p53 Controls Meiotic Prophase Progression and Crossover Formation

Meiosis initiates with the formation of double strand breaks (DSBs) throughout the genome. To avoid genomic instability, these DSBs need to be correctly repaired by homologous recombination. Surveillance mechanisms involving the DNA damage response (DDR) pathway ATM-CHK2-p53 can detect the persistence of unrepaired DBSs and activate the recombination-dependent arrest at the pachytene stage. However, a complete understanding of p53 functions under normal physiological conditions remains lacking. Here, we report a detailed analysis of the p53 role during meiotic prophase in mice spermatocytes. We show that the absence of p53 regulates prophase progression by slowing down the pachytene stage when the recombination-dependent arrest occurs. Furthermore, our results show that p53 is necessary for proper crossover (CO) formation and localization. Our study contributes to a deeper understanding of p53 roles during the meiotic prophase

ATR is required to complete meiotic recombination in mice

Precise execution of recombination during meiosis is essential for forming chromosomally-balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these remain poorly understood, particularly in mammals. Here we report that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated resected DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.We thank M. A. Handel (The Jackson Laboratory, Bar Harbor, USA) for the anti-H1T antibody; E. Marcon for the anti-RPA antibody (University of Toronto, Canada); A. Toth for the anti-pHORMAD2 antibody (U. Dresden, Germany) and N. Hunter for the anti- RNF212 antibody (UC Davis, USA); J. Turner (National Institute for Medical Research,London, UK) for assistance in the RNA-FISH experiments, for the X chromosome probe,for providing AtrFL/−testis samples and for sharing unpublished data; L. Kauppi(University of Helsinki, Finland) for providing us with protocols for the testis cultures;and members of the Roig lab and the Spanish Ministerio de Ciencia e Innovación-funded Network of Spanish groups working on Meiosis (MeioNet, BFU201‐71786‐REDT) and Enrique Martínez Pérez (Imperial College, London, UK) for helpful discussions. M.M.O. was supported by a FPI fellowship from the Ministerio de Ciencia e Innovación (BES-2011-045381). J.L. was supported in part by American Cancer Society post-doctoral fellowship (PF-12-157-01-DMC). S.K. is an Investigator of the Howard Hughes Medical Institute. This work was supported by the Ministerio de Ciencia e Innovación (BFU2010-18965, BFU2013-43965-P and BFU2016-80370-P, I.R.), by the UAB-Aposta award to young investigators (APOSTA2011-03, I.R.) and by the NIH (R35 GM118175, to M.J.and R35 GM118092 to S.K.).S

