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1.
Elife ; 102021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34328417

RESUMO

The molecular events that drive hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV-infected organoids produced covalently closed circular DNA (cccDNA) and HBV early antigen (HBeAg), expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV-infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity and drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor sodium taurocholate co-transporting polypeptide (NTCP) after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the hepatocellular carcinoma (HCC) cohort on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV-infected patients.


Assuntos
Carcinoma Hepatocelular/virologia , Hepatite B/virologia , Neoplasias Hepáticas/virologia , Organoides/virologia , Células Hep G2 , Hepatite B/complicações , Vírus da Hepatite B/patogenicidade , Humanos , Fígado/patologia , Fígado/virologia , Doadores Vivos , Modelos Biológicos , Replicação Viral
2.
Nucleic Acids Res ; 49(5): 2609-2628, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619545

RESUMO

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.


Assuntos
Quebras de DNA de Cadeia Dupla , Meiose/genética , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/fisiologia , Pareamento Cromossômico , Retroalimentação Fisiológica , Gametogênese , Camundongos , Estágio Paquíteno , Cromossomos Sexuais , Transdução de Sinais
3.
PLoS Genet ; 16(6): e1008595, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502153

RESUMO

The recombinase RAD51, and its meiosis-specific paralog DMC1 localize at DNA double-strand break (DSB) sites in meiotic prophase. While both proteins are required during meiotic prophase, their spatial organization during meiotic DSB repair is not fully understood. Using super-resolution microscopy on mouse spermatocyte nuclei, we aimed to define their relative position at DSB foci, and how these vary in time. We show that a large fraction of meiotic DSB repair foci (38%) consisted of a single RAD51 nanofocus and a single DMC1 nanofocus (D1R1 configuration) that were partially overlapping with each other (average center-center distance around 70 nm). The vast majority of the rest of the foci had a similar large RAD51 and DMC1 nanofocus, but in combination with additional smaller nanofoci (D2R1, D1R2, D2R2, or DxRy configuration) at an average distance of around 250 nm. As prophase progressed, less D1R1 and more D2R1 foci were observed, where the large RAD51 nanofocus in the D2R1 foci elongated and gradually oriented towards the distant small DMC1 nanofocus. D1R2 foci frequency was relatively constant, and the single DMC1 nanofocus did not elongate, but was frequently observed between the two RAD51 nanofoci in early stages. D2R2 foci were rare (<10%) and nearest neighbour analyses also did not reveal cofoci formation between D1R1 foci. However, overall, foci localized nonrandomly along the SC, and the frequency of the distance distributions peaked at 800 nm, indicating interference and/or a preferred distance between two ends of a DSB. DMC1 nanofoci where somewhat further away from the axial or lateral elements of the synaptonemal complex (SC, connecting the chromosomal axes of homologs) compared to RAD51 nanofoci. In the absence of the transverse filament of the SC, early configurations were more prominent, and RAD51 nanofocus elongation occurred only transiently. This in-depth analysis of single cell landscapes of RAD51 and DMC1 accumulation patterns at DSB repair sites at super-resolution revealed the variability of foci composition, and defined functional consensus configurations that change over time.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Prófase , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Animais , Quebras de DNA de Cadeia Dupla , Masculino , Camundongos , Espermatócitos/citologia , Espermatócitos/metabolismo
4.
Chromosoma ; 127(3): 341-359, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29582139

RESUMO

During mammalian meiotic prophase, homologous chromosomes connect through the formation of the synaptonemal complex (SC). SYCP3 is a component of the lateral elements of the SC. We have generated transgenic mice expressing N- or C-terminal fluorescent-tagged SYCP3 (mCherry-SYCP3 (CSYCP) and SYCP3-mCherry (SYCPC)) to study SC dynamics and chromosome movements in vivo. Neither transgene rescued meiotic aberrations in Sycp3 knockouts, but CSYCP could form short axial element-like structures in the absence of endogenous SYCP3. On the wild-type background, both fusion proteins localized to the axes of the SC together with endogenous SYCP3, albeit with delayed initiation (from pachytene) in spermatocytes. Around 40% of CSYCP and SYCPC that accumulated on the SC was rapidly exchanging with other tagged proteins, as analyzed by fluorescent recovery after photobleaching (FRAP) assay. We used the CSYCP transgenic mice for further live cell analyses and observed synchronized bouquet configurations in living cysts of two or three zygotene oocyte nuclei expressing CSYCP, which presented cycles of telomere clustering and dissolution. Rapid chromosome movements were observed in both zygotene oocytes and pachytene spermatocytes, but rotational movements of the nucleus were more clear in oocytes. In diplotene spermatocytes, desynapsis was found to proceed in a discontinuous manner, whereby even brief chromosome re-association events were observed. Thus, this live imaging approach can be used to follow changes in the dynamic behavior of the nucleus and chromatin, in normal mice and different infertile mouse models.


Assuntos
Cromossomos de Mamíferos , Ovário/metabolismo , Túbulos Seminíferos/metabolismo , Complexo Sinaptonêmico/genética , Animais , Biomarcadores , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Masculino , Meiose/genética , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Oócitos/metabolismo , Fenótipo , Espermatócitos/metabolismo , Testículo , Transgenes
5.
DNA Repair (Amst) ; 63: 25-38, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29414051

RESUMO

Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11YF/YF mice, 140-160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11YF/YF compared to Spo11+/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Reparo de DNA por Recombinação , Animais , Proteínas de Ciclo Celular/genética , DNA/metabolismo , DNA/efeitos da radiação , Endodesoxirribonucleases/genética , Feminino , Masculino , Meiose/efeitos da radiação , Camundongos , Camundongos Mutantes , Radiação Ionizante
6.
PLoS Genet ; 9(6): e1003538, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23754961

RESUMO

In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.


Assuntos
Pareamento Cromossômico/genética , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Meiose/genética , Inativação do Cromossomo X/genética , Animais , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Feminino , Masculino , Camundongos , Oogênese/genética , Espermatócitos/citologia , Espermatócitos/metabolismo , Cromossomo X/genética , Cromossomo Y/genética
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