RESUMO
Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.
Assuntos
Fatores de Transcrição , Animais , Humanos , Camundongos , Regulação da Expressão Gênica , Mutação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Linhagem CelularRESUMO
Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Interferon Tipo I/metabolismo , Neoplasias Pulmonares/imunologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação/genética , Proteínas Repressoras/metabolismo , Evasão Tumoral/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunidade Inata/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Group 1 innate lymphoid cells (ILC1s) are cytotoxic and interferon gamma-producing lymphocytes lacking antigen-specific receptors, which include ILC1s and natural killer (NK) cells. In mice, ILC1s differ from NK cells, as they develop independently of the NK-specifying transcription factor EOMES, while requiring the repressor ZFP683 (ZNF683 in humans) for tissue residency. Here we identify highly variable ILC1 subtypes across tissues through investigation of human ILC1 diversity by single-cell RNA sequencing and flow cytometry. The intestinal epithelium contained abundant mature EOMES- ILC1s expressing PRDM1 rather than ZNF683, alongside a few immature TCF7+PRDM1- ILC1s. Other tissues harbored NK cells expressing ZNF683 and EOMES transcripts; however, EOMES protein content was variable. These ZNF683+ NK cells are tissue-imprinted NK cells phenotypically resembling ILC1s. The tissue ILC1-NK spectrum also encompassed conventional NK cells and NK cells distinguished by PTGDS expression. These findings establish a foundation for evaluating phenotypic and functional changes within the NK-ILC1 spectrum in diseases.
Assuntos
Imunidade Inata , Células Matadoras Naturais , Linfócitos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas com Domínio T , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Linfócitos/imunologia , Linfócitos/metabolismo , Análise de Célula Única , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Animais , Camundongos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genéticaRESUMO
Behavioral plasticity is key to animal survival. Harpegnathos saltator ants can switch between worker and queen-like status (gamergate) depending on the outcome of social conflicts, providing an opportunity to study how distinct behavioral states are achieved in adult brains. Using social and molecular manipulations in live ants and ant neuronal cultures, we show that ecdysone and juvenile hormone drive molecular and functional differences in the brains of workers and gamergates and direct the transcriptional repressor Kr-h1 to different target genes. Depletion of Kr-h1 in the brain caused de-repression of "socially inappropriate" genes: gamergate genes were upregulated in workers, whereas worker genes were upregulated in gamergates. At the phenotypic level, loss of Kr-h1 resulted in the emergence of worker-specific behaviors in gamergates and gamergate-specific traits in workers. We conclude that Kr-h1 is a transcription factor that maintains distinct brain states established in response to socially regulated hormones.
Assuntos
Formigas/genética , Ecdisterona/farmacologia , Hierarquia Social , Proteínas de Insetos/metabolismo , Neurônios/metabolismo , Sesquiterpenos/farmacologia , Comportamento Social , Transcriptoma/genética , Animais , Formigas/efeitos dos fármacos , Formigas/fisiologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Neurônios/efeitos dos fármacos , Fenótipo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
The striosome compartment within the dorsal striatum has been implicated in reinforcement learning and regulation of motivation, but how striosomal neurons contribute to these functions remains elusive. Here, we show that a genetically identified striosomal population, which expresses the Teashirt family zinc finger 1 (Tshz1) and belongs to the direct pathway, drives negative reinforcement and is essential for aversive learning in mice. Contrasting a "conventional" striosomal direct pathway, the Tshz1 neurons cause aversion, movement suppression, and negative reinforcement once activated, and they receive a distinct set of synaptic inputs. These neurons are predominantly excited by punishment rather than reward and represent the anticipation of punishment or the motivation for avoidance. Furthermore, inhibiting these neurons impairs punishment-based learning without affecting reward learning or movement. These results establish a major role of striosomal neurons in behaviors reinforced by punishment and moreover uncover functions of the direct pathway unaccounted for in classic models.
Assuntos
Aprendizagem da Esquiva/fisiologia , Corpo Estriado/fisiologia , Proteínas de Homeodomínio/genética , Proteínas Repressoras/genética , Animais , Gânglios da Base , Feminino , Proteínas de Homeodomínio/metabolismo , Aprendizagem/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motivação , Neurônios/fisiologia , Punição , Reforço Psicológico , Proteínas Repressoras/metabolismoRESUMO
Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.
Assuntos
Ensaios de Triagem em Larga Escala , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sistemas CRISPR-Cas/genética , Feminino , Inativação Gênica , Genes Reporter , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células K562 , Lentivirus/fisiologia , Anotação de Sequência Molecular , Mutação/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Transcrição Gênica , Dedos de ZincoRESUMO
To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento/genética , Córtex Pré-Frontal/embriologia , Telencéfalo/embriologia , Animais , Transtorno Autístico/genética , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Eucromatina/genética , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Ontologia Genética , Predisposição Genética para Doença , Idade Gestacional , Humanos , Camundongos , Camundongos Transgênicos , Motivos de Nucleotídeos , Mutação Puntual , Córtex Pré-Frontal/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise Espaço-Temporal , Telencéfalo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.
