RESUMO
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
Assuntos
Processamento de Proteína Pós-Traducional , Fosforilação , Humanos , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Transdução de Sinais , Células HEK293 , Proteômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Linfócitos T/metabolismo , Células Jurkat , NF-kappa B/metabolismoRESUMO
Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes, but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a method to profile Th cell identity using Cas9-targeted single-molecule nanopore sequencing. Targeting as few as 10 selected genomic loci, we were able to distinguish major in vitro polarized murine T cell subtypes, as well as intermediate phenotypes, by their native DNA 5mCpG patterns. Moreover, by using off-target sequences, we were able to infer transcription factor activities relevant to each cell subtype. Detection of 5mCpG and 5hmCpG was validated on intestinal Th17 cells escaping transforming growth factor ß control, using single-molecule adaptive sampling. A total of 21 differentially methylated regions mapping to the 10-gene panel were identified in pathogenic Th17 cells relative to their nonpathogenic counterpart. Hence, our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological transition states of Th cells.
Assuntos
Metilação de DNA , Epigênese Genética , Animais , Camundongos , Citosina , DNA/metabolismo , Células Th17/metabolismoRESUMO
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
RESUMO
Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways and intracellular metabolites. Here we show that cell-permeable α-ketoglutarate (αKG) alters the DNA methylation profile of naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating FoxP3+ Treg differentiation and increasing inflammatory cytokines. Adoptive transfer of these T cells into tumor-bearing mice results in enhanced tumor infiltration, decreased FoxP3 expression, and delayed tumor growth. Mechanistically, αKG leads to an energetic state that is reprogrammed toward a mitochondrial metabolism, with increased oxidative phosphorylation and expression of mitochondrial complex enzymes. Furthermore, carbons from ectopic αKG are directly utilized in the generation of fatty acids, associated with lipidome remodeling and increased triacylglyceride stores. Notably, inhibition of either mitochondrial complex II or DGAT2-mediated triacylglyceride synthesis restores Treg differentiation and decreases the αKG-induced inflammatory phenotype. Thus, we identify a crosstalk between αKG, mitochondrial metabolism and triacylglyceride synthesis that controls Treg fate.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ácidos Cetoglutáricos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Fibrossarcoma/genética , Fibrossarcoma/imunologia , Fibrossarcoma/metabolismo , Fibrossarcoma/terapia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Humanos , Imunoterapia Adotiva , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fenótipo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplante , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismoRESUMO
In this issue of Molecular Cell, Byun et al. (2020) find that the dual targeting of glutamine metabolism and the PD-L1 checkpoint inhibitor augments anti-tumor immunity. Mechanistically, decreased glutamine availability attenuated S-glutathionylation of SERCA, resulting in an increase in cytosolic calcium, enhanced NF-κB activity, and upregulation of programmed death-ligand 1.
Assuntos
Antígeno B7-H1 , Neoplasias , Antígeno B7-H1/genética , Glutamina , Humanos , NF-kappa B/genética , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
The polyphenol resveratrol activates the deacetylase Sirt1, resulting in various antioxidant, chemoprotectant, neuroprotective, cardioprotective, and anti-inflammatory properties. We found that at high concentrations of resveratrol, human CD4+ T cells showed defective antigen receptor signaling and arrest at the G1 stage of the cell cycle, whereas at low concentrations, cells were readily activated and exhibited enhanced Sirt1 deacetylase activity. Nevertheless, low-dose resveratrol rapidly stimulated genotoxic stress in the T cells, which resulted in engagement of a DNA damage response pathway that depended on the kinase ATR [ataxia telangiectasia-mutated (ATM) and Rad3-related], but not ATM, and subsequently in premitotic cell cycle arrest. The concomitant activation of p53 was coupled to the expression of gene products that regulate cell metabolism, leading to a metabolic reprogramming that was characterized by decreased glycolysis, increased glutamine consumption, and a shift to oxidative phosphorylation. These alterations in the bioenergetic homeostasis of CD4+ T cells resulted in enhanced effector function, with both naïve and memory CD4+ T cells secreting increased amounts of the inflammatory cytokine interferon-γ. Thus, our data highlight the wide range of metabolic adaptations that CD4+ T lymphocytes undergo in response to genomic stress.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Dano ao DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Adulto , Antioxidantes/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Perfilação da Expressão Gênica/métodos , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Fosforilação Oxidativa/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Resveratrol , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
T cell activation requires that the cell meet increased energetic and biosynthetic demands. We showed that exogenous nutrient availability regulated the differentiation of naïve CD4(+) T cells into distinct subsets. Activation of naïve CD4(+) T cells under conditions of glutamine deprivation resulted in their differentiation into Foxp3(+) (forkhead box P3-positive) regulatory T (Treg) cells, which had suppressor function in vivo. Moreover, glutamine-deprived CD4(+) T cells that were activated in the presence of cytokines that normally induce the generation of T helper 1 (TH1) cells instead differentiated into Foxp3(+) Treg cells. We found that α-ketoglutarate (αKG), the glutamine-derived metabolite that enters into the mitochondrial citric acid cycle, acted as a metabolic regulator of CD4(+) T cell differentiation. Activation of glutamine-deprived naïve CD4(+) T cells in the presence of a cell-permeable αKG analog increased the expression of the gene encoding the TH1 cell-associated transcription factor Tbet and resulted in their differentiation into TH1 cells, concomitant with stimulation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Together, these data suggest that a decrease in the intracellular amount of αKG, caused by the limited availability of extracellular glutamine, shifts the balance between the generation of TH1 and Treg cells toward that of a Treg phenotype.