Estudio de las funciones de TRIP13 en la profase meiótica de mamíferos

A l'inici de la profase meiòtica, la proteïna SPO11 produeix trencaments de doble cadena (DSBs), i la seva reparació per recombinació homòloga (HR) promou que els cromosomes homòlegs s'aparellin i sinapsen. La reparació dels DSBs mitjançant HR o per unió d'extrems no homòlegs (NHEJ) a la meiosi precisa d'una regulació molt estricta. Les proteïnes del complex sinaptonèmic (SC) són reclutades als eixos cromosòmics, la qual cosa és fonamental per a que es pugui completar la recombinació meiòtica. TRIP13, també coneguda com Pch2 en espècies de no vertebrats, és una proteïna que pertany a la família de les AAA+ ATPases. Algunes de les funcions de TRIP13 s'expliquen per la seva capacitat de canviar la conformació de proteïnes que posseeixen el domini FORMA, com MAD2, HORMAD1 i REV7. Hem localitzat TRIP13 a les porcions sinapsades del SC i als telòmers, una localització que no havia estat descrita en cap altre organisme model. Per a aprendre més sobre les funcions de TRIP13 en la reparació dels DSBs i en la sinapsi dels cromosomes homòlegs, hem emprat els mutants Trip13mod/mod, Trip13sev/sev, hem generat els models murins Trip13-/- i Trip13ATPasaDead i també hem realitzat un tractament usant un inhibidor de TRIP13. També es va inmunoprecipitar TRIP13 i les seves proteïnes interactores a partir d'extractes proteics de testicle i es van identificar mitjançant espectrometria de masses. Els resultats de l'espectrometria de masses van revelar que la xaperona HSPA2, un factor que controla la desinapsi, és un interactor de TRIP13. HSPA2 i PLK1, una altra proteïna que promou la desinapsi, es troben desregulades en els mutants de Trip13. Els espermatòcits de mutants de Trip13 presenten defectes en la sinapsi i acumulen DSBs sense reparar. En aquest treball proposem que TRIP13 evita la desinapsi prematura del SC durant la profase meiòtica, regulant la presència de factors desinàptics. D'altra banda, a cèl·lules somàtiques, TRIP13 regula l'elecció de la via de reparació dels DSBs desacoblant el complex Shieldin, en el qual es troba REV7. Aquest complex protegeix els DSBs de la resecció, inhibint la seva reparació per HR i promovent-la per NHEJ. En espermatòcits hem observat com en absència de TRIP13, els marcadors de HR es troben reduïts i els marcadors de NHEJ es troben augmentats. A més, els espermatòcits deficients de Trip13 presenten més cromosomes units pels telòmers, un efecte dependent de 53BP1.Al inicio de la profase meiótica, la proteína SPO11 produce roturas de doble cadena (DSBs), y su reparación por recombinación homóloga (HR) promueve que los cromosomas homólogos se apareen y sinapsen. La reparación de los DSBs mediante HR o por unión de extremos no homólogos (NHEJ) en meiosis precisa de una regulación muy estricta. Las proteínas del complejo sinaptonémico (SC) son reclutadas a los ejes cromosómicos, lo cual es fundamental para que se pueda completar la recombinación meiótica. TRIP13, también conocida como Pch2 en especies de no vertebrados, es una proteína que pertenece a la familia de las AAA+ ATPasas. Algunas de las funciones de TRIP13 se explican por su capacidad de cambiar la conformación de proteínas que poseen el dominio HORMA, como MAD2, HORMAD1 y REV7. Hemos localizado TRIP13 en los en las porciones sinapsadas del SC y en los telómeros, una localización que no había sido descrita en ningún otro organismo modelo. Para aprender más acerca de las funciones de TRIP13 en la reparación de los DSBs y en la sinapsis de los cromosomas homólogos, hemos usado los mutantes Trip13mod/mod, Trip13sev/sev, hemos generado los modelos murinos Trip13-/- y Trip13ATPasaDead y también hemos realizado un tratamiento farmacológico usando un inhibidor de TRIP13. También se inmunoprecipitó TRIP13 y sus proteínas interactoras a partir de extractos proteicos de testículo y se identificaron mediante espectrometría de masas. Los resultados de la espectrometría de masas revelaron que la chaperona HSPA2, un factor que controla la desinapsis, es un interactor de TRIP13. HSPA2 y PLK1, otra proteína que promueve la desinapsis, se encuentran desreguladas en los mutantes de Trip13. Los espermatocitos de mutantes de Trip13 presentan defectos en la sinapsis y acumulan DSBs sin reparar. En este trabajo proponemos que TRIP13 evita la desinapsis prematura del SC durante la profase meiótica regulando la presencia de factores desinápticos. Por otro lado, en células somáticas, TRIP13 regula la elección de la vía de reparación de los DSBs desensamblando el complejo Shieldin, en el cual se encuentra REV7. Este complejo protege a los DSBs de la resección, inhibiendo su reparación por HR y promoviéndola por NHEJ. En espermatocitos hemos observado como en ausencia de TRIP13, los marcadores de HR se encuentran reducidos y los marcadores de NHEJ se encuentran aumentados. Además, los espermatocitos deficientes de Trip13 presentan más cromosomas unidos por los telómeros, un efecto dependiente de 53BP1.At the onset of meiotic prophase, SPO11 produces double-stranded breaks (DSBs), and its repair by homologous recombination (HR) promotes homologous chromosomes to pair and synapse. During meiosis, repairing DSBs by HR or non-homologous end joining (NHEJ) requires very strict regulation. Synaptonemal complex (SC) proteins are recruited into chromosome axes, which is essential for meiotic recombination to be completed. TRIP13, also known as Pch2 in nonvertebrate species, belongs to the AAA+ ATPases family of proteins. Some of TRIP13's functions are explained by its ability to change the conformation of proteins that possess the HORMA domain, such as MAD2, HORMAD1 and REV7. TRIP13 is located in the synapsed portions of SC and at the telomeres, a location not described yet in any other model organism. To learn more about TRIP13 functions in DSBs repair and homologous chromosome synapsis, we have used several Trip13 mutants, like Trip13mod/mod, Trip13sev/sev, we generated Trip13-/- and Trip13ATPaseDead murine mutants and we have also performed pharmacological treatment using a TRIP13 inhibitor. TRIP13 and its interacting proteins were also immunoprecipitated from testis protein extracts and identified by mass spectrometry. The results of mass spectrometry revealed that the HSPA2 chaperone, a factor that controls desynapsis, is a TRIP13 interactor. HSPA2 and PLK1, another protein that promotes desynapsis, are dysregulated in Trip13 mutants. Trip13 mutant spermatocytes exhibit defects in synapsis and accumulate unrepaired DSBs. In this work we propose that TRIP13 avoids premature SC desynapsis during meiotic prophase, regulating the presence of desynaptic factors. On the other hand, in somatic cells, TRIP13 regulates the choice of DSBs repair pathway by disassembling the Shieldin complex, in which REV7 takes part in the complex. Shieldin complex protects DSBs from resection, inhibiting their repair by HR and promoting it by NHEJ. In spermatocytes, we have observed that in the absence of TRIP13, HR markers are reduced and NHEJ markers are increased. In addition, Trip13 deficient spermatocytes have more fused chromosomes by the telomeres, a 53BP1-dependent effect

