35 results found
Jobbins AM, Yu S, Paterson HAB, et al., 2023, Pre-RNA splicing in metabolic homeostasis and liver disease., Trends Endocrinol Metab
The liver plays a key role in sensing nutritional and hormonal inputs to maintain metabolic homeostasis. Recent studies into pre-mRNA splicing and alternative splicing (AS) and their effects on gene expression have revealed considerable transcriptional complexity in the liver, both in health and disease. While the contribution of these mechanisms to cell and tissue identity is widely accepted, their role in physiological and pathological contexts within tissues is just beginning to be appreciated. In this review, we showcase recent studies on the splicing and AS of key genes in metabolic pathways in the liver, the effect of metabolic signals on the spliceosome, and therapeutic intervention points based on RNA splicing.
Nagy D, Maude H, Birdsey GM, et al., 2023, Liver sinusoidal endothelial transcription factors in metabolic homeostasis and disease, Journal of Molecular Endocrinology, Vol: 71, Pages: 1-20, ISSN: 0952-5041
Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells that form the liver microvasculature. LSECs maintain liver homeostasis, scavenging bloodborne molecules, regulating immune response, and actively promoting hepatic stellate cell quiescence. These diverse functions are underpinned by a suite of unique phenotypical attributes distinct from other blood vessels. In recent years, studies have begun to reveal the specific contributions of LSECs to liver metabolic homeostasis and how LSEC dysfunction associates with disease aetiology. This has been particularly evident in the context of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, which is associated with loss of key LSEC phenotypical characteristics and molecular identity. Comparative transcriptome studies of LSECs and other endothelial cells, together with rodent knockout models, have revealed that loss of LSEC identity through disruption of core transcription factor activity leads to impaired metabolic homeostasis and to hallmarks of liver disease. This review explores the current knowledge of LSEC transcription factors, covering their roles in LSEC development and maintenance of key phenotypic features, which, when disturbed, lead to loss of liver metabolic homeostasis and promote features of chronic liver diseases, such as non-alcoholic liver disease.
Maude H, Cebola I, 2023, Zooming into process-specific risk., Nature Metabolism, Vol: 5, Pages: 730-731, ISSN: 2522-5812
Ramos-Pittol J, Fernandes-Freitas I, Milona A, et al., 2023, Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice, Nature Communications, Vol: 14, Pages: 1-10, ISSN: 2041-1723
Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ER may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.
Walters R, Vasilaki E, Aman J, et al., 2023, SOX17 enhancer variants disrupt transcription factor binding and enhancer inactivity drives pulmonary hypertension, Circulation, Vol: 147, Pages: 1606-1621, ISSN: 0009-7322
BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. METHODS: CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. RESULTS: CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of t
McAllan L, Baranasic D, Villicaña S, et al., 2023, Integrative genomic analyses in adipocytes implicate DNA methylation in human obesity and diabetes, Nature Communications, Vol: 14, Pages: 1-20, ISSN: 2041-1723
DNA methylation variations are prevalent in human obesity but evidence of a causative role in disease pathogenesis is limited. Here, we combine epigenome-wide association and integrative genomics to investigate the impact of adipocyte DNA methylation variations in human obesity. We discover extensive DNA methylation changes that are robustly associated with obesity (N = 190 samples, 691 loci in subcutaneous and 173 loci in visceral adipocytes, P < 1 × 10-7). We connect obesity-associated methylation variations to transcriptomic changes at >500 target genes, and identify putative methylation-transcription factor interactions. Through Mendelian Randomisation, we infer causal effects of methylation on obesity and obesity-induced metabolic disturbances at 59 independent loci. Targeted methylation sequencing, CRISPR-activation and gene silencing in adipocytes, further identifies regional methylation variations, underlying regulatory elements and novel cellular metabolic effects. Our results indicate DNA methylation is an important determinant of human obesity and its metabolic complications, and reveal mechanisms through which altered methylation may impact adipocyte functions.
