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Remeseiro S Cuadrado A Carretero M Martínez P Drosopoulos WC Cañamero M Schildkraut CL Blasco MA Losada A 《The EMBO journal》2012,31(9):2076-2089
Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis. 相似文献
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Xiangduo Kong Alexander R. Ball Jr. Hoang Xuan Pham Weihua Zeng Hsiao-Yuan Chen John A. Schmiesing Jong-Soo Kim Michael Berns Kyoko Yokomori 《Molecular and cellular biology》2014,34(4):685-698
Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G2-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin''s intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells. 相似文献
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Peters JM 《The EMBO journal》2012,31(9):2061-2063
EMBO J
31
9, 2076–2089
March132012EMBO J
31
9, 2090–2102
March132012It is well known that somatic and germ cells use different cohesin complexes to
mediate sister chromatid cohesion, but why different isoforms of cohesin also
co-exist within somatic vertebrate cells has remained a mystery. Two papers in this
issue of The EMBO Journal have begun to address this question by analysing
mouse cells lacking SA1, an isoform of a specific cohesin subunit.When one cell divides into two, many things have to go right for the two daughter cells
to receive identical copies of their mother cell''s genome. It has long been
recognized that sister chromatid cohesion, the physical connection established during
DNA replication between newly synthesized sister DNA molecules, is one of these
essential prerequisites for proper chromosome segregation. It is this cohesion that
enables the bi-orientation of chromosomes on the mitotic or meiotic spindle, and thus
makes their symmetrical segregation possible. Cohesion is mediated by cohesin, a
multi-subunit protein complex, which is thought to connect sister DNA molecules by
embracing them as a ring (Figure 1; reviewed in Peters et al, 2008). It is well established that cohesin complexes
differ between somatic and germ cells, where they are needed for the proper separation
of sister chromatids and of homologous chromosomes, respectively. What has been largely
ignored, however, is that even within somatic vertebrate cells there are different forms
of cohesin, containing mutually exclusive variable subunits: either SA1 or the closely
related SA2 protein (also known as STAG1 and STAG2, respectively), and either Pds5A or
the related Pds5B subunit (Peters et al, 2008). Why
is that? Two papers from the Losada lab (Remeseiro et al,
2012a, 2012b) have begun to address this
question by generating mouse cells lacking the SA1 gene, revealing unexpected
insights into the functions of SA1 subunit-containing cohesin complexes
(cohesin-SA1).Open in a separate windowFigure 1Schematic drawing illustrating how the SA1 and SA2 proteins interact in a
mutually exclusive manner with three core subunits of cohesin (Smc1, Smc3,
Rad21) that form a ring-like structure. It has been proposed that these
complexes mediate cohesion by trapping the two sister DNA molecules inside
the cohesin ring (above), and that cohesin rings might affect chromatin
structure by forming or stabilizing intra-chromatid loops (below). Cohesin
is thought to influence gene regulation at least in part by mediating
chromatin looping.Although cohesin is best known for its role in sister chromatid cohesion, it is clearly
also needed for homologous recombination-mediated DNA repair and for gene regulation.
Much of what we know about these functions comes from studies in yeast and fruit flies,
organisms with only a single SA1/SA2-related mitotic subunit (Scc3 in budding yeast),
and only a single Pds5 subunit. It is therefore plausible that, like many other genes
during vertebrate evolution, SA1/SA2 and Pds5A/Pds5B have arisen by gene duplication to
constitute paralogs, with functional differences between them assumed to be subtle.
Consistently, absence of either Pds5A or Pds5B causes only mild, if any, defects in
sister chromatid cohesion, and mice lacking either protein can develop to term, although
they die shortly after birth owing to multiple organ defects (Zhang
et al, 2007, 2009). First
indications that the situation may be different for the Scc3-related subunits came from
Canudas and Smith (2009), who reported that RNAi
depletion of SA1 and SA2 from HeLa cells caused defects in telomere and centromere
cohesion, respectively. The generation of mice lacking either one or both alleles of the
SA1 gene has now allowed a more systematic and thorough analysis of SA1
function (Remeseiro et al, 2012a, 2012b).One of the most striking results obtained in these studies is that most mice lacking SA1
die around day 12 of embryonic development, clearly showing that the function of SA1
cannot be fulfilled by SA2, despite the fact that SA2 is substantially more abundant in
somatic cells than SA1 (Holzmann et al, 2010). What
could this SA1-specific function be? Losada and colleagues report observations, which
imply that SA1 does not have just one, but possibly several important functions in
different processes. First, the authors confirm the previous observation that SA1 is
required for cohesion specifically at telomeres, while likely collaborating with SA2 in
chromosome arms or centromeric regions. Furthermore, telomeres have an unusual
morphology in mitotic chromosomes lacking SA1 (Remeseiro et al,
2012a), reminiscent of a fragile-site phenotype previously reported in
telomeres with DNA replication defects (Sfeir et al,
2009), and SA1 is indeed required for efficient telomere duplication.
