RESUMO
Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.
Assuntos
Aptâmeros de Nucleotídeos , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Inativação de Genes , Linfócitos T , Humanos , Edição de Genes/métodos , Aptâmeros de Nucleotídeos/genética , Linfócitos T/metabolismo , Linfócitos T/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Técnicas de Introdução de Genes/métodos , TransgenesRESUMO
WNT signalling has multiple roles. It maintains pluripotency of embryonic stem cells, assigns posterior identity in the epiblast and induces mesodermal tissue. Here we provide evidence that these distinct functions are conducted by the transcription factor SOX2, which adopts different modes of chromatin interaction and regulatory element selection depending on its level of expression. At high levels, SOX2 displaces nucleosomes from regulatory elements with high-affinity SOX2 binding sites, recruiting the WNT effector TCF/ß-catenin and maintaining pluripotent gene expression. Reducing SOX2 levels destabilizes pluripotency and reconfigures SOX2/TCF/ß-catenin occupancy to caudal epiblast expressed genes. These contain low-affinity SOX2 sites and are co-occupied by T/Bra and CDX. The loss of SOX2 allows WNT-induced mesodermal differentiation. These findings define a role for Sox2 levels in dictating the chromatin occupancy of TCF/ß-catenin and reveal how context-specific responses to a signal are configured by the level of a transcription factor.
Assuntos
Cromatina , beta Catenina , Animais , Mesoderma/metabolismo , Fatores de Transcrição , Vertebrados/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Fate decisions in developing tissues involve cells transitioning between discrete cell states, each defined by distinct gene expression profiles. The Waddington landscape, in which the development of a cell is viewed as a ball rolling through a valley filled terrain, is an appealing way to describe differentiation. To construct and validate accurate landscapes, quantitative methods based on experimental data are necessary. We combined principled statistical methods with a framework based on catastrophe theory and approximate Bayesian computation to formulate a quantitative dynamical landscape that accurately predicts cell fate outcomes of pluripotent stem cells exposed to different combinations of signaling factors. Analysis of the landscape revealed two distinct ways in which cells make a binary choice between one of two fates. We suggest that these represent archetypal designs for developmental decisions. The approach is broadly applicable for the quantitative analysis of differentiation and for determining the logic of developmental decisions.
Assuntos
Teorema de Bayes , Diferenciação CelularRESUMO
Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.
Assuntos
Cromossomos de Mamíferos/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/deficiência , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Sobrevivência Celular , Cromossomos de Mamíferos/genética , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
Regulation of Smoothened by PTCH1 is central to Hedgehog signal transduction. Reporting recently in PNAS, Myers et al. (2017) provide evidence that a transmembrane flux of sodium ions drives PTCH1 activity and that cholesterol regulates Smoothened via its transmembrane domain.
Assuntos
Proteínas Hedgehog , Esteróis , Colesterol , Receptores Acoplados a Proteínas G , Transdução de Sinais , Receptor Smoothened , SódioRESUMO
Signaling pathways direct organogenesis, often through concentration-dependent effects on cells. The hedgehog pathway enables cells to sense and respond to hedgehog ligands, of which the best studied is sonic hedgehog. Hedgehog signaling is essential for development, proliferation, and stem cell maintenance, and it is a driver of certain cancers. Lipid metabolism has a profound influence on both hedgehog signal transduction and the properties of the ligands themselves, leading to changes in the strength of hedgehog signaling and cellular functions. Here we review the evolving understanding of the relationship between lipids and hedgehog signaling.
Assuntos
Proteínas Hedgehog/fisiologia , Metabolismo dos Lipídeos , Transdução de SinaisRESUMO
Smith-Lemli-Opitz syndrome (SLOS) is a common autosomal-recessive disorder that results from mutations in the gene encoding the cholesterol biosynthetic enzyme 7-dehydrocholesterol reductase (DHCR7). Impaired DHCR7 function is associated with a spectrum of congenital malformations, intellectual impairment, epileptiform activity and autism spectrum disorder. Biochemically, there is a deficit in cholesterol and an accumulation of its metabolic precursor 7-dehydrocholesterol (7DHC) in developing tissues. Morphological abnormalities in SLOS resemble those seen in congenital Sonic Hedgehog (SHH)-deficient conditions, leading to the proposal that the pathogenesis of SLOS is mediated by aberrant SHH signalling. SHH signalling is transduced through the transmembrane protein Smoothened (SMO), which localizes to the primary cilium of a cell on activation and is both positively and negatively regulated by sterol molecules derived from cholesterol biosynthesis. One proposed mechanism of SLOS involves SMO dysregulation by altered sterol levels, but the salient sterol species has not been identified. Here, we clarify the relationship between disrupted cholesterol metabolism and reduced SHH signalling in SLOS by modelling the disorder in vitro. Our results indicate that a deficit in cholesterol, as opposed to an accumulation of 7DHC, impairs SMO activation and its localization to the primary cilium.