De Vas M, Boulet F, Joshi SS, et al., 2023, Regulatory de novo mutations underlying intellectual disability, Life Science Alliance, Vol: 6, Pages: 1-16, ISSN: 2575-1077
The genetic aetiology of a major fraction of patients with intellectual disability (ID) remains unknown. De novo mutations (DNMs) in protein-coding genes explain up to 40% of cases, but the potential role of regulatory DNMs is still poorly understood. We sequenced 63 whole genomes from 21 ID probands and their unaffected parents. In addition, we analysed 30 previously sequenced genomes from exome-negative ID probands. We found that regulatory DNMs were selectively enriched in fetal brain-specific enhancers as compared with adult brain enhancers. DNM-containing enhancers were associated with genes that show preferential expression in the prefrontal cortex. Furthermore, we identified recurrently mutated enhancer clusters that regulate genes involved in nervous system development (CSMD1, OLFM1, and POU3F3). Most of the DNMs from ID probands showed allele-specific enhancer activity when tested using luciferase assay. Using CRISPR-mediated mutation and editing of epigenomic marks, we show that DNMs at regulatory elements affect the expression of putative target genes. Our results, therefore, provide new evidence to indicate that DNMs in fetal brain-specific enhancers play an essential role in the aetiology of ID.
Costanzo MC, von Grotthuss M, Massung J, et al., 2023, The Type 2 Diabetes Knowledge Portal: an open access genetic resource dedicated to type 2 diabetes and related traits, Cell Metabolism, Vol: 35, Pages: 695-710.e6, ISSN: 1550-4131
Associations between human genetic variation and clinical phenotypes have become a foundation of biomedical research. Most repositories of these data seek to be disease-agnostic and therefore lack disease-focused views. The Type 2 Diabetes Knowledge Portal (T2DKP) is a public resource of genetic datasets and genomic annotations dedicated to type 2 diabetes (T2D) and related traits. Here, we seek to make the T2DKP more accessible to prospective users and more useful to existing users. First, we evaluate the T2DKP's comprehensiveness by comparing its datasets with those of other repositories. Second, we describe how researchers unfamiliar with human genetic data can begin using and correctly interpreting them via the T2DKP. Third, we describe how existing users can extend their current workflows to use the full suite of tools offered by the T2DKP. We finally discuss the lessons offered by the T2DKP toward the goal of democratizing access to complex disease genetic results.
Paterson HAB, Yu S, Artigas N, et al., 2022, Liver RBFOX2 regulates cholesterol homeostasis via Scarb1 alternative splicing in mice, Nature Metabolism, Vol: 4, Pages: 1812-1829, ISSN: 2522-5812
RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.
De Vas MG, Boulet F, Joshi SS, et al., 2022, Regulatory<i>de novo</i>mutations underlying intellectual disability
<jats:title>Abstract</jats:title><jats:p>The genetic aetiology of a major fraction of patients with intellectual disability (ID) remains unknown.<jats:italic>De novo</jats:italic>mutations (DNMs) in protein-coding genes explain up to 40% of cases, but the potential role of regulatory DNMs is still poorly understood. We sequenced 63 whole genomes from 21 ID probands and their unaffected parents (trio). Additionally, we analysed 30 previously sequenced genomes from exome-negative ID probands. We found that regulatory DNMs were selectively enriched in fetal brain-specific and human-gained enhancers. DNM-containing enhancers were associated with genes that show preferential expression in the pre-frontal cortex, have been previously implicated in ID or related disorders, and exhibit intolerance to loss of function mutations. Moreover, we found that highly interacting regulatory regions from intermediate progenitor cells of the developing human cortex were strongly enriched for ID DNMs. Furthermore, we identified recurrently mutated enhancer clusters that regulate genes involved in nervous system development (<jats:italic>CSMD1, OLFM1</jats:italic>, and<jats:italic>POU3F3)</jats:italic>. The majority of the DNMs from ID probands showed allele-specific enhancer activity when tested using luciferase assay. Using CRISPR-mediated mutation and editing of epigenomic marks, we show that regulatory elements harbouring DNMs indeed function as enhancers and DNMs at regulatory elements affect the expression of putative target genes. Our results, therefore, provide new evidence to indicate that DNMs in fetal brain-specific enhancers play an essential role in the aetiology of ID.</jats:p>
Dixon PH, Levine AP, Cebola I, et al., 2022, GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements, Nature Communications, Vol: 13, ISSN: 2041-1723
Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1,138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility.
