RESUMEN
CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.
Asunto(s)
Exones , Humanos , Exones/genética , Sistemas CRISPR-Cas , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Aptitud Genética , Células HEK293 , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Sitios de Empalme de ARN , Mutación , Regulación de la Expresión Génica , Empalme AlternativoRESUMEN
Inflammatory bowel disease (IBD) results from a dysregulated interaction between the microbiota and a genetically susceptible host. Genetic studies have linked TNFSF15 polymorphisms and its protein TNF-like ligand 1A (TL1A) with IBD, but the functional role of TL1A is not known. Here, we found that adherent IBD-associated microbiota induced TL1A release from CX3CR1+ mononuclear phagocytes (MNPs). Using cell-specific genetic deletion models, we identified an essential role for CX3CR1+MNP-derived TL1A in driving group 3 innate lymphoid cell (ILC3) production of interleukin-22 and mucosal healing during acute colitis. In contrast to this protective role in acute colitis, TL1A-dependent expression of co-stimulatory molecule OX40L in MHCII+ ILC3s during colitis led to co-stimulation of antigen-specific T cells that was required for chronic T cell colitis. These results identify a role for ILC3s in activating intestinal T cells and reveal a central role for TL1A in promoting ILC3 barrier immunity during colitis.
Asunto(s)
Colitis/inmunología , Inmunidad Innata/inmunología , Linfocitos/inmunología , Microbiota/inmunología , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Adulto , Anciano , Animales , Colitis/genética , Colitis/metabolismo , Femenino , Humanos , Inmunidad Innata/genética , Interleucinas/genética , Interleucinas/inmunología , Interleucinas/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microbiota/fisiología , Persona de Mediana Edad , Fagocitos/citología , Fagocitos/inmunología , Fagocitos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Adulto Joven , Interleucina-22RESUMEN
OBJECTIVE: To determine clinical outcomes in patients with stage IA polyp-limited versus endometrium-limited high-grade (type II) endometrial carcinoma (EC). METHODS: We identified all cases of stage IA polyp-limited or endometrium-limited high-grade EC (FIGO grade 3 endometrioid, serous, clear cell, or mixed) who underwent simple hysterectomy, bilateral salpingo-oophorectomy, peritoneal washings, omental biopsy, and pelvic and para-aortic lymph node dissection and received adjuvant treatment at our institution from October 1995 to November 2012. Progression-free survival (PFS) and overall survival (OS) by histology, adjuvant therapy, and polyp-limited versus endometrium-limited disease status were determined using log-rank test. We analyzed 3 treatment groups: patients who received chemotherapy with or without radiation therapy (RT) (intravaginal or pelvic); patients who received RT (intravaginal RT or pelvic RT) alone; and patients who received no adjuvant treatment. RESULTS: In all, 85 women underwent hysterectomy/salpingo-oophorectomy; all were surgically staged with lymph node assessment and had stage IA EC with no lymphovascular or myometrial invasion. Median follow-up for survivors was 46.5 months (range, 1.98-188.8 months). Forty-nine patients (57.6%) had polyp-limited disease, and 36 (42.4%) had endometrium-limited disease. There were no significant differences in clinicopathologic characteristics between patients within the 3 treatment groups with regard to age at diagnosis, mean body mass index, ECOG (Eastern Cooperative Oncology Group) performance status, polyp-limited or endometrium-limited disease, diabetes, or race. The 3-year PFS rate was 94.9% and the 3-year OS rate was 98.8%. Univariate PFS and OS analysis revealed that age was a relevant prognostic factor (PFS hazard ratio [95% confidence interval], 1.13 [1.02-1.25]; P = 0.022; OS hazard ratio [95% confidence interval], 1.19 [1.02-1.38]; P = 0.03). Adjuvant treatment did not impact outcomes. CONCLUSIONS: Clinical outcomes of surgical stage IA type II polyp- or endometrium-limited high-grade epithelial EC are equally favorable regardless of histologic subtype or adjuvant therapy received. The benefit of adjuvant therapy in this select group remains to be determined.
Asunto(s)
Adenocarcinoma de Células Claras/terapia , Quimioterapia Adyuvante , Cistadenocarcinoma Seroso/terapia , Neoplasias Endometriales/terapia , Recurrencia Local de Neoplasia/terapia , Pólipos , Radioterapia Adyuvante , Adenocarcinoma de Células Claras/patología , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Terapia Combinada , Cistadenocarcinoma Seroso/patología , Neoplasias Endometriales/patología , Femenino , Estudios de Seguimiento , Humanos , Histerectomía , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient's primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term "genome shredding." Importantly, in the patient's recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. "Cancer shredding" leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Temozolomida/farmacología , Temozolomida/uso terapéutico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Glioblastoma/patología , Glioma/genética , Glioma/tratamiento farmacológico , Antineoplásicos Alquilantes/farmacologíaAsunto(s)
Resistencia a Antineoplásicos/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación Puntual , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Persona de Mediana Edad , Fosforilación , Piperidinas , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Pirazoles/metabolismo , Pirimidinas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Recurrencia , Análisis de Secuencia de ADNRESUMEN
Perturbations to the epigenome are known drivers of tumorigenesis. In melanoma, alterations in histone methyltransferases that catalyze methylation at histone 3 lysine 9 and histone 3 lysine 27-two sites of critical post-translational modification-have been reported. To study the function of these methyltransferases in melanoma, we engineered melanocytes to express histone 3 lysine-to-methionine mutations at lysine 9 and lysine 27, which are known to inhibit the activity of histone methyltransferases, in a zebrafish melanoma model. Using this system, we found that loss of histone 3 lysine 9 methylation dramatically suppressed melanoma formation and that inhibition of histone 3 lysine 9 methyltransferases in human melanoma cells increased innate immune response signatures. In contrast, loss of histone 3 lysine 27 methylation significantly accelerated melanoma formation. We identified FOXD1 as a top target of PRC2 that is silenced in melanocytes and found that aberrant overexpression of FOXD1 accelerated melanoma onset. Collectively, these data demonstrate how histone 3 lysine-to-methionine mutations can be used to uncover critical roles for methyltransferases.
RESUMEN
Off-target effects are well established confounders of CRISPR negative selection screens that impair the identification of essential genomic loci. In particular, non-coding regulatory elements and repetitive regions are often difficult to target with specific gRNAs, effectively precluding the unbiased screening of a large portion of the genome. To address this, we developed CRISPR Specificity Correction (CSC), a computational method that corrects for the effect of off-targeting on gRNA depletion. We benchmark CSC with data from the Cancer Dependency Map and show that it significantly improves the overall sensitivity and specificity of viability screens while preserving known essentialities, particularly for genes targeted by highly promiscuous gRNAs. We believe this tool will further enable the functional annotation of the genome as it represents a robust alternative to the traditional filtering strategy of discarding unspecific guides from the analysis. CSC is an open-source software that can be seamlessly integrated into current CRISPR analysis pipelines.
Asunto(s)
ARN Guía de Kinetoplastida/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Biología Computacional/métodos , Edición Génica , Humanos , ARN Guía de Kinetoplastida/genética , Programas InformáticosRESUMEN
We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools.
Asunto(s)
Algoritmos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Silenciador del Gen , Aprendizaje Automático , ARN Interferente Pequeño/genética , Programas Informáticos , Sistemas CRISPR-Cas/genética , Mapeo Cromosómico/métodos , Análisis de Secuencia de ARN/métodosRESUMEN
Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease.