RESUMO
In this issue of Cell, Nuñez et al. develop CRISPRoff, a programmable epigenetic memory writer capable of establishing specific gene silencing programs that are stably maintained across cell division and differentiation. The singular dCas9 fusion offers a simple, reliable, and general tool for genome-wide screens, multiplexed editing, and potential therapeutics.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Epigenômica , Regiões Promotoras Genéticas , RedaçãoRESUMO
Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.
Assuntos
Engenharia Genética/métodos , MicroRNAs/genética , Fatores de Transcrição/genética , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/genética , Sítios de Ligação , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Células HEK293 , Humanos , Plasmídeos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sequências de Repetição em Tandem , Transfecção , Xanthomonas , Dedos de Zinco/genéticaRESUMO
BACKGROUND: Aberrant activation of the androgen receptor (AR) is a major factor highly relevant to castration-resistant progression of prostate cancer (PCa). FOXO1, a key downstream effector of PTEN, inhibits androgen-independent activation of the AR. However, the underlying mechanism remains elusive. METHODS: The inhibitory effect of FOXO1 on full-length and constitutively active splice variants of the AR was examined by luciferase reporter assays and real-time reverse transcription polymerase chain reaction (RT-qPCR). In vitro protein binding assays and western blot analyses were used to determine the regions in FOXO1 and AR responsible for their interaction. RESULTS: We found that a putative transcription repression domain in the NH2-terminus of FOXO1 is dispensable for FOXO1 inhibition of the AR. In vitro protein binding assays showed that FOXO1 binds to the transcription activation unit 5 (TAU5) motif in the AR NH2-terminal domain (NTD), a region required for recruitment of p160 activators including SRC-1. Ectopic expression of SRC-1 augmented transcriptional activity of some, but not all AR splice variants examined. Forced expression of FOXO1 blocked the effect of SRC-1 on AR variants' transcriptional activity by decreasing the binding of SRC-1 to the AR NTD. Ectopic expression of FOXO1 inhibited expression of endogenous genes activated primarily by alternatively spliced AR variants in human castration-resistant PCa 22Rv1 cells. CONCLUSIONS: FOXO1 binds to the TAU5 motif in the AR NTD and inhibits ligand-independent activation of AR splice variants, suggesting the PTEN/FOXO1 pathway as a potential therapeutic target for inhibition of aberrant AR activation and castration-resistant PCa growth.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais/fisiologia , Processamento Alternativo , Linhagem Celular Tumoral , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Humanos , Masculino , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgênicos/genética , Transcrição Gênica/fisiologia , Ativação TranscricionalRESUMO
The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.
Assuntos
Anemia Falciforme/terapia , Endonucleases/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Anemia Falciforme/genética , Células Cultivadas , Endonucleases/genética , Marcação de Genes/métodos , Terapia Genética/métodos , Humanos , Cariotipagem , Dedos de Zinco/genética , Dedos de Zinco/fisiologia , Globinas beta/genética , Globinas beta/metabolismoRESUMO
Genome-wide studies have defined cell type-specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.