Jobbins AM, Haberman N, Artigas N, et al., 2022, Dysregulated RNA polyadenylation contributes to metabolic impairment in non-alcoholic fatty liver disease, Nucleic Acids Research, Vol: 50, Pages: 3379-3393, ISSN: 0305-1048
Pre-mRNA processing is an essential mechanism for the generation of mature mRNA and the regulation of gene expression in eukaryotic cells. While defects in pre-mRNA processing have been implicated in a number of diseases their involvement in metabolic pathologies is still unclear. Here, we show that both alternative splicing and alternative polyadenylation, two major steps in pre-mRNA processing, are significantly altered in non-alcoholic fatty liver disease (NAFLD). Moreover, we find that Serine and Arginine Rich Splicing Factor 10 (SRSF10) binding is enriched adjacent to consensus polyadenylation motifs and its expression is significantly decreased in NAFLD, suggesting a role mediating pre-mRNA dysregulation in this condition. Consistently, inactivation of SRSF10 in mouse and human hepatocytes in vitro, and in mouse liver in vivo, was found to dysregulate polyadenylation of key metabolic genes such as peroxisome proliferator-activated receptor alpha (PPARA) and exacerbate diet-induced metabolic dysfunction. Collectively our work implicates dysregulated pre-mRNA polyadenylation in obesity-induced liver disease and uncovers a novel role for SRSF10 in this process.
Cebola I, 2021, Deletion of regulatory elements with all-in-one CRISPR-Cas9 vectors, Enhancers and Promoters - Methods and Protocols, Editors: Benetto, Tilman, Publisher: Springer Nature
Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning by Golden Gate, by adapting the primer design.
Maude H, Sanchez Cabanillas C, Cebola I, 2021, Epigenetics of hepatic insulin resistance, Frontiers in Endocrinology, Vol: 12, Pages: 1-24, ISSN: 1664-2392
Insulin resistance (IR) is largely recognized as a unifying feature that underlies metabolic dysfunction. Both lifestyle and genetic factors contribute to IR. Work from recent years has demonstrated that the epigenome may constitute an interface where different signals may converge to promote IR gene expression programs. Here, we review the current knowledge of the role of epigenetics in hepatic IR, focusing on the roles of DNA methylation and histone post-translational modifications. We discuss the broad epigenetic changes observed in the insulin resistant liver and its associated pathophysiological states and leverage on the wealth of ‘omics’ studies performed to discuss efforts in pinpointing specific loci that are disrupted by these changes. We envision that future studies, with increased genomic resolution and larger cohorts, will further the identification of biomarkers of early onset hepatic IR and assist the development of targeted interventions. Furthermore, there is growing evidence to suggest that persistent epigenetic marks may be acquired over prolonged exposure to disease or deleterious exposures, highlighting the need for preventative medicine and long-term lifestyle adjustments to avoid irreversible or long-term alterations in gene expression.
