Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA

Objective: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa “SAM” system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations. Methods: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes. Results: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations. Conclusions: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions

Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes

BACKGROUND: Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. RESULTS: The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α. CONCLUSIONS: UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA

Regulation of glycolysis in brown adipocytes by HIF-1α

Brown adipose tissue takes up large amounts of glucose during cold exposure in mice and humans. Here we report an induction of glucose transporter 1 expression and increased expression of several glycolytic enzymes in brown adipose tissue from cold-exposed mice. Accordingly, these genes were also induced after β-adrenergic activation of cultured brown adipocytes, concomitant with accumulation of hypoxia inducible factor-1α (HIF-1α) protein levels. HIF-1α accumulation was dependent on uncoupling protein 1 and generation of mitochondrial reactive oxygen species. Expression of key glycolytic enzymes was reduced after knockdown of HIF-1α in mature brown adipocytes. Glucose consumption, lactate export and glycolytic capacity were reduced in brown adipocytes depleted of Hif-1α. Finally, we observed a decreased β-adrenergically induced oxygen consumption in Hif-1α knockdown adipocytes cultured in medium with glucose as the only exogenously added fuel. These data suggest that HIF-1α-dependent regulation of glycolysis is necessary for maximum glucose metabolism in brown adipocytes.ISSN:2045-232

White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

Objective: Increasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. Methods: In this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. Results: Our proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. Conclusion: Our study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage

Loss-of-function variants in CUL3 cause a syndromic neurodevelopmental disorder

Purpose De novovariants inCUL3(Cullin-3 ubiquitin ligase) have been strongly associated with neurodevelopmental disorders (NDDs), but no large case series have been reported so far. Here we aimed to collect sporadic cases carrying rare variants inCUL3,describe the genotype-phenotype correlation, and investigate the underlying pathogenic mechanism.MethodsGenetic data and detailed clinical records were collected via multi-center collaboration. Dysmorphic facial features were analyzed using GestaltMatcher. Variant effects on CUL3 protein stability were assessed using patient-derived T-cells.ResultsWe assembled a cohort of 35 individuals with heterozygousCUL3variants presenting a syndromic NDD characterized by intellectual disability with or without autistic features. Of these, 33 have loss-of-function (LoF) and two have missense variants.CUL3LoF variants in patients may affect protein stability leading to perturbations in protein homeostasis, as evidenced by decreased ubiquitin-protein conjugatesin vitro. Specifically, we show that cyclin E1 (CCNE1) and 4E-BP1 (EIF4EBP1), two prominent substrates of CUL3, fail to be targeted for proteasomal degradation in patient-derived cells.ConclusionOur study further refines the clinical and mutational spectrum ofCUL3-associated NDDs, expands the spectrum of cullin RING E3 ligase-associated neuropsychiatric disorders, and suggests haploinsufficiency via LoF variants is the predominant pathogenic mechanism

CUX1-related neurodevelopmental disorder: deep insights into phenotype-genotype spectrum and underlying pathology

Heterozygous, pathogenic CUX1 variants are associated with global developmental delay or intellectual disability. This study delineates the clinical presentation in an extended cohort and investigates the molecular mechanism underlying the disorder in a Cux1+/− mouse model. Through international collaboration, we assembled the phenotypic and molecular information for 34 individuals (23 unpublished individuals). We analyze brain CUX1 expression and susceptibility to epilepsy in Cux1+/− mice. We describe 34 individuals, from which 30 were unrelated, with 26 different null and four missense variants. The leading symptoms were mild to moderate delayed speech and motor development and borderline to moderate intellectual disability. Additional symptoms were muscular hypotonia, seizures, joint laxity, and abnormalities of the forehead. In Cux1+/− mice, we found delayed growth, histologically normal brains, and increased susceptibility to seizures. In Cux1+/− brains, the expression of Cux1 transcripts was half of WT animals. Expression of CUX1 proteins was reduced, although in early postnatal animals significantly more than in adults. In summary, disease-causing CUX1 variants result in a non-syndromic phenotype of developmental delay and intellectual disability. In some individuals, this phenotype ameliorates with age, resulting in a clinical catch-up and normal IQ in adulthood. The post-transcriptional balance of CUX1 expression in the heterozygous brain at late developmental stages appears important for this favorable clinical course.CAG was supported by the Eunice Kennedy Shriver National Institute Of Child Health & Human Development of the National Institutes of Health under Award Number P50 HD103525. This work was funded by PID2020-112831GB-I00 AEI /10.13039/501100011033 (MN). SS was supported by a grant from the NIH/NINDS (K23NS119666). SWS is supported by the Hospital for Sick Children Foundation, Autism Speaks, and the University of Toronto McLaughlin Center. EM-G was supported by a grant from MICIU FPU18/06240. EVS. was supported by a grant from the NIH (EY025718). CRF was supported by the fund to support clinical research careers in the Region of Southern Denmark (Region Syddanmarks pulje for kliniske forskerkarriereforløb).Peer reviewe

Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder

PURPOSE: Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype, but with limited neuroradiological data and insufficient evidence for causality of the variants. METHODS: Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays, and a zebrafish model. RESULTS: We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs and increased blood lactate. In vitro studies showed that variants within the TARS2301-381 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model. CONCLUSION: We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity

Comprehensive study of 28 individuals with SIN3A-related disorder underscoring the associated mild cognitive and distinctive facial phenotype

Witteveen-Kolk syndrome (OMIM 613406) is a recently defined neurodevelopmental syndrome caused by heterozygous loss-of-function variants in SIN3A. We define the clinical and neurodevelopmental phenotypes related to SIN3A-haploinsufficiency in 28 unreported patients. Patients with SIN3A variants adversely affecting protein function have mild intellectual disability, growth and feeding difficulties. Involvement of a multidisciplinary team including a geneticist, paediatrician and neurologist should be considered in managing these patients. Patients described here were identified through a combination of clinical evaluation and gene matching strategies (GeneMatcher and Decipher). All patients consented to participate in this study. Mean age of this cohort was 8.2 years (17 males, 11 females). Out of 16 patients ≥ 8 years old assessed, eight (50%) had mild intellectual disability (ID), four had moderate ID (22%), and one had severe ID (6%). Four (25%) did not have any cognitive impairment. Other neurological symptoms such as seizures (4/28) and hypotonia (12/28) were common. Behaviour problems were reported in a minority. In patients ≥2 years, three were diagnosed with Autism Spectrum Disorder (ASD) and four with Attention Deficit Hyperactivity Disorder (ADHD). We report 27 novel variants and one previously reported variant. 24 were truncating variants; three were missense variants and one large in-frame gain including exons 10–12

Effects of eight neuropsychiatric copy number variants on human brain structure

Many copy number variants (CNVs) confer risk for the same range of neurodevelopmental symptoms and psychiatric conditions including autism and schizophrenia. Yet, to date neuroimaging studies have typically been carried out one mutation at a time, showing that CNVs have large effects on brain anatomy. Here, we aimed to characterize and quantify the distinct brain morphometry effects and latent dimensions across 8 neuropsychiatric CNVs. We analyzed T1-weighted MRI data from clinically and non-clinically ascertained CNV carriers (deletion/duplication) at the 1q21.1 (n = 39/28), 16p11.2 (n = 87/78), 22q11.2 (n = 75/30), and 15q11.2 (n = 72/76) loci as well as 1296 non-carriers (controls). Case-control contrasts of all examined genomic loci demonstrated effects on brain anatomy, with deletions and duplications showing mirror effects at the global and regional levels. Although CNVs mainly showed distinct brain patterns, principal component analysis (PCA) loaded subsets of CNVs on two latent brain dimensions, which explained 32 and 29% of the variance of the 8 Cohen’s d maps. The cingulate gyrus, insula, supplementary motor cortex, and cerebellum were identified by PCA and multi-view pattern learning as top regions contributing to latent dimension shared across subsets of CNVs. The large proportion of distinct CNV effects on brain morphology may explain the small neuroimaging effect sizes reported in polygenic psychiatric conditions. Nevertheless, latent gene brain morphology dimensions will help subgroup the rapidly expanding landscape of neuropsychiatric variants and dissect the heterogeneity of idiopathic conditions