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The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.
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Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismoRESUMEN
Spatiotemporal control of gene expression during development requires orchestrated activities of numerous enhancers, which are cis-regulatory DNA sequences that, when bound by transcription factors, support selective activation or repression of associated genes. Proper activation of enhancers is critical during embryonic development, adult tissue homeostasis, and regeneration, and inappropriate enhancer activity is often associated with pathological conditions such as cancer. Multiple consortia [e.g., the Encyclopedia of DNA Elements (ENCODE) Consortium and National Institutes of Health Roadmap Epigenomics Mapping Consortium] and independent investigators have mapped putative regulatory regions in a large number of cell types and tissues, but the sequence determinants of cell-specific enhancers are not yet fully understood. Machine learning approaches trained on large sets of these regulatory regions can identify core transcription factor binding sites and generate quantitative predictions of enhancer activity and the impact of sequence variants on activity. Here, we review these computational methods in the context of enhancer prediction and gene regulatory network models specifying cell fate.
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Biología Computacional/métodos , Elementos de Facilitación Genéticos , Redes Reguladoras de Genes , Genoma Humano , HumanosRESUMEN
Genome editing has the potential to revolutionize many investigative and therapeutic strategies in biology and medicine. In the field of regenerative medicine, one of the leading applications of genome engineering technology is the generation of immune evasive pluripotent stem cell-derived somatic cells for transplantation. In particular, as more functional and therapeutically relevant human pluripotent stem cell-derived islets (SCDI) are produced in many labs and studied in clinical trials, there is keen interest in studying the immunogenicity of these cells and modulating allogeneic and autoimmune immune responses for therapeutic benefit. Significant experimental work has already suggested that elimination of Human Leukocytes Antigen (HLA) expression and overexpression of immunomodulatory genes can impact survival of a variety of pluripotent stem cell-derived somatic cell types. Limited work published to date focuses on stem cell-derived islets and work in a number of labs is ongoing. Rapid progress is occurring in the genome editing of human pluripotent stem cells and their progeny focused on evading destruction by the immune system in transplantation models, and while much research is still needed, there is no doubt the combined technologies of genome editing and stem cell therapy will profoundly impact transplantation medicine in the future.
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Islotes Pancreáticos , Células Madre Pluripotentes , Humanos , Ingeniería Genética , Edición Génica , Trasplante de Células MadreRESUMEN
Developmental biology has long benefited from studies of classic model organisms. Recently, human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, have emerged as a new model system that offers unique advantages for developmental studies. Here, we discuss how studies of hPSCs can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development 'in a dish'. We also summarize some of the recently developed genetic tools that greatly facilitate the interrogation of gene function during hPSC differentiation. With the development of high-throughput screening technologies, hPSCs have the potential to revolutionize gene discovery in mammalian development.
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Diferenciación Celular/fisiología , Biología Evolutiva/tendencias , Desarrollo Embrionario/fisiología , Marcación de Gen/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Modelos Biológicos , Células Madre Pluripotentes/fisiología , HumanosRESUMEN
Mouse models are extensively used in metabolic studies. However, inherent differences between the species, notably their blood glucose levels, hampered data translation into clinical settings. In this study, we confirmed GLUT1 to be the predominantly expressed glucose transporter in both adult and fetal human ß-cells. In comparison, GLUT2 is detected in a small yet significant subpopulation of adult ß-cells and is expressed to a greater extent in fetal ß-cells. Notably, GLUT1/2 expression in INS+ cells from human stem cell-derived islet-like clusters (SC-islets) exhibited a closer resemblance to that observed in fetal islets. Transplantation of primary human islets or SC-islets, but not murine islets, lowered murine blood glucose to the human glycemic range, emphasizing the critical role of ß-cells in establishing species-specific glycemia. We further demonstrate the functional requirements of GLUT1 and GLUT2 in glucose uptake and insulin secretion through chemically inhibiting GLUT1 in primary islets and SC-islets and genetically disrupting GLUT2 in SC-islets. Finally, we developed a mathematical model to predict changes in glucose uptake and insulin secretion as a function of GLUT1/2 expression. Collectively, our findings illustrate the crucial roles of GLUTs in human ß-cells, and identify them as key components in establishing species-specific glycemic set points.