Hu M, Cebola I, Carrat G, et al., 2021, Chromatin 3D interaction analysis of the STARD10 locus unveils FCHSD2 as a regulator of insulin secretion., Cell Research, Vol: 34, Pages: 1-1, ISSN: 1001-0602
Using chromatin conformation capture, we show that an enhancer cluster in the STARD10 type 2 diabetes (T2D) locus forms a defined 3-dimensional (3D) chromatin domain. A 4.1-kb region within this locus, carrying 5 T2D-associated variants, physically interacts with CTCF-binding regions and with an enhancer possessing strong transcriptional activity. Analysis of human islet 3D chromatin interaction maps identifies the FCHSD2 gene as an additional target of the enhancer cluster. CRISPR-Cas9-mediated deletion of the variant region, or of the associated enhancer, from human pancreas-derived EndoC-βH1 cells impairs glucose-stimulated insulin secretion. Expression of both STARD10 and FCHSD2 is reduced in cells harboring CRISPR deletions, and lower expression of STARD10 and FCHSD2 is associated, the latter nominally, with the possession of risk variant alleles in human islets. Finally, CRISPR-Cas9-mediated loss of STARD10 or FCHSD2, but not ARAP1, impairs regulated insulin secretion. Thus, multiple genes at the STARD10 locus influence β cell function.
Hu M, Cebola I, Carrat G, et al., 2020, Chromatin 3D interaction analysis of the STARD10 locus unveils FCHSD2 as a new regulator of insulin secretion, Publisher: SPRINGER
Cebola I, 2020, Liver gene regulatory networks: Contributing factors to nonalcoholic fatty liver disease, Wiley Interdisciplinary Reviews: Systems Biology and Medicine, Vol: 12, Pages: 1-29, ISSN: 1939-005X
Metabolic diseases such as nonalcoholic fatty liver disease (NAFLD) result from complex interactions between intrinsic and extrinsic factors, including genetics and exposure to obesogenic environments. These risk factors converge in aberrant gene expression patterns in the liver, which are underlined by altered cis-regulatory networks. In homeostasis and in disease states, liver cis-regulatory networks are established by coordinated action of liver-enriched transcription factors (TFs), which define enhancer landscapes, activating broad gene programs with spatiotemporal resolution. Recent advances in DNA sequencing have dramatically expanded our ability to map active transcripts, enhancers and TF cistromes, and to define the 3D chromatin topology that contains these elements. Deployment of these technologies has allowed investigation of the molecular processes that regulate liver development and metabolic homeostasis. Moreover, genomic studies of NAFLD patients and NAFLD models have demonstrated that the liver undergoes pervasive regulatory rewiring in NAFLD, which is reflected by aberrant gene expression profiles. We have therefore achieved an unprecedented level of detail in the understanding of liver cis-regulatory networks, particularly in physiological conditions. Future studies should aim to map active regulatory elements with added levels of resolution, addressing how the chromatin landscapes of different cell lineages contribute to and are altered in NAFLD and NAFLD-associated metabolic states. Such efforts would provide additional clues into the molecular factors that trigger this disease. This article is categorized under: Biological Mechanisms > Metabolism Biological Mechanisms > Regulatory Biology Laboratory Methods and Technologies > Genetic/Genomic Methods.
