Sequence of Centromere Separation: Role of Centromeric Heterochromatin

The late metaphase-early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late replicating X chromosome appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separation of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of constitutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the genome separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quantitative differences in controlling the separation of centromeres.     

High Resolution Techniques for Study of Human Centromeric Heterochromatin

A high resolution procedure for analyzing human centromeric heterochromatin is described. The combined use of decondensation agents of pericentromeric heterochromatin and electron microscopy of whole mounted chromosomes allows a more precise identification of centromere and pericentromeric regions.     

MYPT1 Sustains Centromeric Cohesion and the Spindle-Assembly Checkpoint

During mitosis, cohesins hold the sister chromatids together until anaphase when arm cohesins are removed （Peters et al., 2008; Yao and Dai, 2012）. The shugoshin （Sgo） proteins play pivotal roles during this stage. There is only one shu- goshin in the fly and budding yeasts, while there are two in other organisms （including fission yeasts）. The two mamma- lian shugoshins, Sgol and Sgo2, carry out distinct functions： Sgol mainly in mitosis, and Sgo2 mainly in meiosis and perturbed mitosis. Mitotic cyclin-dependent kinase 1 （CDKI） phosphorylates Sgol, and targets the Sgol-protein phospha- tase 2A （PP2A） complex to protect centromeric cohesin （Kitajima et al., 2006; Tang et al., 2006; Liu et al., 2012）,     

Mutational Analysis of the Drosophila Sister-Chromatid Cohesion Protein Ord and Its Role in the Maintenance of Centromeric Cohesion

The ord gene is required for proper segregation of all chromosomes in both male and female Drosophila meiosis. Here we describe the isolation of a null ord allele and examine the consequences of ablating ord function. Cytologically, meiotic sister-chromatid cohesion is severely disrupted in flies lacking ORD protein. Moreover, the frequency of missegregation in genetic tests is consistent with random segregation of chromosomes through both meiotic divisions, suggesting that sister cohesion may be completely abolished. However, only a slight decrease in viability is observed for ord null flies, indicating that ORD function is not essential for cohesion during somatic mitosis. In addition, we do not observe perturbation of germ-line mitotic divisions in flies lacking ORD activity. Our analysis of weaker ord alleles suggests that ORD is required for proper centromeric cohesion after arm cohesion is released at the metaphase I/anaphase I transition. Finally, although meiotic cohesion is abolished in the ord null fly, chromosome loss is not appreciable. Therefore, ORD activity appears to promote centromeric cohesion during meiosis II but is not essential for kinetochore function during anaphase.     

Sequential Assembly of Centromeric Proteins in Male Mouse Meiosis

The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E.     

Chromosomal G-dark Bands Determine the Spatial Organization of Centromeric Heterochromatin in the Nucleus

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric alpha-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric alpha-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that "chromosomal environment" plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.     

Biotin-Tag Affinity Purification of a Centromeric Nucleosome Assembly Complex

Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, we used controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a singlechaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. Here, we highlight the benefits of our improved biotin-mediated purification method, and address the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres.     

Regulation of Centromeric Cohesion by Sororin Independently of the APC/C

Regulated separation of sister chromatids is the key event of mitosis. Sister chromatids remain cohered from the moment of DNA duplication until anaphase. Two known factors account for cohesion: DNA catenations and cohesin complexes. Premature loss of centromeric cohesion is prevented by the spindle checkpoint. Here we show that sororin, a protein implicated in promoting cohesion through effects on cohesin complexes, is involved in maintenance of cohesion in response to the spindle checkpoint. Sororin-depleted cells reach prometaphase with cohered sister chromatids and are able to form metaphase plates. However, loss of cohesion in anaphase is asynchronous and cells are unresponsive to the spindle checkpoint, accumulating with separated sisters scattered throughout the cytoplasm. These phenotypes are similar to those seen after Shugoshin depletion, suggesting that sororin and Shugoshin might act in concert. Furthermore, sororin-depleted and Shugoshin-depleted cells lose cohesion independently of the APC/C. Therefore, sororin and Shugoshin protect centromeric cohesion in response to the spindle checkpoint, but prevent the removal of cohesion by a mechanism independent of the APC/C.     

