Chromatid Cohesion Research Articles

AREM: aligning short reads from ChIP-sequencing by expectation maximization.

High-throughput sequencing coupled to chromatin immunoprecipitation (ChIP-Seq) is widely used in characterizing genome-wide binding patterns of transcription factors, cofactors, chromatin modifiers, and other DNA binding proteins. A key step in ChIP-Seq data analysis is to map short reads from high-throughput sequencing to a reference genome and identify peak regions enriched with short reads. Although several methods have been proposed for ChIP-Seq analysis, most existing methods only consider reads that can be uniquely placed in the reference genome, and therefore have low power for detecting peaks located within repeat sequences. Here, we introduce a probabilistic approach for ChIP-Seq data analysis that utilizes all reads, providing a truly genome-wide view of binding patterns. Reads are modeled using a mixture model corresponding to K enriched regions and a null genomic background. We use maximum likelihood to estimate the locations of the enriched regions, and implement an expectation-maximization (E-M) algorithm, called AREM (aligning reads by expectation maximization), to update the alignment probabilities of each read to different genomic locations. We apply the algorithm to identify genome-wide binding events of two proteins: Rad21, a component of cohesin and a key factor involved in chromatid cohesion, and Srebp-1, a transcription factor important for lipid/cholesterol homeostasis. Using AREM, we were able to identify 19,935 Rad21 peaks and 1,748 Srebp-1 peaks in the mouse genome with high confidence, including 1,517 (7.6%) Rad21 peaks and 227 (13%) Srebp-1 peaks that were missed using only uniquely mapped reads. The open source implementation of our algorithm is available at http://sourceforge.net/projects/arem.

CDK11p58 Is Required for Centriole Duplication and Plk4 Recruitment to Mitotic Centrosomes

BackgroundCDK11p58 is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis.Methodology/Principal FindingsIn addition to these previously described roles, our study shows that CDK11p58 inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11p58, which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression.Conclusion/SignificanceWe thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11p58 could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.

Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation

PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome. This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution. Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.

Regulation of endoderm development by zebrafish Nipbl

Program/Abstract # 270 Regulation of endoderm development by zebrafish Nipbl Akihiko Muto a , Thomas F. Schilling b , Anne L. Calof b , Arthur D. Lander a a Dev & Cell Biol., Univ. of California, Irvine, CA, USA b Anatomy & Neurobiol., Univ. of California, Irvine, CA, USA Heterozygous loss of the Nipped-B-like gene (Nipbl) is the most common cause of Cornelia de Lange Syndrome (CdLS). Although Nipbl is a cohesin-associated protein conserved among all eukaryotes, recent studies suggest that it regulates gene expression through mechanisms independent of cohesin's established role in chromatid cohesion. There are 2 Nipbl genes in zebrafish, zNipbl-1 and zNipbl-2, and embryos injected with morpholinos targeting either gene (zNipbl-morphants) show defects in formation of the gut/visceral organs and heart with a range of severity from looping defects to organs duplication. Such morphants show significant reductions in sox32, sox17 and foxa2 expressions in endoderm cells, and we found that combined sox17/foxa2-double morphants exhibited gut bifurca- tions similar to zNipbl-morphants. Interestingly, the degree of sox17 and foxa2 suppression in zNipbl-morphants was greater than could be explained by the reduction in sox32 (a known positive regulator of these genes), and the response of these genes to ectopic sox32 was significantly blunted. These data suggest that zNipbls may influence sox17 and foxa2 transcription directly. In zNipbl-morphants, we also observed abnormal spatial expression of the left–right patterning genes, lefty2 and southpaw, although Kuppfer's vesicle (KV), a critical organ for left–right patterning that depends on sox32 function, formed normally, implying that zNipbls act downstream of KV formation. These results support the idea that zNipbls regulate embryonic development through modulating gene expression at multiple levels. Supported by NIH P01-HD052860. doi:10.1016/j.ydbio.2010.05.433

The different ties that bind

Two homologues of the cohesin protein Scc3 have specialized roles in chromatid cohesion.