Cebola I, 2019, Pancreatic Islet Transcriptional Enhancers and Diabetes, Current Diabetes Reports, Vol: 19, ISSN: 1534-4827
<jats:title>Abstract</jats:title><jats:sec><jats:title>Purpose of Review</jats:title><jats:p>Common genetic variants that associate with type 2 diabetes risk are markedly enriched in pancreatic islet transcriptional enhancers. This review discusses current advances in the annotation of islet enhancer variants and their target genes.</jats:p></jats:sec><jats:sec><jats:title>Recent Findings</jats:title><jats:p>Recent methodological advances now allow genetic and functional mapping of diabetes causal variants at unprecedented resolution. Mapping of enhancer-promoter interactions in human islets has provided a unique appreciation of the complexity of islet gene regulatory processes and enabled direct association of noncoding diabetes risk variants to their target genes.</jats:p></jats:sec><jats:sec><jats:title>Summary</jats:title><jats:p>The recently improved human islet enhancer annotations constitute a framework for the interpretation of diabetes genetic signals in the context of pancreatic islet gene regulation. In the future, integration of existing and yet to come regulatory maps with genetic fine-mapping efforts and in-depth functional characterization will foster the discovery of novel diabetes molecular risk mechanisms.</jats:p></jats:sec>
Cebola I, Miguel-Escalada I, Bonas-Guarch S, et al., 2019, Unravelling of new type 2 diabetes genes with 3D chromatin topology analysis and CRISPR-Cas9 perturbations, BES 2019, ISSN: 1470-3947
Genome-wide association studies have identified nearly 250 loci carrying genetic variants associated with type 2 diabetes (T2D) susceptibility, which are often located within pancreatic islet transcriptional enhancers. Due to the complex nature of transcriptional enhancers, assigning risk variants to true disease susceptibility effector genes has remained a challenge. In this study, we applied promoter capture Hi-C to create a genome-wide map of promoter-enhancer interactions in adult human pancreatic islets. We then set out to investigate which genes are regulated by enhancers carrying T2D risk variants, observing that T2D variants often interact with more than one gene, and that, unlike what has been assumed until now, the nearest genes are not always the true targets of T2D susceptibility variants. We validated our in silico predictions by applying CRISPR-Cas9-based methods to perturb T2D enhancers in the human pancreatic ß cell line EndoC-ßH3, demonstrating that the detected enhancer-promoter interactions reflect functional chromatin interactions in human islets. This study reveals 3D chromatin architecture analysis coupled with genome editing as a powerful framework for interpretation of T2D genetic association signals. Furthermore, the results shed light into unexpected regulatory links that may affected by T2D susceptibility variants, bringing to our attention new players in T2D aetiology.
Cheung R, Pizza G, Nguyen-Tu M-S, et al., 2019, Glucose-controlled miR-125b regulates beta cell function, 55th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), Publisher: SPRINGER, Pages: S219-S219, ISSN: 0012-186X
Miguel-Escalada I, Bonàs-Guarch S, Cebola I, et al., 2019, Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes., Nature genetics, Vol: 51, Pages: 1137-1148, ISSN: 1061-4036
Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer clusters or super-enhancers. So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in three-dimensional (3D) space. Furthermore, their target genes are often unknown. We have created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers to their target genes, often located hundreds of kilobases away. It also revealed >1,300 groups of islet enhancers, super-enhancers and active promoters that form 3D hubs, some of which show coordinated glucose-dependent activity. We demonstrate that genetic variation in hubs impacts insulin secretion heritability, and show that hub annotations can be used for polygenic scores that predict T2D risk driven by islet regulatory variants. Human islet 3D chromatin architecture, therefore, provides a framework for interpretation of T2D genome-wide association study (GWAS) signals.
Beucher A, Cebola I, 2019, One-step dual CRISPR/Cas9 guide RNA cloning protocol, Publisher: Protocol Exchange
Existing protocols for dual guide RNA cloning rely on synthesised DNA oligonucleotides of >100 bp that contain both guide RNA sequences, and are therefore not reusable in alternative experimental designs. Here, we describe a single-step protocol to rapidly and inexpensively generate vectors expressing two guide RNAs (gRNAs) simultaneously, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning gRNAs into virtually any CRISPR/Cas9 backbone that allows cloning by Golden Gate, by adapting the primer design. Here, we provide details for cloning gRNAs into vectors with BbsI and BsmBI sites, two of the most frequently found enzymes in CRISPR/Cas9 gRNA expression cassettes.This protocol has been successfully applied to delete pancreatic islet enhancers that harbour type 2 diabetes variants and to validate enhancer-promoter interactions (Miguel-Escalada et al., Nature Genetics 2019).In the future, we foresee that this simple protocol may also be applied to target coding sequences, as well as to target other important kinds of noncoding regulatory elements, including lncRNAs, miRNAs, and chromatin structural anchor points.
