Identification and molecular characterization of the mammalian α-kleisin RAD21L

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.     

Dynamics of cohesin subunits in grasshopper meiotic divisions

The cohesin complex plays a key role for the maintenance of sister chromatid cohesion and faithful chromosome segregation in both mitosis and meiosis. This complex is formed by two structural maintenance of chromosomes protein family (SMC) subunits and two non-SMC subunits: an α-kleisin subunit SCC1/RAD21/REC8 and an SCC3-like protein. Several studies carried out in different species have revealed that the distribution of the cohesin subunits along the chromosomes during meiotic prophase I is not regular and that some subunits are distinctly incorporated at different cell stages. However, the accurate distribution of the different cohesin subunits in condensed meiotic chromosomes is still controversial. Here, we describe the dynamics of the cohesin subunits SMC1α, SMC3, RAD21 and SA1 during both meiotic divisions in grasshoppers. Although these subunits show a similar patched labelling at the interchromatid domain of metaphase I bivalents, SMCs and non-SMCs subunits do not always colocalise. Indeed, SA1 is the only cohesin subunit accumulated at the centromeric region of all metaphase I chromosomes. Additionally, non-SMC subunits do not appear at the interchromatid domain in either single X or B chromosomes. These data suggest the existence of several cohesin complexes during metaphase I. The cohesin subunits analysed are released from chromosomes at the beginning of anaphase I, with the exception of SA1 which can be detected at the centromeres until telophase II. These observations indicate that the cohesin components may be differentially loaded and released from meiotic chromosomes during the first and second meiotic divisions. The roles of these cohesin complexes for the maintenance of chromosome structure and their involvement in homologous segregation at first meiotic division are proposed and discussed.     

Meiosis-Specific Cohesin Component,Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion,and Required for DNA Repair and Synapsis between Homologous Chromosomes

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.     

Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.     

Quantitative analysis of cohesin complex stoichiometry and SMC3 modification-dependent protein interactions

Cohesin is a protein complex that plays an essential role in pairing replicated sister chromatids during cell division. The vertebrate cohesin complex consists of four core components including structure maintenance of chromosomes proteins SMC1 and SMC3, RAD21, and SA2/SA1. Extensive research suggests that cohesin traps the sister chromatids by a V-shaped SMC1/SMC3 heterodimer bound to the RAD21 protein that closes the ring. Accordingly, the single "ring" model proposes that two sister chromatids are trapped in a single ring that is composed of one molecule each of the 4 subunits. However, evidence also exists for alternative models. The hand-cuff model suggests that each sister chromatid is trapped individually by two rings that are joined through the shared SA1/SA2 subunit. We report here the determination of cohesin subunit stoichiometry of endogenous cohesin complex by quantitative mass spectrometry. Using qConCAT-based isotope labeling, we show that the cohesin core complex contains equimolar of the 4 core components, suggesting that each cohesin ring is closed by one SA1/SA2 molecule. Furthermore, we applied this strategy to quantify post-translational modification-dependent cohesin interactions. We demonstrate that quantitative mass spectrometry is a powerful tool for measuring stoichiometry of endogenous protein core complex.     

Cohesin's DNA Exit Gate Is Distinct from Its Entrance Gate and Is Regulated by Acetylation

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin. Our results reveal?a "releasing" activity inherent to cohesin complexes transiently associated with Wapl that catalyzes their dissociation from chromosomes. Fusion of Smc3's nucleotide binding domain to α-kleisin's N-terminal domain also reduces cohesin turnover within pericentric chromatin and permits establishment of Wapl-resistant cohesion in the absence of Eco1. We suggest that releasing activity opens the Smc3/α-kleisin interface, creating a DNA exit gate distinct from its proposed entry gate at the Smc1/3 interface. According to this notion, the function of Smc3 acetylation is to block its dissociation from α-kleisin. The functional implications of regulated ring opening are discussed.     

Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.     

