RESUMO
In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.
Assuntos
Metilação de DNA , RNA de Interação com Piwi , Masculino , Humanos , Animais , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas/metabolismo , Células Germinativas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Elementos de DNA Transponíveis/genética , Mamíferos/metabolismoRESUMO
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
Assuntos
Arginina , Proteínas Argonautas , Estágio Paquíteno , RNA Interferente Pequeno , Espermatogênese , Animais , Masculino , Camundongos , Arginina/metabolismo , Arginina/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Elementos de DNA Transponíveis , RNA de Interação com Piwi , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Domínio TudorRESUMO
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. Surprisingly, the loss of TDRD5 and TDRKH interaction with MIWI results in defective piRNA amplification, rather than an expected failure of piRNA biogenesis. We find that piRNA amplification is necessary for both transposon control and for sustaining levels of select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as autonomous genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
RESUMO
The paradigmatic hematopoietic tree model is increasingly recognized to be limited, as it is based on heterogeneous populations largely defined by non-homeostatic assays testing cell fate potentials. Here, we combine persistent labeling with time-series single-cell RNA sequencing to build a real-time, quantitative model of in vivo tissue dynamics for murine bone marrow hematopoiesis. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of differentiation at specific stages of erythroid and neutrophil production, illustrating how the model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a kinetoscope allows sequential images to merge into a movie. We posit that this approach is generally applicable to understanding tissue-scale dynamics at high resolution.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Hematopoese/genética , Diferenciação CelularRESUMO
Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.
Assuntos
Progressão da Doença , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Leucemia Mieloide Aguda , Inibidores de Prolil-Hidrolase , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Inibidores de Prolil-Hidrolase/uso terapêutico , Animais , Camundongos , Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Linhagem Celular Tumoral , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com PontesRESUMO
piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.