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1.
Cell ; 187(3): 733-749.e16, 2024 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-38306984

RESUMEN

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.


Asunto(s)
Autoanticuerpos , Enfermedades Autoinmunes , ARN Largo no Codificante , Animales , Femenino , Humanos , Masculino , Ratones , Autoanticuerpos/genética , Enfermedades Autoinmunes/genética , Autoinmunidad/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismo , Inactivación del Cromosoma X , Caracteres Sexuales
2.
Cell ; 185(26): 4904-4920.e22, 2022 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-36516854

RESUMEN

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.


Asunto(s)
Receptores de Antígenos de Linfocitos T , Internalización del Virus , Humanos , Biología , Epítopos , Ligandos , Péptidos , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de la Célula Individual , Genómica
3.
Cell ; 185(11): 1924-1942.e23, 2022 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-35525247

RESUMEN

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.


Asunto(s)
Ganglios Linfáticos , Melanoma , Animales , Tolerancia Inmunológica , Inmunoterapia , Metástasis Linfática/patología , Melanoma/patología , Ratones
4.
Cell ; 184(9): 2394-2411.e16, 2021 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-33743211

RESUMEN

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.


Asunto(s)
Interacciones Huésped-Patógeno , ARN Viral/genética , SARS-CoV-2/genética , Animales , COVID-19/virología , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Genoma Viral , Humanos , Pulmón/virología , Masculino , Espectrometría de Masas , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteoma/metabolismo , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/ultraestructura , Células Vero
5.
Nat Immunol ; 23(6): 848-860, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35624210

RESUMEN

Chronic antigen stimulation during viral infections and cancer can lead to T cell exhaustion, which is characterized by reduced effector function and proliferation, and the expression of inhibitory immune checkpoint receptors. Recent studies have demonstrated that T cell exhaustion results in wholescale epigenetic remodeling that confers phenotypic stability to these cells and prevents T cell reinvigoration by checkpoint blockade. Here, we review foundational technologies to profile the epigenome at multiple scales, including mapping the locations of transcription factors and histone modifications, DNA methylation and three-dimensional genome conformation. We discuss how these technologies have elucidated the development and epigenetic regulation of exhausted T cells and functional implications across viral infection, cancer, autoimmunity and engineered T cell therapies. Finally, we cover emerging multi-omic and genome engineering technologies, current and upcoming opportunities to apply these to T cell exhaustion, and therapeutic opportunities for T cell engineering in the clinic.


Asunto(s)
Neoplasias , Virosis , Linfocitos T CD8-positivos , Metilación de ADN , Epigénesis Genética , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Linfocitos T/metabolismo , Virosis/metabolismo
6.
Nat Immunol ; 23(11): 1614-1627, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36289450

RESUMEN

Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.


Asunto(s)
Linfocitos T CD8-positivos , Virosis , Humanos , Ratones , Animales , Receptores de Antígenos de Linfocitos T/genética , Diferenciación Celular , Linfocitos Infiltrantes de Tumor
7.
Immunity ; 57(9): 2202-2215.e6, 2024 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-39043184

RESUMEN

The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Linfocitos T CD8-positivos , Diferenciación Celular , Epigénesis Genética , Epigenómica , Memoria Inmunológica , Células T de Memoria , Animales , Diferenciación Celular/inmunología , Diferenciación Celular/genética , Ratones , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Linfocitos T CD8-positivos/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Epigenómica/métodos , Ratones Endogámicos C57BL , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Transcriptoma , Cromatina/metabolismo
8.
Immunity ; 56(5): 1027-1045.e8, 2023 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-36791722

RESUMEN

Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.


Asunto(s)
Macrófagos , Microglía , Ratones , Animales , Microglía/metabolismo , Macrófagos/metabolismo , Integrasas/genética , Integrasas/metabolismo , Encéfalo/metabolismo
9.
Mol Cell ; 83(1): 121-138.e7, 2023 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-36521490

RESUMEN

Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings in vivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.


Asunto(s)
Cromatina , Interleucina-4 , Humanos , Ratones , Animales , Interleucina-4/genética , Interleucina-4/farmacología , Cromatina/genética , Cromatina/metabolismo , Macrófagos/metabolismo , Interferón gamma/genética , Interferón gamma/farmacología , Ciclo Celular/genética , División Celular
10.
Genes Dev ; 37(7-8): 291-302, 2023 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-36990511

RESUMEN

Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.


Asunto(s)
ARN Largo no Codificante , Animales , Ratones , Elementos de Facilitación Genéticos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Regiones Promotoras Genéticas
11.
Immunity ; 54(7): 1417-1432.e7, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34004142

RESUMEN

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Δ-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Δ-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Δ-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Hematopoyesis/genética , Transcripción Genética/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Células Dendríticas/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/fisiología
12.
Nature ; 632(8023): 201-208, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39020172

RESUMEN

Telomerase is intimately associated with stem cells and cancer, because it catalytically elongates telomeres-nucleoprotein caps that protect chromosome ends1. Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferation of cells in a telomere-independent manner2-8, but so far, loss-of-function studies have provided no evidence that TERT has a direct role in stem cell function. In many tissues, homeostasis is shaped by stem cell competition, a process in which stem cells compete on the basis of inherent fitness. Here we show that conditional deletion of Tert in the spermatogonial stem cell (SSC)-containing population in mice markedly impairs competitive clone formation. Using lineage tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that clonal inactivation of TERT promotes stem cell differentiation and a genome-wide reduction in open chromatin. This role for TERT in competitive clone formation occurs independently of both its reverse transcriptase activity and the canonical telomerase complex. Inactivation of TERT causes reduced activity of the MYC oncogene, and transgenic expression of MYC in the TERT-deleted pool of SSCs efficiently rescues clone formation. Together, these data reveal a catalytic-activity-independent requirement for TERT in enhancing stem cell competition, uncover a genetic connection between TERT and MYC and suggest that a selective advantage for stem cells with high levels of TERT contributes to telomere elongation in the male germline during homeostasis and ageing.