Depletion of sororin, a protein that is required for cohesin''s ability to mediate
sister chromatid cohesion, also causes a fragile-site phenotype at telomeres. These
findings imply that SA1''s role in telomere cohesion is important for efficient
telomere replication, perhaps, as the authors speculate, because telomere cohesion may
help to stabilize or re-start stalled replication forks, or because cohesion-dependent
homologous recombination might be involved in repair of DNA double strand breaks created
by collapsed replication forks. Interestingly, cells lacking SA1 frequently show
chromosome bridges in anaphase, often fail to divide, and either die or become
bi-nucleated. The exact origin of chromosome bridges is difficult to determine, but
previous studies have found such bridges often associated with fragile sites on
chromosomes; treatment with low doses of DNA replication inhibitors was shown to
increase the frequency of such bridges (Chan et al,
2009), and similar observations were indeed made by Remeseiro et al (2012a) in mouse embryonic fibroblasts. It is
therefore plausible that the telomere cohesion defect observed in SA1-lacking cells
leads to incomplete telomere replication, which in turn results in the formation of
anaphase chromosome bridges and subsequent cytokinesis defects. Losada and colleagues
further speculate that these chromosome segregation defects could underlie the increased
frequency of spontaneous development of various tumours in mice containing just one
instead of two SA1 alleles (Remeseiro et al, 2012a).
This is an attractive interpretation since tetraploidy and aneuploidy are thought to
contribute to the rate with which tumour cells can evolve; however, Losada and
colleagues report SA1 deficiency to cause defects also in other cohesin functions, which
may therefore as well contribute to tumour formation.To further understand why SA1 cannot be fulfilled by SA2, Losada and colleagues also
analysed the distribution of these proteins in the non-repetitive parts of the mouse
genome by chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq). The
results of these experiments, published in the second of the two papers (Remeseiro et al, 2012b), raise the interesting possibility
that cohesin-SA1 associates more frequently with gene promoters than cohesin-SA2.
However, the fact that different antibodies have to be used for any ChIP-based
comparison of the distribution of two proteins makes it difficult to know to what degree
observed differences might be due to different antibody efficiency. Obviously, such
limitations do not exist if the distribution of one and the same protein is analysed
under different conditions, and in such an experimental setting, Remeseiro et al
indeed make some striking observations. When SA1 is absent, SA2 does not detectably
change in abundance, but its distribution in the genome does, in that more than half of
all SA2-binding sites in SA1-deficient cells differ from those bound in wild-type cells.
Most SA2-binding sites in SA1-deficient cells are in intergenic regions, and CTCF, a
zinc finger protein often co-localizing with cohesin and implicated in its gene
regulation function (Peters et al, 2008), appears to
be absent at many of these sites. It presently remains a mystery why cohesin-SA2 changes
its distribution so dramatically in the absence of SA1, but the observation that gene
promoters are more frequently occupied by cohesin in the presence of SA1 than in its
absence raises the possibility that cohesin-SA1 may have a specific role in gene
regulation. This possibility is particularly interesting in light of a recent study that
found hardly any change in gene expression upon re-expression of SA2 in SA2-deficient
human glioblastoma cells (Solomon et al, 2011),
despite the fact that cohesin is thought to regulate numerous genes. With this in mind,
Remeseiro et al analysed gene expression in mouse cells and indeed found 549
genes to be mis-regulated in the absence of SA1, in striking contrast to the
above-mentioned comparison of human SA2-deficient or proficient cells that found only 19
genes to change in expression levels (Solomon et al,
2011). Obviously direct comparisons will be essential to analyse further
the specific roles of SA1 and SA2 in gene regulation, but the current evidence raises
the interesting possibility that SA1 may have a particularly important role in gene
regulation, whereas cohesin-SA2 is dedicated to creating arm and centromeric cohesive
structures for chromosome segregation.That is not to say that cohesin-SA1 cannot mediate sister chromatid cohesion. It almost
certainly can, as it is essential for cohesion at telomeres (Canudas
and Smith, 2009; Remeseiro et al,
2012a). Likewise, it would be wrong to assume that we now fully understand why
SA1 and SA2 co-exist in somatic vertebrate cells, and what their precise functions is.