Assuntos
Colesterol/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Receptor Smoothened/metabolismo , Animais , Células Cultivadas , Desidrocolesteróis/metabolismo , Humanos , Camundongos , Mutação , Células NIH 3T3 , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fenótipo , Síndrome de Smith-Lemli-Opitz/genética , Receptor Smoothened/genéticaRESUMO
In the vertebrate neural tube, the morphogen Sonic Hedgehog (Shh) establishes a characteristic pattern of gene expression. Here we quantify the Shh gradient in the developing mouse neural tube and show that while the amplitude of the gradient increases over time, the activity of the pathway transcriptional effectors, Gli proteins, initially increases but later decreases. Computational analysis of the pathway suggests three mechanisms that could contribute to this adaptation: transcriptional upregulation of the inhibitory receptor Ptch1, transcriptional downregulation of Gli and the differential stability of active and inactive Gli isoforms. Consistent with this, Gli2 protein expression is downregulated during neural tube patterning and adaptation continues when the pathway is stimulated downstream of Ptch1. Moreover, the Shh-induced upregulation of Gli2 transcription prevents Gli activity levels from adapting in a different cell type, NIH3T3 fibroblasts, despite the upregulation of Ptch1. Multiple mechanisms therefore contribute to the intracellular dynamics of Shh signalling, resulting in different signalling dynamics in different cell types.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Fatores de Transcrição Kruppel-Like/genética , Tubo Neural/embriologia , Receptores de Superfície Celular/genética , Animais , Regulação para Baixo , Embrião de Mamíferos , Proteínas de Fluorescência Verde , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Tubo Neural/metabolismo , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Regulação para Cima , Proteína GLI1 em Dedos de ZincoRESUMO
The spatial organization of cell fates in developing tissues often involves the control of transcriptional networks by morphogen gradients. A well-studied example of this is the Sonic-hedgehog (Shh) controlled pattern of neuronal subtype differentiation in the vertebrate neural tube. Here we discuss recent studies involving genome wide analyses, functional experiments and theoretical models that have begun to characterise the molecular logic by which neural cells interpret Shh signalling. The view that emerges from this work is that cell identity results from the combined input of Shh signalling, uniformly expressed neural factors and the cross-regulatory network of downstream Shh target genes. A similar logic is also likely to underpin the patterning of many developing tissues.
Assuntos
Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Tubo Neural/crescimento & desenvolvimento , Animais , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Camundongos , Neurônios/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Recent advances in a number of systems suggest many genes involved in orchestrating regeneration are redeployed from similar processes in development, with others being novel to the regeneration process in particular lineages. Of particular importance will be understanding the architecture of regenerative genetic regulatory networks and whether they are conserved across broad phylogenetic distances. Here, we describe the role of the conserved TALE class protein PBX/Extradenticle in planarians, a representative member of the Lophotrocozoa. PBX/Extradenticle proteins play central roles in both embryonic and post-embryonic developmental patterning in both vertebrates and insects, and we demonstrate a broad requirement during planarian regeneration. We observe that Smed-pbx has pleiotropic functions during regeneration, with a primary role in patterning the anterior-posterior (AP) axis and AP polarity. Smed-pbx is required for expression of polarity determinants notum and wnt1 and for correct patterning of the structures polarized along the AP axis, such as the brain, pharynx and gut. Overall, our data suggest that Smed-pbx functions as a central integrator of positional information to drive patterning of regeneration along the body axis.
Assuntos
Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Proteínas de Homeodomínio/fisiologia , Planárias/fisiologia , Regeneração/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Hibridização In Situ , Microscopia de Fluorescência , Dados de Sequência Molecular , Faringe/cirurgia , Interferência de RNA , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
The GPI-anchor is an established determinant of molecular localisation and various functional roles have been attributed to it. The newt GPI-anchored three-finger protein (TFP) Prod1 is an important regulator of cell behaviour during limb regeneration, but it is unclear how it signals to the interior of the cell. Prod1 was expressed by transfection in cultured newt limb cells and activated transcription and expression of matrix metalloproteinase 9 (MMP9) by a pathway involving ligand-independent activation of epidermal growth factor receptor (EGFR) signalling and phosphorylation of extracellular regulated kinase 1 and 2 (ERK1/2). This was dependent on the presence of the GPI-anchor and critical residues in the α-helical region of the protein. Interestingly, Prod1 in the axolotl, a salamander species that also regenerates its limbs, was shown to activate ERK1/2 signalling and MMP9 transcription despite being anchorless, and both newt and axolotl Prod1 co-immunoprecipitated with the newt EGFR after transfection. The substitution of the axolotl helical region activated a secreted, anchorless version of the newt molecule. The activity of the newt molecule cannot therefore depend on a unique property conferred by the anchor. Prod1 is a salamander-specific TFP and its interaction with the phylogenetically conserved EGFR has implications for our view of regeneration as an evolutionary variable.