Asunto(s)
Competencia Celular , Células Clonales , Células Madre , Telomerasa , Animales , Masculino , Ratones , Diferenciación Celular , Linaje de la Célula , Cromatina/metabolismo , Cromatina/genética , Células Clonales/citología , Células Clonales/enzimología , Células Clonales/metabolismo , Eliminación de Gen , Genes myc , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Espermatogonias/citología , Espermatogonias/metabolismo , Células Madre/citología , Células Madre/enzimología , Células Madre/metabolismo , Telomerasa/deficiencia , Telomerasa/genética , Telomerasa/metabolismo , Transcripción Reversa , Biocatálisis , Homeostasis , Envejecimiento
13.
Immunity ; 53(5): 1078-1094.e7, 2020 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-33010224

RESUMEN

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.


Asunto(s)
Linfocitos B/inmunología , Reacciones Cruzadas/inmunología , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/virología , Flavivirus/inmunología , Interacciones Huésped-Patógeno/inmunología , Memoria Inmunológica , Animales , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Infecciones por Flavivirus/metabolismo , Inmunización , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Especificidad de la Especie
14.
Nature ; 616(7958): 755-763, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37046083

RESUMEN

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, but this effect was not seen in clones with driver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.


Asunto(s)
Hematopoyesis Clonal , Células Madre Hematopoyéticas , Animales , Humanos , Ratones , Alelos , Hematopoyesis Clonal/genética , Estudio de Asociación del Genoma Completo , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Mutación , Regiones Promotoras Genéticas
15.
Nature ; 600(7890): 731-736, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34819668

RESUMEN

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.


Asunto(s)
Neoplasias , Proteínas Nucleares , Azepinas/farmacología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Oncogenes/genética , Factores de Transcripción/genética
16.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-34921117

RESUMEN

Over the past five decades, tremendous effort has been devoted to computational methods for predicting properties of ligands-i.e., molecules that bind macromolecular targets. Such methods, which are critical to rational drug design, fall into two categories: physics-based methods, which directly model ligand interactions with the target given the target's three-dimensional (3D) structure, and ligand-based methods, which predict ligand properties given experimental measurements for similar ligands. Here, we present a rigorous statistical framework to combine these two sources of information. We develop a method to predict a ligand's pose-the 3D structure of the ligand bound to its target-that leverages a widely available source of information: a list of other ligands that are known to bind the same target but for which no 3D structure is available. This combination of physics-based and ligand-based modeling improves pose prediction accuracy across all major families of drug targets. Using the same framework, we develop a method for virtual screening of drug candidates, which outperforms standard physics-based and ligand-based virtual screening methods. Our results suggest broad opportunities to improve prediction of various ligand properties by combining diverse sources of information through customized machine-learning approaches.


Asunto(s)
Antipsicóticos/química , Antipsicóticos/farmacología , Diseño de Fármacos/métodos , Inteligencia Artificial , Sitios de Unión , Regulación de la Expresión Génica/efectos de los fármacos , Ligandos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Relación Estructura-Actividad
17.
Nat Biotechnol ; 2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38760566

RESUMEN

CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.

18.
bioRxiv ; 2024 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-39282425

RESUMEN

FOXP3 is a lineage-defining transcription factor that controls differentiation and maintenance of suppressive function of regulatory T cells (Tregs). Foxp3 is exclusively expressed in Tregs in mice. However, in humans, FOXP3 is not only constitutively expressed in Tregs; it is also transiently expressed in stimulated CD4+CD25- conventional T cells (Tconvs)1-3. Mechanisms governing the expression of FOXP3 in human Tconvs are not understood. Here, we performed CRISPR interference (CRISPRi) screens using a 15K-member gRNA library tiling 39 kb downstream of the FOXP3 transcriptional start site (TSS) to 85 kb upstream of the TSS in Treg and Tconvs. The FOXP3 promoter and conserved non-coding sequences (CNS0, CNS1, CNS2 and CNS3), characterized as enhancer elements in murine Tregs, were required for maintenance of FOXP3 in human Tregs. In contrast, FOXP3 in human Tconvs depended on regulation at CNS0 and a novel Tconv-specific noncoding sequence (TcNS+) located upstream of CNS0. Arrayed validations of these sites identified an additional repressive cis-element overlapping with the PPP1R3F promoter (TcNS-). Pooled CRISPR knockouts revealed multiple transcription factors required for proper expression of FOXP3 in Tconvs, including GATA3, STAT5, IRF4, ETS1 and DNA methylation-associated regulators DNMT1 and MBD2. Analysis of ChIP-seq and ATAC-seq paired with knock-out (KO) of GATA3, STAT5, IRF4, and ETS1 revealed regulation of CNS0 and TcNS+ accessibility. Collectively, this work identified Treg-shared and Tconv-specific cis-elements and the trans-factors that interact with them, building a network of regulators controlling FOXP3 expression in human Tconvs.

19.
Cancer Discov ; 2024 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-39109936

RESUMEN

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show non-homologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

20.
Nat Biotechnol ; 41(2): 262-272, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-35851375

RESUMEN

Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5' and 3' untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes.


Asunto(s)
ARN Circular , ARN , ARN Circular/genética , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Empalme del ARN
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