De Vas MG, Garstang MG, Joshi SS, et al., 2019, <i>De novo</i> variants in population constrained fetal brain enhancers and intellectual disability
<jats:title>Abstract</jats:title><jats:sec><jats:title>Purpose</jats:title><jats:p>The genetic aetiology of a major fraction of patients with intellectual disability (ID) remains unknown. <jats:italic>De novo</jats:italic> mutations (DNMs) in protein-coding genes explain up to 40% of cases, but the potential role of regulatory DNMs is still poorly understood.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We sequenced 70 whole genomes from 24 ID probands and their unaffected parents and analyzed 30 previously sequenced genomes from exome-negative ID probands.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>We found that DNVs were selectively enriched in fetal brain-specific enhancers that show purifying selection in human population. DNV containing enhancers were associated with genes that show preferential expression in the pre-frontal cortex, have been previously implicated in ID or related disorders, and exhibit intolerance to loss of function variants. DNVs from ID probands preferentially disrupted putative binding sites of neuronal transcription factors, as compared to DNVs from healthy individuals and most showed allele-specific enhancer activity. In addition, we identified recurrently mutated enhancer clusters that regulate genes involved in nervous system development (<jats:italic>CSMD1</jats:italic>, <jats:italic>OLFM1</jats:italic> and <jats:italic>POU3F3)</jats:italic>. Moreover, CRISPR-based perturbation of a DNV-containing enhancer caused <jats:italic>CSMD1</jats:italic> overexpression and abnormal expression of neurodevelopmental regulators.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>Our results, therefore, provide new evidence to indicate that DNVs in constrained fetal brain-speci
Rhodes CJ, Batai K, Bleda M, et al., 2019, Genetic determinants of risk in pulmonary arterial hypertension: international genome-wide association studies and meta-analysis, The Lancet Respiratory Medicine, Vol: 7, Pages: 227-238, ISSN: 2213-2600
Martinez-Sanchez A, Nguyen-Tu M-S, Cebola I, et al., 2018, MiR-184 expression is regulated by AMPK in pancreatic islets., FASEB Journal, Vol: 32, Pages: 2587-2600, ISSN: 0892-6638
AMPK is a critical energy sensor and target for widely used antidiabetic drugs. In β-cells, elevated glucose concentrations lower AMPK activity, and the ablation of both catalytic subunits (βAMPKdKO mice) impairs insulin secretion in vivo and β-cell identity. MicroRNAs (miRNAs) are small RNAs that silence gene expression that are essential for pancreatic β-cell function and identity and altered in diabetes. Here, we have explored the miRNAs acting downstream of AMPK in mouse and human β-cells. We identified 14 down-regulated and 9 up-regulated miRNAs in βAMPKdKO vs. control islets. Gene ontology analysis of targeted transcripts revealed enrichment in pathways important for β-cell function and identity. The most down-regulated miRNA was miR-184 (miR-184-3p), an important regulator of β-cell function and compensatory expansion that is controlled by glucose and reduced in diabetes. We demonstrate that AMPK is a potent regulator and an important mediator of the negative effects of glucose on miR-184 expression. Additionally, we reveal sexual dimorphism in miR-184 expression in mouse and human islets. Collectively, these data demonstrate that glucose-mediated changes in AMPK activity are central for the regulation of miR-184 and other miRNAs in islets and provide a link between energy status and gene expression in β-cells.-Martinez-Sanchez, A., Nguyen-Tu, M.-S., Cebola, I., Yavari, A., Marchetti, P., Piemonti, L., de Koning, E., Shapiro, A. M. J., Johnson, P., Sakamoto, K., Smith, D. M., Leclerc, I., Ashrafian, H., Ferrer, J., Rutter, G. A. MiR-184 expression is regulated by AMPK in pancreatic islets.