Cohesin gene mutations in tumorigenesis: from discovery to clinical significance

Cohesin is a multi-protein complex composed of four core subunits (SMC1A, SMC3, RAD21, and either STAG1 or STAG2) that is responsible for the cohesion of sister chromatids following DNA replication until its cleavage during mitosis thereby enabling faithful segregation of sister chromatids into two daughter cells. Recent cancer genomics analyses have discovered a high frequency of somatic mutations in the genes encoding the core cohesin subunits as well as cohesin regulatory factors (e.g. NIPBL, PDS5B, ESPL1) in a select subset of human tumors including glioblastoma, Ewing sarcoma, urothelial carcinoma, acute myeloid leukemia, and acute megakaryoblastic leukemia. Herein we review these studies including discussion of the functional significance of cohesin inactivation in tumorigenesis and potential therapeutic mechanisms to selectively target cancers harboring cohesin mutations. [BMB Reports 2014; 47(6): 299-310]     

A new meiosis-specific cohesin complex implicated in the cohesin code for homologous pairing

We identify a new mammalian cohesin subunit, RAD21-like protein (RAD21L), with sequence similarity to RAD21 and REC8. RAD21L localizes along axial elements in early meiotic prophase, in a manner that is spatiotemporally different to either REC8 or RAD21. Remarkably, RAD21L and REC8 have symmetrical, mutually exclusive localization on the not-yet-synapsed homologues, implying that the cohesin patterning could provide a code for homologue recognition. RAD21 transiently localizes to axial elements after the dissociation of RAD21L and REC8 in late pachytene, a period of recombination repair. Further, we show that the removal of cohesins and synaptonemal complex during late meiotic prophase is promoted by Polo-like kinase 1, which is similar to the mitotic prophase pathway.     

Cohesin axis maturation and presence of RAD51 during first meiotic prophase in a true bug

We have analyzed in a true bug, Graphosoma italicum (Pentatomidae, Hemiptera), the temporal and functional relationships between recombination events, synapsis progression, and SMC1α and SMC3 cohesin axis maturation throughout the male first meiotic prophase. The localization of the histone variant histone H3 trimethylated at lysine 9 at chromosome ends has allowed us to determine the association of these heterochromatic domains through prophase I stages. Results highlighted that cohesins provide to be good markers for synapsis progression since the formation, morphology, and development of the SMC1α and SMC3 cohesin axes resemble the synaptonemal complex dynamics and, also, that in this species the initiation of recombination precedes synapsis. In addition, we have carried out an accurate cytological characterization of the diffuse stage, which takes place after pachytene, and also analyzed the presence of the cohesin subunits, SMC1α and SMC3, and the recombinase RAD51 at this stage. The mechanisms underlying the absence of SMC1α and SMC3 axes from the diffuse stage onwards are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance,but Not Sister Chromatid Cohesion,during Drosophila Female Meiosis

Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.     

Arabidopsis thaliana WAPL Is Essential for the Prophase Removal of Cohesin during Meiosis

Sister chromatid cohesion, which is mediated by the cohesin complex, is essential for the proper segregation of chromosomes in mitosis and meiosis. The establishment of stable sister chromatid cohesion occurs during DNA replication and involves acetylation of the complex by the acetyltransferase CTF7. In higher eukaryotes, the majority of cohesin complexes are removed from chromosomes during prophase. Studies in fly and human have shown that this process involves the WAPL mediated opening of the cohesin ring at the junction between the SMC3 ATPase domain and the N-terminal domain of cohesin''s α-kleisin subunit. We report here the isolation and detailed characterization of WAPL in Arabidopsis thaliana. We show that Arabidopsis contains two WAPL genes, which share overlapping functions. Plants in which both WAPL genes contain T-DNA insertions show relatively normal growth and development but exhibit a significant reduction in male and female fertility. The removal of cohesin from chromosomes during meiotic prophase is blocked in Atwapl mutants resulting in chromosome bridges, broken chromosomes and uneven chromosome segregation. In contrast, while subtle mitotic alterations are observed in some somatic cells, cohesin complexes appear to be removed normally. Finally, we show that mutations in AtWAPL suppress the lethality associated with inactivation of AtCTF7. Taken together our results demonstrate that WAPL plays a critical role in meiosis and raises the possibility that mechanisms involved in the prophase removal of cohesin may vary between mitosis and meiosis in plants.     