Estudio de las funciones de TRIP13 en la profase meiótica de mamíferos

A l’inici de la profase meiòtica, la proteïna SPO11 produeix trencaments de doble cadena (DSBs), i la seva reparació per recombinació homòloga (HR) promou que els cromosomes homòlegs s’aparellin i sinapsen. La reparació dels DSBs mitjançant HR o per unió d’extrems no homòlegs (NHEJ) a la meiosi precisa d’una regulació molt estricta. Les proteïnes del complex sinaptonèmic (SC) són reclutades als eixos cromosòmics, la qual cosa és fonamental per a que es pugui completar la recombinació meiòtica. TRIP13, també coneguda com Pch2 en espècies de no vertebrats, és una proteïna que pertany a la família de les AAA+ ATPases. Algunes de les funcions de TRIP13 s’expliquen per la seva capacitat de canviar la conformació de proteïnes que posseeixen el domini FORMA, com MAD2, HORMAD1 i REV7. Hem localitzat TRIP13 a les porcions sinapsades del SC i als telòmers, una localització que no havia estat descrita en cap altre organisme model. Per a aprendre més sobre les funcions de TRIP13 en la reparació dels DSBs i en la sinapsi dels cromosomes homòlegs, hem emprat els mutants Trip13mod/mod, Trip13sev/sev, hem generat els models murins Trip13-/- i Trip13ATPasaDead i també hem realitzat un tractament usant un inhibidor de TRIP13. També es va inmunoprecipitar TRIP13 i les seves proteïnes interactores a partir d’extractes proteics de testicle i es van identificar mitjançant espectrometria de masses. Els resultats de l’espectrometria de masses van revelar que la xaperona HSPA2, un factor que controla la desinapsi, és un interactor de TRIP13. HSPA2 i PLK1, una altra proteïna que promou la desinapsi, es troben desregulades en els mutants de Trip13. Els espermatòcits de mutants de Trip13 presenten defectes en la sinapsi i acumulen DSBs sense reparar. En aquest treball proposem que TRIP13 evita la desinapsi prematura del SC durant la profase meiòtica, regulant la presència de factors desinàptics. D’altra banda, a cèl·lules somàtiques, TRIP13 regula l’elecció de la via de reparació dels DSBs desacoblant el complex Shieldin, en el qual es troba REV7. Aquest complex protegeix els DSBs de la resecció, inhibint la seva reparació per HR i promovent-la per NHEJ. En espermatòcits hem observat com en absència de TRIP13, els marcadors de HR es troben reduïts i els marcadors de NHEJ es troben augmentats. A més, els espermatòcits deficients de Trip13 presenten més cromosomes units pels telòmers, un efecte dependent de 53BP1.Al inicio de la profase meiótica, la proteína SPO11 produce roturas de doble cadena (DSBs), y su reparación por recombinación homóloga (HR) promueve que los cromosomas homólogos se apareen y sinapsen. La reparación de los DSBs mediante HR o por unión de extremos no homólogos (NHEJ) en meiosis precisa de una regulación muy estricta. Las proteínas del complejo sinaptonémico (SC) son reclutadas a los ejes cromosómicos, lo cual es fundamental para que se pueda completar la recombinación meiótica. TRIP13, también conocida como Pch2 en especies de no vertebrados, es una proteína que pertenece a la familia de las AAA+ ATPasas. Algunas de las funciones de TRIP13 se explican por su capacidad de cambiar la conformación de proteínas que poseen el dominio HORMA, como MAD2, HORMAD1 y REV7. Hemos localizado TRIP13 en los en las porciones sinapsadas del SC y en los telómeros, una localización que no había sido descrita en ningún otro organismo modelo. Para aprender más acerca de las funciones de TRIP13 en la reparación de los DSBs y en la sinapsis de los cromosomas homólogos, hemos usado los mutantes Trip13mod/mod, Trip13sev/sev, hemos generado los modelos murinos Trip13-/- y Trip13ATPasaDead y también hemos realizado un tratamiento farmacológico usando un inhibidor de TRIP13. También se inmunoprecipitó TRIP13 y sus proteínas interactoras a partir de extractos proteicos de testículo y se identificaron mediante espectrometría de masas. Los resultados de la espectrometría de masas revelaron que la chaperona HSPA2, un factor que controla la desinapsis, es un interactor de TRIP13. HSPA2 y PLK1, otra proteína que promueve la desinapsis, se encuentran desreguladas en los mutantes de Trip13. Los espermatocitos de mutantes de Trip13 presentan defectos en la sinapsis y acumulan DSBs sin reparar. En este trabajo proponemos que TRIP13 evita la desinapsis prematura del SC durante la profase meiótica regulando la presencia de factores desinápticos. Por otro lado, en células somáticas, TRIP13 regula la elección de la vía de reparación de los DSBs desensamblando el complejo Shieldin, en el cual se encuentra REV7. Este complejo protege a los DSBs de la resección, inhibiendo su reparación por HR y promoviéndola por NHEJ. En espermatocitos hemos observado como en ausencia de TRIP13, los marcadores de HR se encuentran reducidos y los marcadores de NHEJ se encuentran aumentados. Además, los espermatocitos deficientes de Trip13 presentan más cromosomas unidos por los telómeros, un efecto dependiente de 53BP1.At the onset of meiotic prophase, SPO11 produces double-stranded breaks (DSBs), and its repair by homologous recombination (HR) promotes homologous chromosomes to pair and synapse. During meiosis, repairing DSBs by HR or non-homologous end joining (NHEJ) requires very strict regulation. Synaptonemal complex (SC) proteins are recruited into chromosome axes, which is essential for meiotic recombination to be completed. TRIP13, also known as Pch2 in nonvertebrate species, belongs to the AAA+ ATPases family of proteins. Some of TRIP13’s functions are explained by its ability to change the conformation of proteins that possess the HORMA domain, such as MAD2, HORMAD1 and REV7. TRIP13 is located in the synapsed portions of SC and at the telomeres, a location not described yet in any other model organism. To learn more about TRIP13 functions in DSBs repair and homologous chromosome synapsis, we have used several Trip13 mutants, like Trip13mod/mod, Trip13sev/sev, we generated Trip13-/- and Trip13ATPaseDead murine mutants and we have also performed pharmacological treatment using a TRIP13 inhibitor. TRIP13 and its interacting proteins were also immunoprecipitated from testis protein extracts and identified by mass spectrometry. The results of mass spectrometry revealed that the HSPA2 chaperone, a factor that controls desynapsis, is a TRIP13 interactor. HSPA2 and PLK1, another protein that promotes desynapsis, are dysregulated in Trip13 mutants. Trip13 mutant spermatocytes exhibit defects in synapsis and accumulate unrepaired DSBs. In this work we propose that TRIP13 avoids premature SC desynapsis during meiotic prophase, regulating the presence of desynaptic factors. On the other hand, in somatic cells, TRIP13 regulates the choice of DSBs repair pathway by disassembling the Shieldin complex, in which REV7 takes part in the complex. Shieldin complex protects DSBs from resection, inhibiting their repair by HR and promoting it by NHEJ. In spermatocytes, we have observed that in the absence of TRIP13, HR markers are reduced and NHEJ markers are increased. In addition, Trip13 deficient spermatocytes have more fused chromosomes by the telomeres, a 53BP1-dependent effect.Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia Cel·lula

