Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase IIα localization and chromosome condensation

Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.     

H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis

Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.     

Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin''s Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin‐PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C‐dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.     

Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in poly (ADP-ribose) polymerase (Parp-1) null oocytes

In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1^(−/−) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1^(−/−) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains.     

Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice

Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects.     

Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy

Mouse oocytes isolated from large antral follicles were exposed to a wide range of concentrations of bisphenol A (BPA) during maturation in vitro (50 ng/ml to 10 microg/ml BPA in medium). Exposure to high concentrations of BPA (10 microg/ml) affected spindle formation, distribution of pericentriolar material and chromosome alignment on the spindle (termed congression failure), and caused a significant meiotic arrest. However, BPA did not increase hyperploidy at meiosis II at any tested concentration. Some but not all meiosis I arrested oocytes had MAD2-positive foci at centromeres of chromosomes in bivalents, suggesting that they had failed to pass the spindle checkpoint control. In a second set of experiments prepubertal mice were exposed sub-chronically for 7 days to low BPA by daily oral administration, followed by in vitro maturation of the denuded oocytes to metaphase II in the absence of BPA, as this treatment protocol was previously reported to induce chromosome congression failure and therefore suspected to cause aneuploidy in oocytes. The sub-chronic exposure subtly affected spindle morphology and oocyte maturation. However, as with the exposure in vitro, there was no evidence that low BPA doses increased hyperploidy at meiosis II. In conclusion, the data suggest that mouse oocytes from mice respond to BPA-induced disturbances in spindle formation by induction of meiotic arrest. This response might result from an effective checkpoint mechanism preventing the occurrence of chromosome malsegregation and aneuploidy. Low chronic BPA exposure in vivo as such does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes. Additional factors besides BPA may have caused the high rate of congression failure and the temporary increase in hyperploidy in mouse metaphase II oocytes reported previously.     

Spo13 facilitates monopolin recruitment to kinetochores and regulates maintenance of centromeric cohesion during yeast meiosis

BACKGROUND: Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication. During the first meiotic division, homologous chromosomes segregate to opposite poles. This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms. Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division. Little is known about how these meiosis I-specific events are regulated. RESULTS: Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores. Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20. CONCLUSION: Spo13 is a key regulator of several meiosis I events. The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions. Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin.     

The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis

Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.     

Hec1 inhibition alters spindle morphology and chromosome alignment in porcine oocytes

Aneuploidy is caused by incorrect chromosome segregation and can result in cancer or birth defects. The spindle assembly checkpoint (SAC) guarantees proper cell cycle progression. Highly Expressed in Cancer protein 1 (Hec1, also called Ndc80) is the core component of the Ndc80 complex and is involved in regulating both kinetochore-microtubule interactions and the SAC during mitosis in multiple cell types. However, its involvement in pig oocyte meiotic maturation remains uncertain. Thus, we investigated Hec1 expression, localization, and possible functions during porcine oocyte meiosis. Immunofluorescent staining showed that Hec1 was expressed in porcine oocytes and was associated with centromeres at both the metaphase I and metaphase II stages. Disrupting Hec1 function with its inhibitor INH1 resulted in polar body extrusion defects in porcine oocytes. Moreover, inhibiting Hec1 activity also resulted in severe chromosome misalignments and aberrant spindle morphology. Our results showed a unique localization pattern for Hec1 in porcine oocytes and suggested that Hec1 was required for chromosome alignment and spindle organization. Thus, Hec1 might regulate spindle checkpoint activity during mammalian oocyte meiosis.     

Phosphorylation of histone H2A is associated with centromere function and maintenance in meiosis

Histone phosphorylation is dynamically regulated during cell division, for example phosphorylation of histone H3 (H3)-Ser10, H3-Thr11 and H3-Ser28. Here we analyzed maize (Zea mays L) for Thr133-phosphorylated histone H2A, which is important for spindle checkpoint control and localization of the centromere cohesion protector Shugoshin in mammals and yeast. Immunostaining results indicate that phosphorylated H2A-Thr133 signals bridged those of the centromeric H3 histone variant CENH3 by using a plant displaying yellow fluorescent protein-CENH3 signals and H2A-Thr133 is phosphorylated in different cell types. During mitosis, H2A-Thr133 phosphorylation becomes strong in metaphase and is specific to centromere regions but drops during later anaphase and telophase. Immunostaining for several maize dicentric chromosomes revealed that the inactive centromeres have lost phosphorylation of H2A-Thr133. During meiosis in maize meiocytes, H2A phosphorylation becomes strong in the early pachytene stage and increases to a maximum at metaphase I. In the maize meiotic mutant afd1 (absence of first division), sister chromatids show equational separation at metaphase I, but there are no changes in H2A-Thr-133 phosphorylation during meiosis compared with the wild type. In sgo1 mutants, sister chromatids segregate randomly during meiosis II, and phosphorylation of H2A-Thr-133 is observed on the centromere regions during meiosis II. The availability of such mutants in maize that lack sister cohesion and Shugoshin indicate that the signals for phosphorylation are not dependent on cohesion but on centromere activity.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

Xkid chromokinesin is required for the meiosis I to meiosis II transition in Xenopus laevis oocytes

