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1.
Cell ; 186(11): 2456-2474.e24, 2023 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-37137305

RESUMEN

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.


Asunto(s)
Edición Génica , Células Madre Hematopoyéticas , Humanos , Diferenciación Celular , Sistemas CRISPR-Cas , Genoma , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Ingeniería Genética , Análisis de la Célula Individual
2.
Nature ; 626(8000): 799-807, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38326615

RESUMEN

Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.


Asunto(s)
Enfermedad de la Arteria Coronaria , Células Endoteliales , Estudio de Asociación del Genoma Completo , Hemangioma Cavernoso del Sistema Nervioso Central , Humanos , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Predisposición Genética a la Enfermedad/genética , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Polimorfismo de Nucleótido Simple , Epigenómica , Transducción de Señal/genética , Herencia Multifactorial
3.
Nature ; 593(7858): 238-243, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33828297

RESUMEN

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease1. Many of the underlying causal variants may affect enhancers2,3, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types4. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Predisposición Genética a la Enfermedad , Variación Genética/genética , Genoma Humano/genética , Estudio de Asociación del Genoma Completo , Enfermedades Inflamatorias del Intestino/genética , Línea Celular , Cromosomas Humanos Par 10/genética , Ciclofilinas/genética , Células Dendríticas , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Mitocondrias/metabolismo , Especificidad de Órganos/genética , Fenotipo
4.
Nature ; 539(7629): 452-455, 2016 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-27783602

RESUMEN

Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators. Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes. However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes. Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis. Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.


Asunto(s)
Regulación de la Expresión Génica/genética , Genes/genética , Sitios Genéticos/genética , Regiones Promotoras Genéticas/genética , Empalme del ARN/genética , ARN Largo no Codificante/genética , Transcripción Genética/genética , Animales , Línea Celular , Secuencia Conservada/genética , Evolución Molecular , Femenino , Genómica , Masculino , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Sitios de Empalme de ARN/genética , ARN Mensajero/genética
5.
Nature ; 477(7364): 295-300, 2011 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-21874018

RESUMEN

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.


Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Animales , Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Ratones , Unión Proteica , Factores de Transcripción/metabolismo
6.
bioRxiv ; 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-39026740

RESUMEN

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes - those with strong distal enhancers - are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

7.
bioRxiv ; 2023 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-38187584

RESUMEN

Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects and programmability of such DNA sequences remain difficult to map or predict because we have lacked scalable methods to precisely edit regulatory DNA and quantify the effects in an endogenous genomic context. Here we present an approach to measure the quantitative effects of hundreds of designed DNA sequence variants on gene expression, by combining pooled CRISPR prime editing with RNA fluorescence in situ hybridization and cell sorting (Variant-FlowFISH). We apply this method to mutagenize and rewrite regulatory DNA sequences in an enhancer and the promoter of PPIF in two immune cell lines. Of 672 variant-cell type pairs, we identify 497 that affect PPIF expression. These variants appear to act through a variety of mechanisms including disruption or optimization of existing transcription factor binding sites, as well as creation of de novo sites. Disrupting a single endogenous transcription factor binding site often led to large changes in expression (up to -40% in the enhancer, and -50% in the promoter). The same variant often had different effects across cell types and states, demonstrating a highly tunable regulatory landscape. We use these data to benchmark performance of sequence-based predictive models of gene regulation, and find that certain types of variants are not accurately predicted by existing models. Finally, we computationally design 185 small sequence variants (≤10 bp) and optimize them for specific effects on expression in silico. 84% of these rationally designed edits showed the intended direction of effect, and some had dramatic effects on expression (-100% to +202%). Variant-FlowFISH thus provides a powerful tool to map the effects of variants and transcription factor binding sites on gene expression, test and improve computational models of gene regulation, and reprogram regulatory DNA.

8.
Nature ; 440(7084): 671-5, 2006 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-16572171

RESUMEN

Here we present a finished sequence of human chromosome 15, together with a high-quality gene catalogue. As chromosome 15 is one of seven human chromosomes with a high rate of segmental duplication, we have carried out a detailed analysis of the duplication structure of the chromosome. Segmental duplications in chromosome 15 are largely clustered in two regions, on proximal and distal 15q; the proximal region is notable because recombination among the segmental duplications can result in deletions causing Prader-Willi and Angelman syndromes. Sequence analysis shows that the proximal and distal regions of 15q share extensive ancient similarity. Using a simple approach, we have been able to reconstruct many of the events by which the current duplication structure arose. We find that most of the intrachromosomal duplications seem to share a common ancestry. Finally, we demonstrate that some remaining gaps in the genome sequence are probably due to structural polymorphisms between haplotypes; this may explain a significant fraction of the gaps remaining in the human genome.


Asunto(s)
Cromosomas Humanos Par 15/genética , Evolución Molecular , Duplicación de Gen , Animales , Secuencia Conservada/genética , Genes , Genoma Humano , Haplotipos/genética , Humanos , Macaca mulatta/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , Polimorfismo Genético/genética , Análisis de Secuencia de ADN , Sintenía/genética
9.
Nat Genet ; 51(12): 1664-1669, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31784727

RESUMEN

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases1-4. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types5,6. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Animales , Factor de Transcripción GATA1/genética , Regulación de la Expresión Génica , Histona Desacetilasa 6/genética , Humanos , Hibridación Fluorescente in Situ , Células K562 , Ratones , Modelos Genéticos , ARN Guía de Kinetoplastida
10.
Science ; 354(6313): 769-773, 2016 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-27708057

RESUMEN

Gene expression in mammals is regulated by noncoding elements that can affect physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase of sequence in the vicinity of two essential transcription factors, MYC and GATA1, and identify nine distal enhancers that control gene expression and cellular proliferation. Quantitative features of chromatin state and chromosome conformation distinguish the seven enhancers that regulate MYC from other elements that do not, suggesting a strategy for predicting enhancer-promoter connectivity. This CRISPRi-based approach can be applied to dissect transcriptional networks and interpret the contributions of noncoding genetic variation to human disease.


Asunto(s)
Mapeo Cromosómico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos de Facilitación Genéticos/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Regiones Promotoras Genéticas/fisiología , Sistemas CRISPR-Cas , Proliferación Celular/genética , Enfermedad/genética , Elementos de Facilitación Genéticos/genética , Factor de Transcripción GATA1/genética , Regulación de la Expresión Génica , Humanos , Células K562 , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Reacción en Cadena en Tiempo Real de la Polimerasa
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