H2AFX and MDC1 promote maintenance of genomic integrity in male germ cells

In somatic cells, H2afx and Mdc1 are close functional partners in DNA repair and damage response. However, it is not known whether they are also involved in the maintenance of genome integrity in meiosis. By analyzing chromosome dynamics in H2afx spermatocytes, we found that the synapsis of autosomes and X-Y chromosomes was impaired in a fraction of cells. Such defects correlated with an abnormal recombination profile. Conversely, Mdc1 was dispensable for the synapsis of the autosomes and played only a minor role in X-Y synapsis, compared with the action of H2afx. This suggested that those genes have non-overlapping functions in chromosome synapsis. However, we observed that both genes play a similar role in the assembly of MLH3 onto chromosomes, a key step in crossover formation. Moreover, we show that H2afx and Mdc1 cooperate in promoting the activation of the recombination-dependent checkpoint, a mechanism that restrains the differentiation of cells with unrepaired DSBs. This occurs by a mechanism that involves P53. Overall, our data show that, in male germ cells, H2afx and Mdc1 promote the maintenance of genome integrity

ATR is required to complete meiotic recombination in mice

Precise execution of recombination during meiosis is essential for forming chromosomally-balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these remain poorly understood, particularly in mammals. Here we report that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated resected DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination

ATR is a multifunctional regulator of male mouse meiosis

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis

ATR is a multifunctional regulator of male mouse meiosis

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis