RESUMEN
Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENs to DNA may interfere with binding of key components of DNA repair complexes, favoring either classical- or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w.
Asunto(s)
Edición Génica/métodos , Sistemas CRISPR-Cas/genética , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Humanos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Nucleasas con Dedos de Zinc/metabolismoRESUMEN
The retinoblastoma protein or its regulators are altered in most human cancers. Although commonly thought of as solely a repressor of E2F-dependent transcription and cell cycle progression, pRb has gained notoriety in recent years as a key actor in cellular differentiation programs. In the June issue of Molecular Cell, Benevolenskaya et al. report that a long-known but poorly understood pRb interactor, RBP2, acts as an inhibitor of differentiation contributing to pRb's role as a coordinator of differentiation and cell cycle exit. Loss of pRb may unleash RBP2, maintaining cells in a poorly differentiated progenitor state that is prerequisite to tumor formation.
Asunto(s)
Diferenciación Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neoplasias/fisiopatología , Proteína de Retinoblastoma/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Proteínas de Ciclo Celular/fisiología , Proliferación Celular , Proteínas de Unión al ADN/fisiología , Factores de Transcripción E2F , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Biológicos , Neoplasias/etiología , Neoplasias/metabolismo , Unión Proteica , Proteína de Retinoblastoma/metabolismo , Proteína 2 de Unión a Retinoblastoma , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
We have previously shown that the retinoblastoma protein (pRb) can activate expression of Runx2-dependent, bone-specific genes in cultured cells. We now show that pRb also plays a role early in osteogenesis, and that in primary RB1(-/-) calvarial cells there is an increased osteoprogenitor pool. To understand pRb's function in vivo, we generated a conditional RB1-KO mouse in which pRb expression is efficiently extinguished in osteoblasts. These animals display an apparent developmental defect in bones, most strikingly in the calvaria. Cultured RB1(-/-) calvarial osteoblasts fail to cease proliferation upon reaching confluence or following differentiation. Re-plating assays of primary RB1(-/-) calvarial cells after differentiation showed a clear adipogenic ability with increased multipotency. RB1(-/-) osteoblasts display a severe reduction in levels of mRNAs expressed late in differentiation. In this study, we present strong evidence that pRb has multiple regulatory roles in osteogenesis. Furthermore, in the absence of RB1(-/-) there is a larger pool of multipotent cells compared with the WT counterpart. This increased pool of osteoprogenitor cells may be susceptible to additional transforming events leading to osteosarcoma, and is therefore key to understanding RB1 as a target in malignancy.
Asunto(s)
Desarrollo Óseo , Células Madre Mesenquimatosas/citología , Proteína de Retinoblastoma/fisiología , Cráneo/citología , Animales , Proliferación Celular , Ratones , Ratones Noqueados , Osteoblastos/citología , Proteína de Retinoblastoma/genéticaRESUMEN
The retinoblastoma gene, RB1, is frequently inactivated in a subset of tumors, including retinoblastoma and osteosarcoma (OS). One characteristic of OS, as well as other tumors in which RB1 is frequently inactivated, is the lack of N-cadherin-mediated cell-cell adhesions. The frequent inactivation of RB1 and parallel loss of N-cadherin expression in OS prompted us to ask whether these observations are directly related to each other. In this study, we observed reduced N-cadherin expression in RB1(-/-) calvarial osteoblasts. In addition, RB1(-/-) cell lines had increased migration potential compared to their RB1(+/+) counterparts. These properties of RB1(-/-) cell lines correlated with an adipogenic potential lacking in RB1(+/+) cell lines, suggesting that each property is present in an immature progenitor cell. The isolation of a cell population with low surface expression of N-cadherin and enhanced adipogenic ability supports this view. Interestingly, the acute loss of pRb does not affect N-cadherin expression or migration or confer adipogenic potential to immortalized RB1(+/+) calvarial cells, suggesting that these traits are not a direct consequence of pRb loss; rather, pRb loss leads to the expansion and immortalization of an immature progenitor pool characterized by these properties.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Cadherinas/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Proteína de Retinoblastoma/deficiencia , Cráneo/citología , Cráneo/metabolismo , Células 3T3 , Adipogénesis , Animales , Secuencia de Bases , Cadherinas/genética , Movimiento Celular , Células Cultivadas , Cartilla de ADN/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Expresión Génica , Genes de Retinoblastoma , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
Retinoblastoma protein (pRb) phosphorylation plays a central role in mediating cell cycle G1/S stage transition, together with E2F transcription factors. The binding of pRb to E2F is thought to be controlled by the sequential and cumulative phosphorylation of pRb at various amino acids. In addition to well characterized roles as a tumor suppressor, pRb has more recently been implicated in osteoprogenitor and other types of stem cell maintenance, proliferation and differentiation, thereby influencing the morphogenesis of developing organs. In this study, we present data characterizing the expression of pRb and three phosphorylated pRb (ppRb) isoforms-ppRbS780, ppRbS795, ppRbS807/811-in developmentally staged mouse molar and incisor teeth. Our results reveal distinct developmental expression patterns for individual ppRb isoforms in dental epithelial and dental mesenchymal cell differentiation, suggesting discrete functions in tooth development.