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Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 2 , Células Secretoras de Insulina , Humanos , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 1/genética , Animales , Ratones , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Glucemia/metabolismo , Glucosa/metabolismo , Secreción de Insulina/fisiología , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/metabolismoRESUMEN
Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer â¼664 kb from the ONECUT1 promoter, since coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
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Haploinsufficiency for GATA6 is associated with congenital heart disease (CHD) with variable comorbidity of pancreatic or diaphragm defects, although the etiology of disease is not well understood. Here, we used cardiac directed differentiation from human embryonic stem cells (hESCs) as a platform to study GATA6 function during early cardiogenesis. GATA6 loss-of-function hESCs had a profound impairment in cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation due to early defects during the mesendoderm and lateral mesoderm patterning stages. Profiling by RNA-seq and CUT&RUN identified genes of the WNT and BMP programs regulated by GATA6 during early mesoderm patterning. Furthermore, interactome analysis detected GATA6 binding with developmental transcription factors and chromatin remodelers suggesting cooperative regulation of cardiac lineage gene accessibility. We show that modulating WNT and BMP inputs during the first 48 hours of cardiac differentiation is sufficient to partially rescue CPC and CM defects in GATA6 heterozygous and homozygous mutant hESCs. This study provides evidence of the regulatory functions for GATA6 directing human precardiac mesoderm patterning during the earliest stages of cardiogenesis to further our understanding of haploinsufficiency causing CHD and the co-occurrence of cardiac and other organ defects caused by human GATA6 mutations.
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Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, â¼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
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Pancreatic beta cell proliferation has emerged as the principal mechanism for homeostatic maintenance of beta cell mass during adult life. This underscores the importance of understanding the mechanisms of beta cell replication and suggests novel approaches for regenerative therapy to treat diabetes. Here we use an in vivo pulse-chase labeling assay to investigate the replication dynamics of adult mouse beta cells. We find that replicated beta cells are able to re-enter the cell division cycle shortly after mitosis and regain their normal proliferative potential after a short quiescence period of several days. This quiescence period is lengthened with advanced age, but shortened during injury-driven beta cell regeneration and following treatment with a pharmacological activator of glucokinase, providing strong evidence that metabolic demand is a key determinant of cell cycle re-entry. Lastly, we show that cyclin D2, a crucial factor in beta cell replication, is downregulated during cell division, and is slowly upregulated post-mitosis by a glucose-sensitive mechanism. These results demonstrate that beta cells quickly regain their capacity to re-enter the cell cycle post-mitosis and implicate glucose control of cyclin D2 expression in the regulation of this process.
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Senescencia Celular , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Envejecimiento , Animales , Ciclo Celular , Proliferación Celular , Ciclina D2/metabolismo , Células Secretoras de Insulina/citología , RatonesRESUMEN
Phenotypic plasticity associated with the hybrid epithelial-mesenchymal transition (EMT) is crucial to metastatic seeding and outgrowth. However, the mechanisms governing the hybrid EMT state remain poorly defined. Here we showed that deletion of the epigenetic regulator MLL3, a tumour suppressor frequently altered in human cancer, promoted the acquisition of hybrid EMT in breast cancer cells. Distinct from other EMT regulators that mediate only unidirectional changes, MLL3 loss enhanced responses to stimuli inducing EMT and mesenchymal-epithelial transition in epithelial and mesenchymal cells, respectively. Consequently, MLL3 loss greatly increased metastasis by enhancing metastatic colonization. Mechanistically, MLL3 loss led to increased IFNγ signalling, which contributed to the induction of hybrid EMT cells and enhanced metastatic capacity. Furthermore, BET inhibition effectively suppressed the growth of MLL3-mutant primary tumours and metastases. These results uncovered MLL3 mutation as a key driver of hybrid EMT and metastasis in breast cancer that could be targeted therapeutically.
