Análisis funcional de las Kleisinas meióticas de mamíferos

[ES] En mamíferos existen parálogos para la mayoría de subunidades del complejo de cohesinas. En nuestro laboratorio se identificó una nueva kleisina específica de meiosis, denominada RAD21L. La generación de dos modelos de ratón con pérdida de su función ha permitido determinar que esta kleisina forma parte de complejos de cohesinas in vivo y que es esencial para la espermatogénesis, ya que su ausencia provoca un bloqueo en zigotena que da lugar a azoospermia. Además, las hembras deficientes para RAD21L presentan una disminución en la cantidad de folículos primordiales y esterilidad precoz sin pérdida aparente de cohesión centromérica. Asimismo, el desarrollo de un modelo murino con pérdida simultánea de las dos kleisinas meioticas RAD21L y REC8 nos ha permitido determinar que los complejos de cohesinas meióticos son esenciales para el ensamblaje del elemento axial (AE), del complejo sinaptonémico (SC) y el inicio de la recombinación en mamíferos. De forma adicional, hemos comprobado que la carga o el mantenimiento de RAD21L en los centrómeros dependen en parte de SGOL2, una shugoshina caracterizada previamente por proteger a REC8 del corte por Separasa en anafase I. Además, el desarrollo de un ratón deficiente para SGOL2 y Securina ha revelado que probablemente estas dos proteínas no llevan a cabo funciones redundantes en la inhibición de Separasa, a diferencia de lo que ocurre en S. cerevisiae. No obstante, la función de Securina en la intercinesis es dependiente de complejos de cohesinas meióticos.[EN] In mammals there paralogous most complex subunits cohesin. Our laboratory identified a novel meiosis-specific kleisina, RAD21L called. The generation of two mouse models with loss of function has kleisina possible to determine that this is part of cohesin complexes in vivo and which is spermatogenesis essential, since its absence causes a blockage in giving zigotena azoospermia place. Furthermore, RAD21L deficient females had a lower in the number of primordial follicles and premature sterility without apparent loss of cohesion centromeric. Also, the development of a murine model with simultaneous loss of the two meiotic kleisinas REC8 RAD21L and allowed us to determine that cohesin complexes meiotic are essential for assembly of the axial element (AE), synaptonemal complex (SC) and the initiation of recombination in mammals. Additionally, we found that the maintaining load or RAD21L at centromeres SGOL2 depend in part a previously characterized shugoshina protect the cutting REC8 separase at anaphase I. Furthermore, the development of a mouse deficient for SGOL2 and securin has revealed that these two proteins likely not perform redundant functions in the inhibition of Separase, unlike what happens in S. cerevisiae. However, the function of securin interkinesis is dependent meiotic cohesin complexes

Análisis funcional de las kleisinas meióticas de mamíferos

Tesis Doctoral presentada por Yurema Herrán Santamaría, licenciada en Biología para optar al grado de Doctor por la Universidad de Salamanca.[ES]: En mamíferos existen parálogos para la mayoría de subunidades del complejo de cohesinas. En nuestro laboratorio se identificó una nueva kleisina específica de meiosis, denominada RAD21L. La generación de dos modelos de ratón con pérdida de su función ha permitido determinar que esta kleisina forma parte de complejos de cohesinas in vivo y que es esencial para la espermatogénesis, ya que su ausencia provoca un bloqueo en zigotena que da lugar a azoospermia. Además, las hembras deficientes para RAD21L presentan una disminución en la cantidad de folículos primordiales y esterilidad precoz sin pérdida aparente de cohesión centromérica. Asimismo, el desarrollo de un modelo murino con pérdida simultánea de las dos kleisinas meioticas RAD21L y REC8 nos ha permitido determinar que los complejos de cohesinas meióticos son esenciales para el ensamblaje del elemento axial (AE), del complejo sinaptonémico (SC) y el inicio de la recombinación en mamíferos. De forma adicional, hemos comprobado que la carga o el mantenimiento de RAD21L en los centrómeros dependen en parte de SGOL2, una shugoshina caracterizada previamente, que protege a REC8 del corte por Separasa en anafase I. Además, el desarrollo de un ratón deficiente para SGOL2 y Securina ha revelado que probablemente estas dos proteínas no llevan a cabo funciones redundantes en la inhibición de Separasa, a diferencia de lo que ocurre en S. cerevisiae. No obstante, la función de Securina en la intercinesis es dependiente de complejos de cohesinas meióticos.[EN]: In mammals there paralogous most complex subunitscohesin. Our laboratory identified a novel meiosis-specific kleisina,RAD21L called. The generation of two mouse models with loss of function haskleisina possible to determine that this is part of cohesin complexes in vivo and which isspermatogenesis essential, since its absence causes a blockage in giving zigotenaazoospermia place. Furthermore, RAD21L deficient females had a lowerin the number of primordial follicles and premature sterility without apparent loss of cohesioncentromeric. Also, the development of a murine model with simultaneous loss of the twomeiotic kleisinas REC8 RAD21L and allowed us to determine that cohesin complexesmeiotic are essential for assembly of the axial element (AE), synaptonemal complex(SC) and the initiation of recombination in mammals. Additionally, we found that themaintaining load or RAD21L at centromeres SGOL2 depend in part apreviously characterized shugoshina protect the cutting REC8 separase at anaphase I.Furthermore, the development of a mouse deficient for SGOL2 and securin has revealed thatthese two proteins likely not perform redundant functions in the inhibition ofSeparase, unlike what happens in S. cerevisiae. However, the function of securininterkinesis is dependent meiotic cohesin complexesPeer Reviewe

