RESUMO
ABSTRACT: Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL), but pan-Notch inhibitors showed excessive toxicity in clinical trials. To find alternative ways to target Notch signals, we investigated cell division cycle 73 (Cdc73), which is a Notch cofactor and key component of the RNA polymerase-associated transcriptional machinery, an emerging target in T-ALL. Although we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, chromatin and nascent gene expression profiling showed that Cdc73 intersects with Ets1 and Notch at chromatin within enhancers to activate expression of known T-ALL oncogenes through its enhancer functions. Cdc73 also intersects with these factors within promoters to activate transcription of genes that are important for DNA repair and oxidative phosphorylation through its gene body functions. Consistently, Cdc73 deletion induced DNA damage and apoptosis and impaired mitochondrial function. The CDC73-induced DNA repair expression program co-opted by NOTCH1 is more highly expressed in T-ALL than in any other cancer. These data suggest that Cdc73 might induce a gene expression program that was eventually intersected and hijacked by oncogenic Notch to augment proliferation and mitigate the genotoxic and metabolic stresses of elevated Notch signaling. Our report supports studying factors such as CDC73 that intersect with Notch to derive a basic scientific understanding on how to combat Notch-dependent cancers without directly targeting the Notch complex.
Assuntos
5'-Nucleotidase , Leucemia de Células T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Camundongos , Linhagem Celular Tumoral , Cromatina , Dano ao DNA/genética , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Fatores de Transcrição/genética , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismoRESUMO
PIWI-interacting RNAs (piRNAs) engage PIWI proteins to silence transposons and promote germ cell development in animals. In diverse species, piRNA biogenesis occurs near the mitochondrial surface, and involves mitochondrial membrane-anchored factors. In mice, two cytoplasmic PIWI proteins, MIWI and MILI, receive processed pachytene piRNAs at intermitochodrial cement (IMC). However, how MIWI and MILI are initially recruited to the IMC to engage multiple steps of piRNA processing is unclear. Here, we show that mitochondria-anchored TDRKH controls multiple steps of pachytene piRNA biogenesis in mice. TDRKH specifically recruits MIWI, but not MILI, to engage the piRNA pathway. It is required for the production of the entire MIWI-bound piRNA population and enables trimming of MILI-bound piRNAs. The failure to recruit MIWI to the IMC with TDRKH deficiency results in loss of MIWI in the chromatoid body, leading to spermiogenic arrest and piRNA-independent retrotransposon LINE1 de-repression in round spermatids. Our findings identify a mitochondrial surface-based scaffolding mechanism separating the entry and actions of two critical PIWI proteins in the same piRNA pathway to drive piRNA biogenesis and germ cell development.
Assuntos
Proteínas Argonautas/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Animais , Masculino , Camundongos , Miose/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Estágio Paquíteno/genética , Retroelementos/genética , Espermatogênese/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismoRESUMO
Ring finger protein 216 (RNF216) belongs to the RING family of E3 ubiquitin ligases that are involved in cellular protein degradation. Mutations in human Rnf216 gene have been identified in Gordon Holmes syndrome, which is defined by ataxia, dementia, and hypogonadotropism. However, the gene function of Rnf216 in mammalian species remains unknown. Here, we show that targeted deletion of Rnf216 in mice results in disruption in spermatogenesis and male infertility. RNF216 is not required for female fertility. These findings reveal an essential function of RNF216 in spermatogenesis and male fertility and suggest a critical role for RNF216 in human gonadal development.
Assuntos
Infertilidade Masculina/genética , Espermatogênese/genética , Ubiquitina-Proteína Ligases/fisiologia , Animais , Fertilidade/genética , Humanos , Hipogonadismo/genética , Hipogonadismo/patologia , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Ubiquitina-Proteína Ligases/genéticaRESUMO
The cytoplasmic droplet is a conserved dilated area of cytoplasm situated at the neck of the sperm flagellum. Viewed as residual cytoplasm inherited from late spermatids, the cytoplasmic droplet contains numerous saccular elements as its key content. However, the origin of these saccules and the function of the cytoplasmic droplet have long been speculative. Here, we identify the molecular origin of these cytoplasmic droplet components by uncovering a vesicle pathway essential for formation and sequestration of saccules within the cytoplasmic droplet. This process is governed by a transmembrane protein SYPL1 and its interaction with VAMP3. Genetic ablation of SYPL1 in mice reveals that SYPL1 dictates the formation and accumulation of saccular elements in the forming cytoplasmic droplet. Derived from the Golgi, SYPL1 vesicles are critical for segregation of key metabolic enzymes within the forming cytoplasmic droplet of late spermatids and epididymal sperm, which are required for sperm development and male fertility. Our results uncover a mechanism to actively form and segregate saccules within the cytoplasmic droplet to promote sperm fertility.
Assuntos
Sêmen , Espermatozoides , Animais , Masculino , Camundongos , Vesícula , Citoplasma , Citosol , FertilidadeRESUMO
Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL) but pan-Notch inhibitors were toxic in clinical trials. To find alternative ways to target Notch signals, we investigated Cell division cycle 73 (Cdc73), which is a Notch cofactor and component of transcriptional machinery, a potential target in T-ALL. While we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the lymphoid transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, Cdc73, Ets1, and Notch intersect chromatin at promoters and enhancers to activate oncogenes and genes that are important for DNA repair and oxidative phosphorylation. Consistently, Cdc73 deletion in T-ALL cells induced DNA damage and impaired mitochondrial function. Our data suggests that Cdc73 might promote a gene expression program that was eventually intersected by Notch to mitigate the genotoxic and metabolic stresses of elevated Notch signaling. We also provide mechanistic support for testing inhibitors of DNA repair, oxidative phosphorylation, and transcriptional machinery. Inhibiting pathways like Cdc73 that intersect with Notch at chromatin might constitute a strategy to weaken Notch signals without directly targeting the Notch complex.
RESUMO
Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Leucemia de Células T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Proteína Proto-Oncogênica c-ets-1 , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinogênese/efeitos dos fármacos , Leucemia de Células T/tratamento farmacológico , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Proteína Proto-Oncogênica c-ets-1/antagonistas & inibidores , Receptor Notch1/antagonistas & inibidores , Transdução de Sinais/fisiologiaRESUMO
Pachytene piRNAs are the most abundant piRNAs in mammalian adult testes. They are generated from long precursor transcripts by the primary piRNA biogenesis pathway but the factors involved in pachytene piRNA precursors processing are poorly understood. Here we show that the Tudor domain-containing 5 (TDRD5) protein is essential for pachytene piRNA biogenesis in mice. Conditional inactivation of TDRD5 in mouse postnatal germ cells reveals that TDRD5 selectively regulates the production of pachytene piRNAs from abundant piRNA-producing precursors, with little effect on low-abundant piRNAs. Unexpectedly, TDRD5 is not required for the 5' end processing of the precursors, but is crucial for promoting production of piRNAs from the other regions of the transcript. Furthermore, we show that TDRD5 is an RNA-binding protein directly associating with piRNA precursors. These observations establish TDRD5 as a piRNA biogenesis factor and reveal two genetically separable steps at the start of pachytene piRNA processing.