Assuntos
Arabidopsis/química , Arabidopsis/citologia , Polaridade Celular/fisiologia , Células Vegetais/fisiologia , Polimerização , Domínios Proteicos , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteína Axina/química , Proteína Axina/metabolismo , Bryopsida/química , Bryopsida/citologia , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Células COS , Chlorocebus aethiops , Proteínas Desgrenhadas/metabolismo , Células HEK293 , Humanos , Marchantia/química , Marchantia/citologia , Marchantia/genética , Marchantia/crescimento & desenvolvimento , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Plantas Geneticamente Modificadas , Proteínas Repressoras/metabolismo , Via de Sinalização WntRESUMO
Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.
Assuntos
Nervo Acessório/fisiologia , Folículo Piloso/citologia , Cabelo/crescimento & desenvolvimento , Proteínas Hedgehog/metabolismo , Norepinefrina/metabolismo , Transdução de Sinais/genética , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Nervo Acessório/citologia , Animais , Ciclo Celular/genética , Temperatura Baixa , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Cabelo/citologia , Cabelo/fisiologia , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piloereção , RNA-Seq , Receptores Adrenérgicos beta 2/deficiência , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Nicho de Células-Tronco , Células-Tronco/citologia , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/fisiologia , Sinapses/fisiologiaRESUMO
The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.
Assuntos
Memória Imunológica/fisiologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas Nucleares/genética , Células Precursoras de Linfócitos B/imunologia , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Cromatina/química , Cromatina/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Histona Desacetilases/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Correpressor 2 de Receptor Nuclear/química , Correpressor 2 de Receptor Nuclear/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
Metabolic conditions affect the developmental tempo of animals. Developmental gene regulatory networks (GRNs) must therefore synchronize their dynamics with a variable timescale. We find that layered repression of genes couples GRN output with variable metabolism. When repressors of transcription or mRNA and protein stability are lost, fewer errors in Drosophila development occur when metabolism is lowered. We demonstrate the universality of this phenomenon by eliminating the entire microRNA family of repressors and find that development to maturity can be largely rescued when metabolism is reduced. Using a mathematical model that replicates GRN dynamics, we find that lowering metabolism suppresses the emergence of developmental errors by curtailing the influence of auxiliary repressors on GRN output. We experimentally show that gene expression dynamics are less affected by loss of repressors when metabolism is reduced. Thus, layered repression provides robustness through error suppression and may provide an evolutionary route to a shorter reproductive cycle.
Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Olho/citologia , Feminino , Insulina/metabolismo , Mutação com Perda de Função , MicroRNAs/metabolismo , Modelos Teóricos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.
Assuntos
Período de Replicação do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina , DNA/genética , Período de Replicação do DNA/genética , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos/genética , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Proteínas Repressoras/metabolismo , Análise Espaço-TemporalRESUMO
The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.
Assuntos
Neoplasias da Mama/genética , Metástase Neoplásica/genética , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteína Homeobox Nanog/metabolismo , Metástase Neoplásica/fisiopatologia , Células Neoplásicas Circulantes/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.
Assuntos
Autoimunidade , Citotoxicidade Imunológica , Imunoterapia Adotiva , Melanoma Experimental/terapia , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Neoplasias Cutâneas/terapia , Linfócitos T/transplante , Ubiquitina-Proteína Ligases/metabolismo , Animais , Feminino , Células HEK293 , Células HeLa , Humanos , Imunidade Humoral , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fenótipo , Ligação Proteica , Proteínas Repressoras/genética , Ribonucleases/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral , Ubiquitina-Proteína Ligases/genéticaRESUMO
Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Imunidade Inata , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Timócitos/metabolismo , Timo/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Técnicas de Cultura de Órgãos , Fenótipo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Timócitos/imunologia , Timo/embriologia , Timo/imunologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.
Assuntos
Surdez/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Processamento Alternativo/genética , Animais , Linhagem Celular , Éxons , Regulação da Expressão Gênica/genética , Células HEK293 , Células Ciliadas Auditivas/fisiologia , Audição/genética , Audição/fisiologia , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Splicing de RNA/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição , Vorinostat/farmacologiaRESUMO
As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to â¼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.
Assuntos
Adenosina Trifosfatases , Cromossomos Bacterianos , Proteínas de Ligação a DNA , Escherichia coli K12 , Complexos Multiproteicos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Escherichia coli K12/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Estrutura Quaternária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Repressoras/metabolismo , Elongação da Transcrição Genética/efeitos dos fármacos , Fatores de Elongação da Transcrição/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Drosophila , Feminino , Células HCT116 , Células HEK293 , Resposta ao Choque Térmico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase II/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Using integrative genomic analysis of 360 metastatic castration-resistant prostate cancer (mCRPC) samples, we identified a novel subtype of prostate cancer typified by biallelic loss of CDK12 that is mutually exclusive with tumors driven by DNA repair deficiency, ETS fusions, and SPOP mutations. CDK12 loss is enriched in mCRPC relative to clinically localized disease and characterized by focal tandem duplications (FTDs) that lead to increased gene fusions and marked differential gene expression. FTDs associated with CDK12 loss result in highly recurrent gains at loci of genes involved in the cell cycle and DNA replication. CDK12 mutant cases are baseline diploid and do not exhibit DNA mutational signatures linked to defects in homologous recombination. CDK12 mutant cases are associated with elevated neoantigen burden ensuing from fusion-induced chimeric open reading frames and increased tumor T cell infiltration/clonal expansion. CDK12 inactivation thereby defines a distinct class of mCRPC that may benefit from immune checkpoint immunotherapy.