RESUMO
Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.
Assuntos
Ferroptose , Células-Tronco Hematopoéticas , Humanos , Endopeptidases/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.
Assuntos
Proteínas de Ligação a RNA , Transativadores , Proteínas de Transporte/metabolismo , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Células HeLa , Células HEK293 , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismoRESUMO
Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of â¼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.
Assuntos
Córtex Auditivo/fisiologia , Síndrome de Williams/fisiopatologia , Animais , Córtex Auditivo/citologia , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Fenótipo , Transativadores/genética , Síndrome de Williams/genéticaRESUMO
Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Transativadores , Fatores de Transcrição , Proteínas Supressoras de Tumor , Proteínas Proto-Oncogênicas/metabolismo , Animais , Transativadores/metabolismo , Transativadores/genética , Camundongos , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Camundongos Endogâmicos C57BL , Proteínas Cromossômicas não Histona/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Camundongos Knockout , Montagem e Desmontagem da Cromatina , Diferenciação Celular/imunologiaRESUMO
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Assuntos
Proteína Fosfatase 2/metabolismo , Apoptose , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ativadores de Enzimas/metabolismo , Fase G1 , Humanos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Fenotiazinas/farmacologia , Fosforilação , Proteína Fosfatase 2/fisiologia , Subunidades Proteicas/metabolismo , Transativadores/efeitos dos fármacos , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.
Assuntos
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Antígenos CD19/genética , Antígenos CD19/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Humanos , Imunoterapia Adotiva/efeitos adversos , Glicoproteínas de Membrana/metabolismo , Poliadenilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Transativadores/metabolismoRESUMO
Understanding transcription factor navigation through the nucleus remains critical for developing targeted therapeutics. The GLI1 transcription factor must maintain maximal Hedgehog pathway output in basal cell carcinomas (BCCs), and we have previously shown that resistant BCCs increase GLI1 deacetylation through atypical protein kinase Cι/λ (aPKC) and HDAC1. Here we identify a lamina-associated polypeptide 2 (LAP2) isoform-dependent nuclear chaperoning system that regulates GLI1 movement between the nuclear lamina and nucleoplasm to achieve maximal activation. LAP2ß forms a two-site interaction with the GLI1 zinc-finger domain and acetylation site, stabilizing an acetylation-dependent reserve on the inner nuclear membrane (INM). By contrast, the nucleoplasmic LAP2α competes with LAP2ß for GLI1 while scaffolding HDAC1 to deacetylate the secondary binding site. aPKC functions to promote GLI1 association with LAP2α, promoting egress off the INM. GLI1 intranuclear trafficking by LAP2 isoforms represents a powerful signal amplifier in BCCs with implications for zinc finger-based signal transduction and therapeutics.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Células 3T3 , Animais , Carcinoma Basocelular/metabolismo , Linhagem Celular , Cromatina , Proteínas de Ligação a DNA/fisiologia , Células HEK293 , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/fisiologia , Histona Desacetilase 1/metabolismo , Humanos , Proteínas de Membrana/fisiologia , Camundongos , Chaperonas Moleculares/metabolismo , Lâmina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco/fisiologia , Dedos de ZincoRESUMO
Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.
Assuntos
Citrobacter rodentium , Infecções por Enterobacteriaceae , Glicólise , Proteínas HMGB , Imunidade Inata , Linfócitos , Camundongos Knockout , Animais , Camundongos , Adaptação Fisiológica/imunologia , Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Hexoquinase/metabolismo , Hexoquinase/genética , Interleucina-17/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Transativadores/metabolismo , Transativadores/genética , Proteínas HMGB/genética , Proteínas HMGB/imunologia , Proteínas HMGB/metabolismoRESUMO
NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Óperon de RNAr/genéticaRESUMO
Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of â¼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.
Assuntos
Variação Genética , Macrófagos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células da Medula Óssea/citologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Análise por Conglomerados , Elementos Facilitadores Genéticos/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.
Assuntos
Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Switching de Imunoglobulina/efeitos dos fármacos , Proteínas Mad2/antagonistas & inibidores , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a Telômeros/antagonistas & inibidores , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Transativadores/deficiência , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Cromatina/genética , Feminino , Hematopoese/genética , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Célula Única , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/fisiologia , Transativadores/genética , TranscriptomaRESUMO
Neutrophils are essential first-line defense cells against invading pathogens, yet when inappropriately activated, their strong immune response can cause collateral tissue damage and contributes to immunological diseases. However, whether neutrophils can intrinsically titrate their immune response remains unknown. Here we conditionally deleted the Spi1 gene, which encodes the myeloid transcription factor PU.1, from neutrophils of mice undergoing fungal infection and then performed comprehensive epigenomic profiling. We found that as well as providing the transcriptional prerequisite for eradicating pathogens, the predominant function of PU.1 was to restrain the neutrophil defense by broadly inhibiting the accessibility of enhancers via the recruitment of histone deacetylase 1. Such epigenetic modifications impeded the immunostimulatory AP-1 transcription factor JUNB from entering chromatin and activating its targets. Thus, neutrophils rely on a PU.1-installed inhibitor program to safeguard their epigenome from undergoing uncontrolled activation, protecting the host against an exorbitant innate immune response.
Assuntos
Epigênese Genética/imunologia , Epigenômica/métodos , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Transativadores/imunologia , Animais , Candida albicans/imunologia , Candida albicans/fisiologia , Candidíase/genética , Candidíase/imunologia , Candidíase/microbiologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Análise de Sobrevida , Transativadores/deficiência , Transativadores/genética , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
Deleterious somatic mutations in DNA methyltransferase 3 alpha (DNMT3A) and TET mehtylcytosine dioxygenase 2 (TET2) are associated with clonal expansion of hematopoietic cells and higher risk of cardiovascular disease (CVD). Here, we investigated roles of DNMT3A and TET2 in normal human monocyte-derived macrophages (MDM), in MDM isolated from individuals with DNMT3A or TET2 mutations, and in macrophages isolated from human atherosclerotic plaques. We found that loss of function of DNMT3A or TET2 resulted in a type I interferon response due to impaired mitochondrial DNA integrity and activation of cGAS signaling. DNMT3A and TET2 normally maintained mitochondrial DNA integrity by regulating the expression of transcription factor A mitochondria (TFAM) dependent on their interactions with RBPJ and ZNF143 at regulatory regions of the TFAM gene. These findings suggest that targeting the cGAS-type I IFN pathway may have therapeutic value in reducing risk of CVD in patients with DNMT3A or TET2 mutations.
Assuntos
Doenças Cardiovasculares , DNA Metiltransferase 3A/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Humanos , Interferons/metabolismo , Macrófagos/metabolismo , Mitocôndrias/genética , Mutação/genética , Nucleotidiltransferases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/patologia , Metilação de DNA , Tumores Neuroectodérmicos/genética , Tumores Neuroectodérmicos/patologia , Sequência de Aminoácidos , Neoplasias do Sistema Nervoso Central/classificação , Neoplasias do Sistema Nervoso Central/diagnóstico , Criança , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Tumores Neuroectodérmicos/classificação , Tumores Neuroectodérmicos/diagnóstico , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transdução de Sinais , Transativadores , Proteínas Supressoras de Tumor/genéticaRESUMO
Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.
Assuntos
Edição de Genes , Transativadores , Camundongos , Animais , Transativadores/metabolismo , Citosina , Mutação , DNA Mitocondrial/genética , Sistemas CRISPR-CasRESUMO
While 19S proteasome regulatory particle (RP) inhibition is a promising new avenue for treating bortezomib-resistant myeloma, the anti-tumor impact of inhibiting 19S RP component PSMD14 could not be explained by a selective inhibition of proteasomal activity. Here, we report that PSMD14 interacts with NSD2 on chromatin, independent of 19S RP. Functionally, PSMD14 acts as a histone H2AK119 deubiquitinase, facilitating NSD2-directed H3K36 dimethylation. Integrative genomic and epigenomic analyses revealed the functional coordination of PSMD14 and NSD2 in transcriptional activation of target genes (e.g., RELA) linked to myelomagenesis. Reciprocally, RELA transactivates PSMD14, forming a PSMD14/NSD2-RELA positive feedback loop. Remarkably, PSMD14 inhibitors enhance bortezomib sensitivity and fosters anti-myeloma synergy. PSMD14 expression is elevated in myeloma and inversely correlated with overall survival. Our study uncovers an unappreciated function of PSMD14 as an epigenetic regulator and a myeloma driver, supporting the pursuit of PSMD14 as a therapeutic target to overcome the treatment limitation of myeloma.
Assuntos
Histonas , Mieloma Múltiplo , Humanos , Histonas/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Bortezomib/farmacologia , Bortezomib/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/metabolismo , Inibidores de Proteassoma/farmacologia , Transativadores/metabolismoRESUMO
Plasmacytoid dendritic cells (pDCs) are an immune subset devoted to the production of high amounts of type 1 interferons in response to viral infections. Whereas conventional dendritic cells (cDCs) originate mostly from a common dendritic cell progenitor (CDP), pDCs have been shown to develop from both CDPs and common lymphoid progenitors. Here, we found that pDCs developed predominantly from IL-7R+ lymphoid progenitor cells. Expression of SiglecH and Ly6D defined pDC lineage commitment along the lymphoid branch. Transcriptional characterization of SiglecH+Ly6D+ precursors indicated that pDC development requires high expression of the transcription factor IRF8, whereas pDC identity relies on TCF4. RNA sequencing of IL-7R+ lymphoid and CDP-derived pDCs mirrored the heterogeneity of mature pDCs observed in single-cell analysis. Both mature pDC subsets are able to secrete type 1 interferons, but only myeloid-derived pDCs share with cDCs their ability to process and present antigen.
Assuntos
Células Dendríticas/imunologia , Células-Tronco/imunologia , Animais , Linfócitos B/citologia , Linhagem da Célula , Células Cultivadas , Células Dendríticas/citologia , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Fatores Reguladores de Interferon/metabolismo , Lectinas/metabolismo , Masculino , Camundongos , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina-7/metabolismo , Transativadores/metabolismo , Transcrição GênicaRESUMO
Regulated protein degradation is essential. The timed destruction of crucial proteins by the ClpXP protease drives cell-cycle progression in the bacterium Caulobacter crescentus. Although ClpXP is active alone, additional factors are inexplicably required for cell-cycle-dependent proteolysis. Here, we show that these factors constitute an adaptor hierarchy wherein different substrates are destroyed based on the degree of adaptor assembly. The hierarchy builds upon priming of ClpXP by the adaptor CpdR, which promotes degradation of one class of substrates and also recruits the adaptor RcdA to degrade a second class of substrates. Adding the PopA adaptor promotes destruction of a third class of substrates and inhibits degradation of the second class. We dissect RcdA to generate bespoke adaptors, identifying critical substrate elements needed for RcdA recognition and uncovering additional cell-cycle-dependent ClpXP substrates. Our work reveals how hierarchical adaptors and primed proteases orchestrate regulated proteolysis during bacterial cell-cycle progression.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Proteólise , Motivos de Aminoácidos , Proteínas de Bactérias/química , Caulobacter crescentus/enzimologia , Proteínas de Ciclo Celular , Endopeptidase Clp/metabolismo , Transativadores/química , Transativadores/metabolismoRESUMO
Aging is a risk factor for chronic diseases, and identifying targets for intervention is a goal of the aging field. Burkewitz et al. now describe a mechanism that mediates the specific role for AMPK in longevity, whereby its activity in neurons modulates metabolism and mitochondrial integrity in peripheral tissues.