018 11;7(21):e009243

1 Department of Experimental Cardiology Heart Centre Academic Medical Center Amsterdam Amsterdam The Netherlands.

Background Electrocardiographic ( ECG ) parameters are regarded as intermediate phenotypes of cardiac arrhythmias. Insight into the genetic underpinnings of these parameters is expected to contribute to the understanding of cardiac arrhythmia mechanisms. Here we used HXB / BXH recombinant inbred rat strains to uncover genetic loci and candidate genes modulating ECG parameters. Methods and Results RR interval, PR interval, QRS duration, and QT c interval were measured from ECG s obtained in 6 male rats from each of the 29 available HXB / BXH recombinant inbred strains. Genes at loci displaying significant quantitative trait loci (QTL) effects were prioritized by assessing the presence of protein-altering variants, and by assessment of cis expression QTL ( eQTL ) effects and correlation of transcript abundance to the respective trait in the heart. Cardiac RNA -seq data were additionally used to generate gene co-expression networks. QTL analysis of ECG parameters identified 2 QTL for PR interval, respectively, on chromosomes 10 and 17. At the chromosome 10 QTL , cis- eQTL effects were identified for Acbd4, Cd300lg, Fam171a2, and Arhgap27; the transcript abundance in the heart of these 4 genes was correlated with PR interval. At the chromosome 17 QTL , a cis- eQTL was uncovered for Nhlrc1 candidate gene; the transcript abundance of this gene was also correlated with PR interval. Co-expression analysis furthermore identified 50 gene networks, 6 of which were correlated with PR interval or QRS duration, both parameters of cardiac conduction. Conclusions These newly identified genetic loci and gene networks associated with the ECG parameters of cardiac conduction provide a starting point for future studies with the potential of identifying novel mechanisms underlying cardiac electrical function.
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http://dx.doi.org/10.1161/JAHA.118.009243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404199PMC
November 2018

Biologically relevant laminin as chemically defined and fully human platform for human epidermal keratinocyte culture.

Nat Commun 2018 10 30;9(1):4432. Epub 2018 Oct 30.

Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, 8 College Road, Singapore, 169857, Singapore.

The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system. Through our characterization efforts of the human skin basement membrane and murine feeder layer 3T3-J2, we identified two biologically relevant recombinant laminins-LN-511 and LN-421- as potential candidates to replace the murine feeder. Herein, we report a completely xeno-free and defined culture system utilizing these laminins which enables robust expansion of adult human skin keratinocytes. We demonstrate that our laminin system is comparable to the 3T3-J2 co-culture system in terms of basal markers' profile, colony-forming efficiency and the ability to form normal stratified epidermal structure in both in vitro and in vivo models. These results show that the proposed system may not only provide safer keratinocyte use in the clinics, but also facilitate the broader use of other cultured human epithelial cells in regenerative medicine.
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http://dx.doi.org/10.1038/s41467-018-06934-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207750PMC
October 2018

Temporal dynamics of Wnt-dependent transcriptome reveal an oncogenic Wnt/MYC/ribosome axis.

J Clin Invest 2018 12 12;128(12):5620-5633. Epub 2018 Nov 12.

Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.

Activating mutations in the Wnt pathway drive a variety of cancers, but the specific targets and pathways activated by Wnt ligands are not fully understood. To bridge this knowledge gap, we performed a comprehensive time-course analysis of Wnt-dependent signaling pathways in an orthotopic model of Wnt-addicted pancreatic cancer, using a porcupine (PORCN) inhibitor currently in clinical trials, and validated key results in additional Wnt-addicted models. The temporal analysis of the drug-perturbed transcriptome demonstrated direct and indirect regulation of more than 3,500 Wnt-activated genes (23% of the transcriptome). Regulation was both via Wnt/β-catenin and through the modulation of protein abundance of important transcription factors, including MYC, via Wnt-dependent stabilization of proteins (Wnt/STOP). Our study identifies a central role of Wnt/β-catenin and Wnt/STOP signaling in controlling ribosome biogenesis, a key driver of cancer proliferation.
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http://dx.doi.org/10.1172/JCI122383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264740PMC
December 2018

A systems-level framework for drug discovery identifies Csf1R as an anti-epileptic drug target.

Nat Commun 2018 09 3;9(1):3561. Epub 2018 Sep 3.

Division of Brain Sciences, Imperial College London, London, W12 0NN, UK.

The identification of drug targets is highly challenging, particularly for diseases of the brain. To address this problem, we developed and experimentally validated a general computational framework for drug target discovery that combines gene regulatory information with causal reasoning ("Causal Reasoning Analytical Framework for Target discovery"-CRAFT). Using a systems genetics approach and starting from gene expression data from the target tissue, CRAFT provides a predictive framework for identifying cell membrane receptors with a direction-specified influence over disease-related gene expression profiles. As proof of concept, we applied CRAFT to epilepsy and predicted the tyrosine kinase receptor Csf1R as a potential therapeutic target. The predicted effect of Csf1R blockade in attenuating epilepsy seizures was validated in three pre-clinical models of epilepsy. These results highlight CRAFT as a systems-level framework for target discovery and suggest Csf1R blockade as a novel therapeutic strategy in epilepsy. CRAFT is applicable to disease settings other than epilepsy.
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http://dx.doi.org/10.1038/s41467-018-06008-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120885PMC
September 2018