Identification and molecular characterization of the mammalian α-kleisin RAD21L

11 páginas, 7 figuras, 1 tabla.-- This is an open access article.Meiosis is a fundamental process that generates new combinations between maternal and paternal genomes and haploid gametes from diploid progenitors. Many of the meiosis-specific events stem from the behavior of the cohesin complex (CC), a proteinaceous ring structure that entraps sister chromatids until the onset of anaphase. CCs ensure chromosome segregation, participate in DNA repair, regulate gene expression, and also contribute to synaptonemal complex (SC) formation at meiosis by keeping long-range distant DNA interactions through its conserved structure. Studies from yeast to humans have led to the assumption that Scc1/RAD21 is the α-kleisin that closes the tripartite CC that entraps two DNA molecules in mitosis, while its paralog REC8 is essential for meiosis. Here we describe the identification of RAD21L, a novel mammalian CC subunit with homology to the RAD21/REC8 α-kleisin subfamily, which is expressed in mouse testis. RAD21L interacts with other cohesin subunits such as SMC1α, SMC1b, SMC3 and with the meiosis-specific STAG3 protein. Thus, our results demonstrate the existence of a new meiotic-specific CC constituted by this α-kleisin and expand the view of REC8 as the only specific meiotic α-kleisin.This work was supported by SAF, J CyLe (SA) and BFU, and BFU/BCM. C.G.C. and Y.H. are supported by FIS and FPI fellowships respectively. E.L. is recipient of a Ramón y Cajal Research contract.Peer reviewe

The cohesin subunit RAD21L functions in meiotic synapsis and exhibits sexual dimorphism in fertility

The cohesin complex is a ring-shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α-kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α-kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L-deficient females are fertile but develop an age-dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural aging.This work was supported by SAF (2008-0317), J CyLe (SA), BFU (2008- 00300/BCM) and BFU (2009-08975/BMC). CGC and YS are supported by FIS and FPI fellowships, respectively. EL is recipient of a Ramo´n y Cajal Research contract.Peer reviewe

The cohesin subunit RAD21L functions in meiotic synapsis and exhibits sexual dimorphism in fertility

The cohesin complex is a ring-shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α-kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α-kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I, which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L-deficient females are fertile but develop an age-dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural agin

Shugoshin-2 is essential for the completion of meiosis but not for mitotic cell division in mice

Shugoshin-2 (SGOL2) is one of the two mammalian orthologs of the Shugoshin/Mei-S322 family of proteins that regulate sister chromatid cohesion by protecting the integrity of the multiprotein cohesin complexes. This protective system is essential for faithful chromosome segregation during mitosis and meiosis, which is the physical basis of Mendelian inheritance. Regardless of its evolutionary conservation from yeast to mammals, little is known about the in vivo relevance and specific role that SGOL2 plays in mammals. Here we show that disruption of the gene encoding mouse SGOL2 does not cause any alteration in sister chromatid cohesion in embryonic cultured fibroblasts and adult somatic tissues. Moreover, mutant mice develop normally and survive to adulthood without any apparent alteration. However, both male and female Sgol2-deficient mice are infertile. We demonstrate that SGOL2 is necessary for protecting centromeric cohesion during mammalian meiosis I. In vivo, the loss of SGOL2 promotes a premature release of the meiosis-specific REC8 cohesin complexes from anaphase I centromeres. This molecular alteration is manifested cytologically by the complete loss of centromere cohesion at metaphase II leading to single chromatids and physiologically with the formation of aneuploid gametes that give rise to infertility

Meiotic cohesin complexes are essential for the formation of the axial element in mice

This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date. After six months it is available under a Creative Commons License.Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.This work was supported by grant SAF2011-25252 and Junta de Castilla y León. C. Gutiérrez-Caballero and Y. Herrán are supported by Fondo de Investigación Sanitaria and Formación de Personal Investigador fellowships, respectively. E. Llano is the recipient of a Ramón y Cajal Research contract.Peer Reviewe