Martinez-Sanchez A, Nguyen-Tu MS, Cebola I, et al., 2018, Adenosine Monophosphate (AMP)-activated protein kinase (AMPK) regulates the expression of miR-184 and other miRNAs important for beta cell function, Publisher: WILEY, Pages: 48-48, ISSN: 0742-3071
Rhodes CJ, Batai K, Bleda M, et al., 2018, Genetic Determinants of Risk and Survival in Pulmonary Arterial Hypertension, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Miguel-Escalada I, Bonàs-Guarch S, Cebola I, et al., 2018, Human pancreatic islet 3D chromatin architecture provides insights into the genetics of type 2 diabetes
Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer regions (enhancer clusters, stretch enhancers or super-enhancers). So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in 3D-space. Furthermore, their target genes are generally unknown. We have now created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers with their target genes, often located hundreds of kilobases away. It further revealed sets of islet enhancers, super-enhancers and active promoters that form 3D higher-order hubs, some of which show coordinated glucose-dependent activity. Hub genetic variants impact the heritability of insulin secretion, and help identify individuals in whom genetic variation of islet function is important for T2D. Human islet 3D chromatin architecture thus provides a framework for interpretation of T2D GWAS signals.
Cebola I, Pasquali L, 2016, Non-coding genome functions in diabetes, Journal of Molecular Endocrinology, Vol: 56, Pages: R1-R20, ISSN: 1479-6813
Most of the genetic variation associated with diabetes, through genome-wide association studies, does not reside in protein-coding regions, making the identification of functional variants and their eventual translation to the clinic challenging. In recent years, high-throughput sequencing-based methods have enabled genome-scale high-resolution epigenomic profiling in a variety of human tissues, allowing the exploration of the human genome outside of the well-studied coding regions. These experiments unmasked tens of thousands of regulatory elements across several cell types, including diabetes-relevant tissues, providing new insights into their mechanisms of gene regulation. Regulatory landscapes are highly dynamic and cell-type specific and, being sensitive to DNA sequence variation, can vary with individual genomes. The scientific community is now in place to exploit the regulatory maps of tissues central to diabetes etiology, such as pancreatic progenitors and adult islets. This giant leap forward in the understanding of pancreatic gene regulation is revolutionizing our capacity to discriminate between functional and non-functional non-coding variants, opening opportunities to uncover regulatory links between sequence variation and diabetes susceptibility. In this review, we focus on the non-coding regulatory landscape of the pancreatic endocrine cells and provide an overview of the recent developments in this field.
Cebola I, Custodio J, Munoz M, et al., 2015, Epigenetics override pro-inflammatory PTGS transcriptomic signature towards selective hyperactivation of PGE(2) in colorectal cancer, Clinical Epigenetics, Vol: 7, ISSN: 1868-7083
BackgroundMisregulation of the PTGS (prostaglandin endoperoxide synthase, also known as cyclooxygenase or COX) pathway may lead to the accumulation of pro-inflammatory signals, which constitutes a hallmark of cancer. To get insight into the role of this signaling pathway in colorectal cancer (CRC), we have characterized the transcriptional and epigenetic landscapes of the PTGS pathway genes in normal and cancer cells.ResultsData from four independent series of CRC patients (502 tumors including adenomas and carcinomas and 222 adjacent normal tissues) and two series of colon mucosae from 69 healthy donors have been included in the study. Gene expression was analyzed by real-time PCR and Affymetrix U219 arrays. DNA methylation was analyzed by bisulfite sequencing, dissociation curves, and HumanMethylation450K arrays. Most CRC patients show selective transcriptional deregulation of the enzymes involved in the synthesis of prostanoids and their receptors in both tumor and its adjacent mucosa. DNA methylation alterations exclusively affect the tumor tissue (both adenomas and carcinomas), redirecting the transcriptional deregulation to activation of prostaglandin E2 (PGE2) function and blockade of other biologically active prostaglandins. In particular, PTGIS, PTGER3, PTGFR, and AKR1B1 were hypermethylated in more than 40 % of all analyzed tumors.ConclusionsThe transcriptional and epigenetic profiling of the PTGS pathway provides important clues on the biology of the tumor and its microenvironment. This analysis renders candidate markers with potential clinical applicability in risk assessment and early diagnosis and for the design of new therapeutic strategies.
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