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Neoplasias de la Mama , Células Madre Mesenquimatosas , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Diferenciación Celular , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Células Madre Mesenquimatosas/patología , Metástasis de la Neoplasia/patologíaRESUMEN
Pluripotent stem cells are defined by both the ability to unlimitedly self-renew and differentiate to any somatic cell lineage, but understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. We performed four parallel genome-scale CRISPR-Cas9 screens to investigate the interplay between these two aspects of pluripotency. Our comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including many mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control stem cell identity. We further discovered a core set of factors that control both stem cell fitness and pluripotency identity, including an interconnected network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus self-renewal, and offer a valuable model for categorizing gene function in broad biological contexts.
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Comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Our network modeling revealed that nonlinear enhancer-gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Utilizing hESC definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core endoderm lineage-specifying transcription factors, and many enhancers had strong effects mid-transition but weak effects post-transition. Through integrating enhancer activity measurements and three-dimensional enhancer-promoter interaction information, we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.
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The mechanisms underlying the ability of embryonic stem cells (ESCs) to rapidly activate lineage-specific genes during differentiation remain largely unknown. Through multiple CRISPR-activation screens, we discovered human ESCs have pre-established transcriptionally competent chromatin regions (CCRs) that support lineage-specific gene expression at levels comparable to differentiated cells. CCRs reside in the same topological domains as their target genes. They lack typical enhancer-associated histone modifications but show enriched occupancy of pluripotent transcription factors, DNA demethylation factors, and histone deacetylases. TET1 and QSER1 protect CCRs from excessive DNA methylation, while HDAC1 family members prevent premature activation. This "push and pull" feature resembles bivalent domains at developmental gene promoters but involves distinct molecular mechanisms. Our study provides new insights into pluripotency regulation and cellular plasticity in development and disease. One sentence summary: We report a class of distal regulatory regions distinct from enhancers that confer human embryonic stem cells with the competence to rapidly activate the expression of lineage-specific genes.
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Comprehensive enhancer discovery is challenging because most enhancers, especially those contributing to complex diseases, have weak effects on gene expression. Our gene regulatory network modeling identified that nonlinear enhancer gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Using human embryonic stem cell definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set of enhancers for each of the core endoderm-specifying transcription factors. Many enhancers had strong effects mid-transition but weak effects post-transition, consistent with the nonlinear temporal responses to enhancer perturbation predicted by the modeling. Integrating three-dimensional genomic information, we were able to develop a CTCF-loop-constrained Interaction Activity model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and systematic enhancer discovery in both normal and pathological cell state transitions.
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Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Humanos , Elementos de Facilitación Genéticos/genética , Diferenciación Celular/genética , Factores de Transcripción/genética , Redes Reguladoras de Genes/genética , Cromatina/genéticaRESUMEN
Understanding diverse responses of individual cells to the same perturbation is central to many biological and biomedical problems. Current methods, however, do not precisely quantify the strength of perturbation responses and, more importantly, reveal new biological insights from heterogeneity in responses. Here we introduce the perturbation-response score (PS), based on constrained quadratic optimization, to quantify diverse perturbation responses at a single-cell level. Applied to single-cell transcriptomes of large-scale genetic perturbation datasets (e.g., Perturb-seq), PS outperforms existing methods for quantifying partial gene perturbation responses. In addition, PS presents two major advances. First, PS enables large-scale, single-cell-resolution dosage analysis of perturbation, without the need to titrate perturbation strength. By analyzing the dose-response patterns of over 2,000 essential genes in Perturb-seq, we identify two distinct patterns, depending on whether a moderate reduction in their expression induces strong downstream expression alterations. Second, PS identifies intrinsic and extrinsic biological determinants of perturbation responses. We demonstrate the application of PS in contexts such as T cell stimulation, latent HIV-1 expression, and pancreatic cell differentiation. Notably, PS unveiled a previously unrecognized, cell-type-specific role of coiled-coil domain containing 6 (CCDC6) in guiding liver and pancreatic lineage decisions, where CCDC6 knockouts drive the endoderm cell differentiation towards liver lineage, rather than pancreatic lineage. The PS approach provides an innovative method for dose-to-function analysis and will enable new biological discoveries from single-cell perturbation datasets.
