Somatic coding mutations in human induced pluripotent stem cells

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use

CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA “bait” oligonucleotides restore poly-GR–associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.This work was supported by the U.S. National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Aging (NIA) grant R01NS104219 (E.K.); NIH/NINDS grant R21NS107761 (E.K.); AFM-Telethon French Muscular Dystrophy Association Trampoline Grant #23648 (J.A.O.); AFM-Telethon postdoctoral fellowship (J.A.O.); Ramon y Cajal fellowships RYC2019-026980-I (J.A.O.) and RYC2021-033294-I (I.R.S.); Gipuzkoa Foru Aldundia 2019-FELL-000017-01 (I.R.S.); Maria de Maeztu Units of Excellence CEX2021-001159-M (J.A.O.) and MDM-2017-0720 (I.R.S.); NINDS grants R01NS097850 and R01NS131409 (J.K.I.); Department of Defense grants PR211919 and W81XWH2110131 (J.K.I.); John Douglas French Alzheimer’s Foundation (J.K.I.); Center for Regenerative Nanomedicine at the Simpson Querrey Institute (S.I.S. and T.D.C.); Intramural Research Program, NIH, National Cancer Institute (NCI), Center for Cancer Research (M.B. and S.L.W.); Les Turner ALS Foundation (E.K.); and New York Stem Cell Foundation (J.K.I. and E.K.).With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2021-001159-M (J.A.O.)).Peer reviewe

Supplementary Materials for CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology

This PDF file includes: Figs. S1 to S10 Legends for tables S1 to S6Other Supplementary Material includes the following: Tables S1 to S6Peer reviewe

RIP140 represses the "brown-in-white" adipocyte program including a futile cycle of triacyclglycerol breakdown and synthesis

Receptor-interacting protein 140 (RIP140) is a corepressor of nuclear receptors that is highly expressed in adipose tissues. We investigated the role of RIP140 in conditionally immortal preadipocyte cell lines prepared from white or brown fat depots. In white adipocytes, a large set of brown fat-associated genes was up-regulated in the absence of RIP140. In contrast, a relatively minor role can be ascribed to RIP140 in the control of basal gene expression in differentiated brown adipocytes because significant changes were observed only in Ptgds and Fabp3. The minor role of RIP140 in brown adipocytes correlates with the similar histology and uncoupling protein 1 and CIDEA staining in knockout compared with wild-type brown adipose tissue (BAT). In contrast, RIP140 knockout sc white adipose tissue (WAT) shows increased numbers of multilocular adipocytes with elevated staining for uncoupling protein 1 and CIDEA. Furthermore in a white adipocyte cell line, the markers of BRITE adipocytes, Tbx1, CD137, Tmem26, Cited1, and Epsti1 were repressed in the presence of RIP140 as was Prdm16. Microarray analysis of wild-type and RIP140-knockout white fat revealed elevated expression of genes associated with cold-induced expression or high expression in BAT. A set of genes associated with a futile cycle of triacylglycerol breakdown and resynthesis and functional assays revealed that glycerol kinase and glycerol-3-phosphate dehydrogenase activity as well as [3H]glycerol incorporation were elevated in the absence of RIP140. Thus, RIP140 blocks the BRITE program in WAT, preventing the expression of brown fat genes and inhibiting a triacylglycerol futile cycle, with important implications for energy homeostasis

Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients

Background: Amyotrophic lateral sclerosis (ALS) is a devastating disease whose complex pathology has been associated with a strong genetic component in the context of both familial and sporadic disease. Herein, we adopted a next-generation sequencing approach to Greek patients suffering from sporadic ALS (together with their healthy counterparts) in order to explore further the genetic basis of sporadic ALS (sALS). Results: Whole-genome sequencing analysis of Greek sALS patients revealed a positive association between FTO and TBC1D1 gene variants and sALS. Further, linkage disequilibrium analyses were suggestive of a specific diseaseassociated haplotype for FTO gene variants. Genotyping for these variants was performed in Greek, Sardinian, and Turkish sALS patients. A lack of association between FTO and TBC1D1 variants and sALS in patients of Sardinian and Turkish descent may suggest a founder effect in the Greek population. FTO was found to be highly expressed in motor neurons, while in silico analyses predicted an impact on FTO and TBC1D1 mRNA splicing for the genomic variants in question. Conclusions: To our knowledge, this is the first study to present a possible association between FTO gene variants and the genetic etiology of sALS. In addition, the next-generation sequencing-based genomics approach coupled with the two-step validation strategy described herein has the potential to be applied to other types of human complex genetic disorders in order to identify variants of clinical significance