Chromosome cohesion – rings, knots, orcs and fellowship

Sister-chromatid cohesion is essential for accurate chromosome segregation. A key discovery towards our understanding of sister-chromatid cohesion was made 10 years ago with the identification of cohesins. Since then, cohesins have been shown to be involved in cohesion in numerous organisms, from yeast to mammals. Studies of the composition, regulation and structure of the cohesin complex led to a model in which cohesin loading during S-phase establishes cohesion, and cohesin cleavage at the onset of anaphase allows sister-chromatid separation. However, recent studies have revealed activities that provide cohesion in the absence of cohesin. Here we review these advances and propose an integrative model in which chromatid cohesion is a result of the combined activities of multiple cohesion mechanisms.

Keeping sisters close

One mechanism for holding sister chromatids together during mitosis isn't enough, report Kenji Shimada and Susan Gasser (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland). They identify a second mechanism that involves the origin recognition complex, which serves earlier in the cell cycle to instigate DNA replication. Figure 1 Chromatids (green) fall apart when ORC vanishes (bottom). The cohesin complex connects sister chromatids until the metaphase-to-anaphase transition. But Shimada and Gasser depleted one of the ORC components from yeast cells and found evidence that it helps out. When production of one ORC component was switched off after replication was complete, the cells stalled in the G2 or M phase. The spindle checkpoint was activated, indicating that chromatid cohesion had gone awry. When the researchers tracked sister chromatid adhesion in cells lacking ORC, they found that the strands were prematurely separating at all three positions they checked. Reinstating the ORC component spurred the chromatids to reunite. Shimada and Gasser also determined that inserting extra copies of an ARS, an ORC-binding locus, into one yeast chromosome could restore normal sister attachment, even in cells lacking functional cohesin. The researchers conclude that, although ORC can't substitute for cohesin, the two complexes operate independently to strap sister chromatids together. Why cells need two methods to secure sister chromatids is a mystery, says Gasser. But, she adds, ORC's sister act may be a side effect of another of its suspected functions: linking heterochromatin domains. Reference: Shimada, K., and S. Gasser. 2007. Cell. 128:85–99. [PubMed]

Genetic Analysis of Saccharomyces cerevisiae H2A Serine 129 Mutant Suggests a Functional Relationship Between H2A and the Sister-Chromatid Cohesion Partners Csm3–Tof1 for the Repair of Topoisomerase I-Induced DNA Damage

Collision between a topoisomerase I-DNA intermediate and an advancing replication fork represents a unique form of replicative damage. We have shown previously that yeast H2A serine 129 is involved in the recovery from this type of damage. We now report that efficient repair also requires proteins involved in chromatid cohesion: Csm3; Tof1; Mrc1, and Dcc1. Epistasis analysis defined several pathways involving these proteins. Csm3 and Tof1 function in a same pathway and downstream of H2A. In addition, the pathway involving H2A/Csm3/Tof1 is distinct from the pathways involving the Ctf8/Ctf18/Dcc1 complex, the Rad9 pathway, and another involving Mrc1. Our genetic studies suggest a role for H2A serine 129 in the establishment of specialized cohesion structure necessary for the normal repair of topoisomerase I-induced DNA damage.

The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

Mouse centric and pericentric satellite repeats form distinct functional heterochromatin.

Heterochromatin is thought to play a critical role for centromeric function. However, the respective contributions of the distinct repetitive sequences found in these regions, such as minor and major satellites in the mouse, have remained largely unsolved. We show that these centric and pericentric repeats on the chromosomes have distinct heterochromatic characteristics in the nucleus. Major satellites from different chromosomes form clusters associated with heterochromatin protein 1α, whereas minor satellites are individual entities associated with centromeric proteins. Both regions contain methylated histone H3 (Me-K9 H3) but show different micrococcal nuclease sensitivities. A dinucleosome repeating unit is found specifically associated with major satellites. These domains replicate asynchronously, and chromatid cohesion is sustained for a longer time in major satellites compared with minor satellites. Such prolonged cohesion in major satellites is lost in the absence of Suv39h histone methyltransferases. Thus, we define functionally independent centromeric subdomains, which spatio-temporal isolation is proposed to be important for centromeric cohesion and dissociation during chromosome segregation.