Inhibitors of Histone Deacetylases Alter Kinetochore Assembly by Disrupting Pericentromeric Heterochromatin

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-? on pericentromeric heterochromatin in S-phase and G2, decreased pericentromeric targeting of Aurora B kinase, resulting in decreased pre-mitotic phosphorylation of pericentromeric histone H3(S10) in G2, followed by assembly of deficient kinetochores in M-phase. HP1-?, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.     

Genetic Analysis of the Centromeric Heterochromatin of Chromosome 2 of DROSOPHILA MELANOGASTER: Deficiency Mapping of Ems-Induced Lethal Complementation Groups

Until recently, little was known of the genetic constitution of the heterochromatic segments of the major autosomes of Drosophila melanogaster . Our previous report described the genetic dissection of the proximal, heterochromatic region of chromosome 2 of Drosophila melanogaster by means of a series of overlapping deficiencies generated by the detachment of compound second autosomes (Hilliker and Holm 1975). Analysis of these deficiencies by inter se complementation, pseudo-dominance tests with proximal mutations and allelism tests with known deficiencies provided evidence for the existence of at least two loci between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. These data in conjunction with cytological observations demonstrated that light and rolled and three loci lying between them are located within the proximal heterochromatin of the second chromosome.——The present report describes the further analysis of this region through the induction with ethyl methanesulphonate (EMS) of recessive lethals allelic to the 2L and 2R proximal deficiencies associated with the detachment products. Analysis of the 118 EMS-induced recessive lethals and visible mutations recovered provided evidence for seven loci in the 2L heterochromatin and six loci in the 2R heterochromatin, with multiple alleles being obtained for most sites. Of these loci, one in 2L and two in 2R fall near the heterochromatic-euchromatic junctions of 2L and 2R respectively. None of the 113 EMS lethals behaved as a deficiency, implying that the heterochromatic loci uncovered in this study represent nonrepetitive cistrons. Thus functional genetic loci are found in heterochromatin, albeit at a very low density relative to euchromatin.     

The Post-anaphase SUMO Pathway Ensures the Maintenance of Centromeric Cohesion through Meiosis I-II Transition in Mammalian Oocytes

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Population Genetics of Tandem Repeats in Centromeric Heterochromatin: Unequal Crossing over and Chromosomal Divergence at the Responder Locus of Drosophila Melanogaster

The Responder (Rsp) locus in Drosophila melanogaster is the target locus of segregation distortion and is known to be comprised of a tandem array of 120-bp repetitive sequences. In this study, we first determined the large scale molecular structure of the Rsp locus, which extends over a region of 600 kb on the standard sensitive (cn bw) chromosome. Within the region, small Rsp repeat arrays are interspersed with non-Rsp sequences and account for 10-20% of the total sequences. We isolated and sequenced 32 Rsp clones from three different chromosomes. The main results are: (1) Rsp repeats isolated from the same chromosome are not more similar than those from different chromosomes. This implies either that there are more homologous exchanges at the Rsp locus than expected or, alternatively, that the second chromosomes of D. melanogaster have diverged from one another more recently at the centromeric heterochromatin than at the nearby euchromatin. (2) The repeats usually have a dimeric structure with an average difference of 16% between the left and right halves. The differences allow us to easily identify the products of unequal exchanges. Despite the large differences between the two halves, exchanges have occurred frequently and the majority of them fall within a 29-bp interval of identity between the two halves. Our data thus support the suggestion that recombination depends on short stretches of complete identity rather than long stretches of general homology. (3) Frequent unequal crossover events obscure the phylogenetic relationships between repeats; therefore, different parts of any single repeat could often have different phylogenetic histories. The high rate of unequal crossing over may also help explain the evolutionary dynamics of the Rsp locus.