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The identification of cell-type-specific 3D chromatin interactions between regulatory elements can help to decipher gene regulation and to interpret the function of disease-associated non-coding variants. However, current chromosome conformation capture (3C) technologies are unable to resolve interactions at this resolution when only small numbers of cells are available as input. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps and regulatory interactions from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility profiles across metacells, and predicted CTCF motif tracks as input features and employs a lightweight architecture to enable training on standard GPUs. Once trained on paired scATAC-seq and Hi-C data in human cell lines and tissues, ChromaFold can accurately predict both the 3D contact map and peak-level interactions across diverse human and mouse test cell types. In benchmarking against a recent deep learning method that uses bulk ATAC-seq, DNA sequence, and CTCF ChIP-seq to make cell-type-specific predictions, ChromaFold yields superior prediction performance when including CTCF ChIP-seq data as an input and comparable performance without. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations. ChromaFold thus achieves state-of-the-art prediction of 3D contact maps and regulatory interactions using scATAC-seq alone as input data, enabling accurate inference of cell-type-specific interactions in settings where 3C-based assays are infeasible.
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The epigenome plays a crucial role in modulating the activity of regulatory elements, thereby orchestrating diverse transcriptional programs during embryonic development. Human (h)PSC stepwise differentiation provides an excellent platform for capturing dynamic epigenomic events during lineage transition in human development. Here we discuss how recent technological advances, from epigenomic mapping to targeted perturbation, are providing a more comprehensive appreciation of remodeling of the chromatin landscape during human development with implications for aberrant rewiring in disease. We predict that the continuous innovation of hPSC differentiation methods, epigenome mapping, and CRISPR (clustered regularly interspaced short palindromic repeats) perturbation technologies will allow researchers to build toward not only a comprehensive understanding of the epigenomic mechanisms governing development, but also a highly flexible way to model diseases with opportunities for translation.
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Epigenoma , Células Madre Pluripotentes , Cromatina/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Epigenoma/genética , Epigenómica , HumanosRESUMEN
Formation of a properly sized and patterned embryo during gastrulation requires a well-coordinated interplay between cell proliferation, lineage specification and tissue morphogenesis. Following transient physical or pharmacological manipulations of embryo size, pre-gastrulation mouse embryos show remarkable plasticity to recover and resume normal development. However, it remains unclear how mechanisms driving lineage specification and morphogenesis respond to defects in cell proliferation during and after gastrulation. Null mutations in DNA replication or cell-cycle-related genes frequently lead to cell-cycle arrest and reduced cell proliferation, resulting in developmental arrest before the onset of gastrulation; such early lethality precludes studies aiming to determine the impact of cell proliferation on lineage specification and morphogenesis during gastrulation. From an unbiased ENU mutagenesis screen, we discovered a mouse mutant, tiny siren (tyrn), that carries a hypomorphic mutation producing an aspartate to tyrosine (D939Y) substitution in Pold1, the catalytic subunit of DNA polymerase δ. Impaired cell proliferation in the tyrn mutant leaves anterior-posterior patterning unperturbed during gastrulation but results in reduced embryo size and severe morphogenetic defects. Our analyses show that the successful execution of morphogenetic events during gastrulation requires that lineage specification and the ordered production of differentiated cell types occur in concordance with embryonic growth.
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ADN Polimerasa III , Gastrulación , Animales , ADN Polimerasa III/genética , Embrión de Mamíferos , Gastrulación/genética , Ratones , Morfogénesis/genética , MutaciónRESUMEN
MODY8 (maturity-onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes associated with mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar cells. MODY8 patients develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes. In the present study, we report the transfer of CEL proteins from acinar cells to ß-cells as a form of cross-talk between exocrine and endocrine cells. Human ß-cells show a relatively higher propensity for internalizing the mutant versus the wild-type CEL protein. After internalization, the mutant protein forms stable intracellular aggregates leading to ß-cell secretory dysfunction. Analysis of pancreas sections from a MODY8 patient reveals the presence of CEL protein in the few extant ß-cells. The present study provides compelling evidence for the mechanism by which a mutant gene expressed specifically in acinar cells promotes dysfunction and loss of ß-cells to cause diabetes.