Evidence of a Transcriptional Co-activator Function of Cohesin STAG/SA/Scc3

Cohesins hold sister chromatids together from DNA replication until they are segregated. Although cohesins Smc1, Smc3, and Scc1/Rad21 are involved in chromatid cohesion and other cellular processes, little is known about the other mitotic cohesin subunit, Scc3/STAG. Here we describe STAG/Scc3, which may act as a transcriptional co-activator. STAG2 is able to enhance the activity of the tumor necrosis factor alpha, the CD69, and the human immunodeficiency virus long terminal repeat promoters in a NF-kappaB-dependent manner. In addition, STAG2 interacts with the viral transactivator Tat and enhances the Tat-mediated activation of the human immunodeficiency virus long terminal repeat promoter. Moreover, STAG2 co-activates a multimeric NF-kappaB reporter construct and enhances the activity of the transactivation domain of p65/RelA in a Gal4 system. This function is dependent on one of the LXXLL co-activation motives present in this cohesin and is substantiated by the interaction of STAG2 with the p65 subunit of NF-kappaB. These results describe a novel activity for cohesins, suggesting a role for STAG/Scc3 in transcriptional regulation.

Structural Proteins of the SMC (Structural Maintenance of Chromosomes) Family and Their Role in Chromatin Reorganization

Structural chromatin proteins of the SMC (Structural Maintenance of Chromosomes) family play an important role in structural DNA reorganization in pro- and eukaryotes. Eukaryotic SMC proteins are the core components of the cohesin and condensin complexes. The cohesin complex is responsible for sister chromatid and homolog cohesion in mitosis and meiosis. The condensin complex uses ATP energy to induce positive coiled-coils in DNA, which results in compaction of the latter and formation of mitotic chromosome scaffold. In addition, the SMC proteins constitute recombination and recombination repair complexes. In hermaphrodites of nematode Caenorhabditis elegans, the SMC protein-containing complex controls dosage compensation and inactivation of the X chromosome genes.

Cis-Acting DNA from Fission Yeast Centromeres Mediates Histone H3 Methylation and Recruitment of Silencing Factors and Cohesin to an Ectopic Site

Background: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin. Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function. The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins. This centromeric heterochromatin is required for cohesion between sister centromeres. Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation. The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established.Results: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast. Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus. This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site. In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9.Conclusions: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.

Mechanisms of non-disjunction in human female meiosis: the co-existence of two modes of malsegregation evidenced by the karyotyping of 1397 in-vitro unfertilized oocytes

Although numerous studies have been published on the chromosomal constitution of in-vitro unfertilized human oocytes, data remain highly variable and controversial because of the size of oocyte samples, technical reservations and potential misinterpretation. A cytogenetic study was undertaken on 3042 unfertilized human oocytes recovered from 792 women participating in an IVF programme for various infertility problems. Both a gradual fixation technique and an R-banding procedure were used. The analysis was successful in 1397 oocytes (45.9%) for which interpretable metaphases were obtained. Of the 1397 oocyte karyotypes, 1088 (77.9%) were normal (23,X). The overall frequency of chromosomal abnormality was 22.1%. No correlation was found between the rate of abnormalities and the type of infertility. Aneuploidy was observed in 151 cells (10.8%), consisting of 5.4% hypohaploidies, 4.1% hyperhaploidies, 0.8% complex aneuploidies and 0.05% extreme aneuploidies with less than 18 chromosomes. Both whole chromosome non-disjunction and chromatid predivision contributed to the formation of aneuploid oocytes, but the numerical abnormalities due to single chromatids significantly exceeded conventional non-disjunctions. Abnormalities also included 5.4% diploid oocytes, 3.8% sets of chromatids alone and 2.1% structural aberrations. Aneuploidy was found in all chromosome groups. However, groups E and G exhibited significantly higher frequencies of non-disjunction than expected, whereas groups A and B showed a significantly low incidence of aneuploidy. The implication of both chromosome and chromatid abnormalities in the occurrence of non-disjunction are discussed in relation to the recent data on chromatid cohesion throughout cell division. The results were consistent with the hypothesis of an unequal occurrence of non-disjunction among the chromosome groups in female meiosis.