Changes in the expression and localization of cohesin subunits during meiosis in a non-mammalian vertebrate, the medaka fish

Until the onset of anaphase, sister chromatids are bound to each other by a multi-subunit protein complex called cohesin. Since chromosomes in meiosis behave differently from those in mitosis, the cohesion and separation of homologous chromosomes and sister chromatids in meiosis are thought to be regulated by meiosis-specific cohesin subunits. Actually, several meiosis-specific cohesin subunits, including Rec8, STAG3 and SMC1beta, are known to exist in mammals; however, there are no reports of meiosis-specific cohesin subunits in other vertebrates. To investigate the protein expression and localization of cohesin subunits during meiosis in non-mammalian species, we isolated cDNA clones encoding SMC1alpha, SMC1beta, SMC3 and Rad21 in the medaka and produced antibodies against recombinant proteins. Medaka SMC1beta was expressed solely in gonads, while SMC1alpha, SMC3 and Rad21 were also expressed in other organs and in cultured cells. SMC1beta forms a complex with SMC3 but not with Rad21, in contrast to SMC1alpha, which forms complexes with both SMC3 and Rad21. SMC1alpha and Rad21 were mainly expressed in mitotically dividing cells in the testis (somatic cells and spermatogonia), although their weak expression was detected in pre-leptotene spermatocytes. SMC1beta was expressed in spermatogonia and spermatocytes. SMC1beta was localized along the chromosomal arms as well as on the centromeres in meiotic prophase I, and its existence on the chromosomes persisted up to metaphase II, a situation different from that reported in the mouse, in which SMC1beta is lost from the chromosome arms in late pachytene despite its universal presence in vertebrates.     

High density of REC8 constrains sister chromatid axes and prevents illegitimate synaptonemal complex formation

During meiosis, cohesin complexes mediate sister chromatid cohesion (SCC), synaptonemal complex (SC) assembly and synapsis. Here, using super‐resolution microscopy, we imaged sister chromatid axes in mouse meiocytes that have normal or reduced levels of cohesin complexes, assessing the relationship between localization of cohesin complexes, SCC and SC formation. We show that REC8 foci are separated from each other by a distance smaller than 15% of the total chromosome axis length in wild‐type meiocytes. Reduced levels of cohesin complexes result in a local separation of sister chromatid axial elements (LSAEs), as well as illegitimate SC formation at these sites. REC8 but not RAD21 or RAD21L cohesin complexes flank sites of LSAEs, whereas RAD21 and RAD21L appear predominantly along the separated sister‐chromatid axes. Based on these observations and a quantitative distribution analysis of REC8 along sister chromatid axes, we propose that the high density of randomly distributed REC8 cohesin complexes promotes SCC and prevents illegitimate SC formation.     

A new role for the mitotic RAD21/SCC1 cohesin in meiotic chromosome cohesion and segregation in the mouse

The evolutionarily conserved cohesin complex is required for the establishment and maintenance of sister chromatid cohesion, in turn essential for proper chromosome segregation. RAD21/SCC1 is a regulatory subunit of the mitotic cohesin complex, as it links together all other subunits of the complex. The destruction of RAD21/SCC1 along chromosomal arms and later at centromeres results in the dissociation of the cohesin complex, facilitating chromosome segregation. Here, we report for the first time that mammalian RAD21/SCC1 associates with the axial/lateral elements of the synaptonemal complex along chromosome arms and on centromeres of mouse spermatocytes. Importantly, RAD21/SCC1 is lost from chromosome arms in late prophase I but persists on centromeres. The loss of centromeric RAD21/SCC1 coincides with the separation of sister chromatids at anaphase II. These findings support a role for mammalian RAD21/SCC1 in maintaining sister chromatid cohesion in meiosis.     

Imbalance of SMC1 and SMC3 Cohesins Causes Specific and Distinct Effects

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.     

Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1alpha, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.