Regulation of Gene Expression by the NR Corepressor RIP140 in Adipocytes

Nuclear receptors (NR) are ligand-induced transcription factors that regulate the expression of genes involved in a number of physiological processes including metabolism. Their ability to regulate transcription is dependent on the recruitment of specific cofactors that remodel chromatin and promote the assembly of the basal transcriptional machinery. Receptor interacting protein 140 (RIP140) is a corepressor for NRs that utilizes four independent repression domains to inhibit gene expression of target genes. While RIP140-null mice develop around 70% less white adipose tissue than wild-type littermates, the process of adipogenesis is unaffected in RIP140-null and RIP140-expressing cells. To elucidate the intrinsic role of RIP140 in adipocytes i used full mouse genome arrays to compare the gene expression profiles of pre-adipocytes and adipocytes with and without RIP140. I demonstrate that the transcriptional corepressor negatively regulates major gene clusters in a number of metabolic pathways including glycolysis, TCA cycle, oxidative phosphorylation, fatty acid oxidation and glycerol metabolism. Additionally, a futile metabolic cycle of triglyceride recycling is activated in RIP140 null adipocytes. In order to determine the molecular mechanism of RIP140 repression i analysed the epigenetic profile of Ucp1, a gene which is typically expressed in brown but not white adipocytes. I utilized RIP140-null and RIP140-expressing adipocytes and found that RIP140 is essential for both DNA and histone methylation to maintain gene repression. RIP140 expression promotes the assembly of DNA and histone methyltransferases on the Ucp1 enhancer and leads to methylation of specific CpG residues and histones as judged by bisulphite genomic sequencing and chromatin immunoprecipitation assays. These results suggest that RIP140 serves as a scaffold for both DNA and histone methyltransferase activities to inhibit gene transcription by two key epigenetic repression systems. RIP140 could potentially act to divert white adipocytes from a brown fat phenotype by repressing genes involved in fatty acid oxidation and mitochondrial respiration and by silencing genes like Ucp1

Integrative analysis of epilepsy-associated genes reveals expression-phenotype correlations

Abstract Epilepsy is a highly prevalent neurological disorder characterized by recurrent seizures. Patients exhibit broad genetic, molecular, and clinical diversity involving mild to severe comorbidities. The factors that contribute to this phenotypic diversity remain unclear. Here we used publicly available datasets to systematically interrogate the expression pattern of 230 epilepsy-associated genes across human tissues, developmental stages, and central nervous system (CNS) cellular subtypes. We grouped genes based on their curated phenotypes into 3 broad classes: core epilepsy genes (CEG), where seizures are the dominant phenotype, developmental and epileptic encephalopathy genes (DEEG) that are associated with developmental and epileptic encephalopathy, and seizure-related genes (SRG), which are characterized by the presence of seizures and gross brain malformations. We find that compared to the other two groups of genes, DEEGs are highly expressed within the adult CNS, exhibit the highest and most dynamic expression in various brain regions across development, and are significantly enriched in GABAergic neurons. Our analysis provides an overview of the expression pattern of epilepsy-associated genes with spatiotemporal resolution and establishes a broad expression-phenotype correlation in epilepsy

Progress toward the clinical application of patient-specific pluripotent stem cells

Induced pluripotent stem (iPS) cells are generated by epigenetic reprogramming of somatic cells through the exogenous expression of transcription factors. These cells, just like embryonic stem cells, are likely to have a major impact on regenerative medicine, because they self-renew and retain the potential to be differentiated into all cell types of the human body. In this Review, we describe the current state of iPS cell technology, including approaches by which they are generated and what is known about their biology, and discuss the potential applications of these cells for disease modeling, drug discovery, and, eventually, cell replacement therapy.Stem Cell and Regenerative Biolog

Identification of BAF57 mutations in human breast cancer cell lines

El pdf del artículo es la versión de autor.Accumulating genetic and biochemical evidences support a role for the SWI/SNF chromatin-remodeling complex in cancer development and multiple core subunits of these complexes have been found to function as tumor suppressor genes. The core SWI/SNF subunit BAF57 mediates direct interactions with estrogen and androgen receptors (ER and AR) regulating their transcriptional activity. BAF57 gene maps to chromosome band 17 q21 in close proximity to the BRCA1 gene. This locus has been associated with frequent loss of heterozygosity (LOH) and allelic imbalance in breast cancers; however, BRCA1 mutations are rare events in sporadic breast cancer with LOH in the region, suggesting that another tumor suppressor gene resides in this area. All these reasons prompted us to screen for mutations in the BAF57 gene using a panel of the most commonly used human breast cancer cell lines. All cell lines analysed contain wild-type copies of BAF57 gene with the only exception of the breast ductal carcinoma cell line BT549. Sequencing of genomic DNA and cDNA generated from BT549 mRNA demonstrated the presence of a CA dinucleotide insertion in exon 5 of BAF57. The absence of wild-type BAF57 alleles indicates that this is a biallelic inactivating mutation that causes a frameshift and as a consequence a premature stop codon leading to a truncated BAF57 protein. A functional characterisation of the truncated BAF57 showed that it has lost the ability to bind to ER but still binds to the nuclear receptor coactivator SRC1e. Furthermore, we observed that the expression of the truncated BAF57 increased the ability of SRC1e to potentiate transcriptional activation by ERα, suggesting that mutations in BAF57 could contribute to the oncogenic transformation in breast cancer cells. © Springer 2006.Our work was funded by the Wellcome Trust (grant 061930), the Association for International Cancer Research (03-098) and Ministerio de Educación y Ciencia (SAF2004-02549).Peer Reviewe