Human Chromatid Cohesin Component hRad21 Is Phosphorylated in M Phase and Associated with Metaphase Centromeres

Sister chromatids duplicated in S phase are connected with each other during G(2) and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells.

Chromosome segregation one hundred years after Mendel's rediscovery

In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This chromatid cohesion is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on a multisubunit protein complex called cohesin, which possibly forms the physical bridges that connect sisters. Proteolytic cleavage of cohesin's Scc1 subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. We show here that separin is a cysteine protease related to caspases and that it alone can cleave Scc1 in vitro. By replacing one of Scc1's cleavage sites by that for a different site specific protease, we show that cleavage of Scc1 in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.

Review: SMCs in the World of Chromosome Biology— From Prokaryotes to Higher Eukaryotes

The study of higher order chromosome structure and how it is modified through the course of the cell cycle has fascinated geneticists, biochemists, and cell biologists for decades. The results from many diverse technical avenues have converged in the discovery of a large superfamily of chromosome-associated proteins known as SMCs, for structural maintenance of chromosomes, which are predicted to have ATPase activity. Now found in all eukaryotes examined, and numerous prokaryotes as well, SMCs play crucial roles in chromatid cohesion, chromosome condensation, sex chromosome dosage compensation, and DNA recombination repair. In eukaryotes, SMCs exist in five subfamilies, which appear to associate with one another in particular pairs to perform their specific functions. In this review, we summarize current progress examining the roles these proteins, and the complexes they form, play in chromosome metabolism. We also present a twist in the SMC story, with the possibility of one SMC moonlighting in an unpredicted location.

Chromosome condensation factor Brn1p is required for chromatid separation in mitosis.

This work describes BRN1, the budding yeast homologue of Drosophila Barren and Xenopus condensin subunit XCAP-H. The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP-G. Overexpression of SMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex.

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatids of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division.(ABSTRACT TRUNCATED AT 250 WORDS)

Alien chromosome fragments conditioning resistance to beet cyst nematode in diploid descendants from monosomic additions of Beta procumbens to B. vulgaris

A monosomic addition plant of Beta vulgaris, containing an extra chromosome of B. procumbens with a gene for resistance to the beet cyst nematode, gave rise to a diploid descendant with an additional chromosome fragment. This fragment was found to bear the gene for resistance. A study of its morphology and behaviour made it likely that it represents the short arm of the alien chromosome and may have originated by centric fission of the single addition chromosome at anaphase I or II. The fragment showed incomplete generative transmission. In part of the somatic cells the fragment was missing and occasionally more than one fragment per cell was observed. The transmission of resistance in the telosome addition material was about 10%, which is 2.5 times lower than that of the monosomic addition. The pattern of transmission can be explained by an incomplete centromere, otherwise disturbed centric activity, or chromatid cohesiveness. Comparison with other monosomic addition plants showed that the resistance gene is located on the short arm of the satellite chromosome of B. procumbens and thus is linked to the nucleoar organizer region. The long arm of this chromosome possesses genes influencing annuality and leaf shape.Key words: Beta vulgaris, Beta procumbens, beet cyst nematode, monosomic addition, centric chromosome fragment.