There are many things we do not understand. For example, if SA2 has little or no role in
gene regulation, as the Solomon et al (2011) study
implies, why does SA2 nevertheless interact directly with CTCF (Xiao
et al, 2011), its gene regulation collaborator? How do
cohesin-SA1 and cohesin-SA2 complexes further differ in their genomic distributions and
their functions depending on whether they contain either Pds5A or Pds5B, constituting
really not just two but four distinct cohesin complexes? The work by Losada and
colleagues represents an important step towards understanding these questions, but there
is still a long and presumably exciting way to go to understand how different cohesin
complexes control the mammalian genome. 相似文献
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A handcuff model for the cohesin complex 总被引:1,自引:0,他引:1
Nenggang Zhang Sergey G. Kuznetsov Shyam K. Sharan Kaiyi Li Pulivarthi H. Rao Debananda Pati 《The Journal of cell biology》2008,183(6):1019-1031
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Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans. 相似文献
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María Carretero Miguel Ruiz‐Torres Miriam Rodríguez‐Corsino Isabel Barthelemy Ana Losada 《The EMBO journal》2013,32(22):2938-2949
Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis‐segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions. 相似文献
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Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis‐independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1–Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring. 相似文献
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Sister chromatid cohesion depends on a multiprotein cohesin complex containing two SMC subunits, Smc1 and Smc3, that dimerize to form V-shaped molecules with ABC-like ATPase heads at the tips of their two arms. Cohesin's Smc1 and Smc3 "heads" are connected by an alpha kleisin subunit called Scc1, forming a tripartite ring with a diameter around 40 nm. We show here that some cohesin remains tightly bound to circular minichromosomes after their purification from yeast cells and that cleavage either of cohesin's ring or of the minichromosome's DNA destroys their association. This suggests that the stable association between cohesin and chromatin detected here is topological rather than physical, which is consistent with the notion that DNA is trapped inside cohesin rings. 相似文献
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Ola Orgil Avi Matityahu Thomas Eng Vincent Guacci Douglas Koshland Itay Onn 《PLoS genetics》2015,11(3)
The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized SCC3 to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation. 相似文献
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The cohesin complex is required for the DNA damage‐induced G2/M checkpoint in mammalian cells
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Jan‐Michael Peters 《The EMBO journal》2009,28(17):2625-2635
Cohesin complexes mediate sister chromatid cohesion. Cohesin also becomes enriched at DNA double‐strand break sites and facilitates recombinational DNA repair. Here, we report that cohesin is essential for the DNA damage‐induced G2/M checkpoint. In contrast to cohesin's role in DNA repair, the checkpoint function of cohesin is independent of its ability to mediate cohesion. After RNAi‐mediated depletion of cohesin, cells fail to properly activate the checkpoint kinase Chk2 and have defects in recruiting the mediator protein 53BP1 to DNA damage sites. Earlier work has shown that phosphorylation of the cohesin subunits Smc1 and Smc3 is required for the intra‐S checkpoint, but Smc1/Smc3 are also subunits of a distinct recombination complex, RC‐1. It was, therefore, unknown whether Smc1/Smc3 function in the intra‐S checkpoint as part of cohesin. We show that Smc1/Smc3 are phosphorylated as part of cohesin and that cohesin is required for the intra‐S checkpoint. We propose that accumulation of cohesin at DNA break sites is not only needed to mediate DNA repair, but also facilitates the recruitment of checkpoint proteins, which activate the intra‐S and G2/M checkpoints. 相似文献
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Chromosomal cohesin forms a ring 总被引:46,自引:0,他引:46
The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils. Smc1 and Smc3 associate to form V-shaped heterodimers. Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules. We address here whether cohesin forms such rings in vivo. Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes. We show that N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer. Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin. 相似文献
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Nenggang Zhang Yunyun Jiang Qilong Mao Borries Demeler Yizhi Jane Tao Debananda Pati 《PloS one》2013,8(7)
The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the ''core'' cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301–750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located at 60–81 aa of the N-terminus and 383–392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, α-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this α-helical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SA-binding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21. 相似文献