Xkid chromokinesin is required for chromosome alignment on the metaphase plate of spindles formed in Xenopus laevis egg extracts. We have investigated the role of Xkid in Xenopus oocyte meiotic maturation, a progesterone-triggered process that reinitiates the meiotic cell cycle in oocytes arrested at the G2/M border of meiosis I. Here we show that Xkid starts to accumulate at the time of germinal vesicle breakdown and reaches its largest quantities at metaphase II in oocytes treated with progesterone. Both germinal vesicle breakdown and spindle assembly at meiosis I can occur normally in the absence of Xkid. But Xkid-depleted oocytes cannot reactivate Cdc2/cyclin B after meiosis I and, instead of proceeding to meiosis II, they enter an interphase-like state and undergo DNA replication. Expression of a Xkid mutant that lacks the DNA-binding domain allows Xkid-depleted oocytes to complete meiotic maturation. Our results show that Xkid has a role in the meiotic cell cycle that is independent from its role in metaphase chromosome alignment.     

Trichlorfon predisposes to aneuploidy and interferes with spindle formation in in vitro maturing mouse oocytes

The pesticide trichlorfon (TCF) has been implicated in human trisomy 21, and in errors in chromosome segregation at male meiosis II in the mouse. We previously provided evidence that TCF interferes with spindle integrity and cell-cycle control during murine oogenesis. To assess the aneugenic activity of TCF in oogenesis, we presently analysed maturation, spindle assembly, and chromosome constitution in mouse oocytes maturing in vitro in the presence of 50 or 100 microg/ml TCF for 16 h or in pulse-chase experiments. TCF stimulated maturation to meiosis II at 50 microg/ml, but arrested meiosis in some oocytes at 100 microg/ml. TCF at 100 microg/ml was aneugenic causing non-disjunction of homologous chromosomes at meiosis I, a significant increase of the hyperploidy rate at metaphase II, and a significant rise in the numbers of oocytes that contained a 'diploid' set of metaphase II chromosomes (dyads). TCF elevated the rate of precocious chromatid segregation (predivision) at 50 and 100 microg/ml. Pulse-chase experiments with 100 microg/ml TCF present during the first 7 h or the last 9 h of maturation in vitro did not affect meiotic progression and induced intermediate levels of hyperploidy at metaphase II. Exposure to > or =50 microg/ml TCF throughout maturation in vitro induced severe spindle aberrations at metaphase II, and over one-third of the oocytes failed to align all chromosomes at the spindle equator (congression failure). These observations suggest that exposure to high concentrations of TCF induces non-disjunction at meiosis I of oogenesis, while lower doses may preferentially cause errors in chromosome segregation at meiosis II due to disturbances in spindle function, and chromosome congression as well as precocious separation of chromatids prior to anaphase II. The data support evidence from other studies that TCF has to be regarded as a germ cell aneugen.     

2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes

2-Methoxyestradiol (2-ME) is a metabolite of 17beta-estradiol and a natural component of follicular fluid. Local concentrations of 2-ME may be increased by exposure to environmental pollutants that activate the expression of enzymes in the metabolic pathway from 17beta-estradiol to 2-ME. It has been suspected that this may have adverse effects on spindle formation in maturing oocytes, which would affect embryo quality. To study the dose-response patterns, we exposed denuded mouse oocytes to 2-ME during in vitro maturation. Meiotic progression, spindle morphology, centrosome integrity, and chromosome congression were examined by immunofluorescence and noninvasive polarizing microscopy (PolScope). Chromosomal constituents were assessed after spreading and C-banding. 2-ME sustained MAD2L1 expression at the centromeres and increased the number of meiosis I-blocked oocytes in a dose-dependent manner. 2-ME also caused dramatic dose-dependent increases in the hyperploidy of metaphase II oocytes. Some of these meiosis II oocytes contained anaphase I-like chromosomes, which suggests that high concentrations of the catecholestradiol interfere with the physical separation of chromosomes. Noninvasive PolScope analysis and tubulin immunofluorescence revealed that perturbations in spindle organization, which resulted in severe disturbances of the chromosome alignment at the spindle equator (congression failure), were caused by 2-ME at meiosis I and II. Pericentrin-positive centrosomes failed to align at the spindle poles, and multipolar spindles and prominent arrays of cytoplasmic microtubule asters were induced in 2-ME-exposed metaphase II oocytes. In conclusion, a micromolar level of 2-ME is aneugenic for mammalian oocytes. Therefore, exposure to 2-ME and conditions that increase the intrinsic local concentration of 2-ME in the ovary may affect fertility and increase risks for chromosomal aberrations in the oocyte and embryo.     

Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein

The present study was designed to investigate the localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at various stages during the first meiosis were fixed and immunostained for MAD1, kinetochores, microtubules, and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes were treated with nocodazole or Taxol before examination. The anti-MAD1 antibody was injected into the oocytes at the germinal vesicle (GV) stage for examination of chromosome alignment and spindle formation. It was found that MAD1 was present in the oocytes from the GV to prometaphase I stages around the nuclei. When the oocytes reached the metaphase I (M-I) to metaphase II (M-II) stages, MAD1 was mainly localized at the spindle poles. However, MAD1 relocated to the vicinity of the chromosomes when spindles were disassembled by nocodazole or cooling, and the relocated MAD1 moved back to the spindle poles during spindle recovery. Taxol treatment did not affect the MAD1 localization. Although anti-MAD1 antibody injection did not affect nuclear maturation, significantly higher proportions of injected oocytes had misaligned chromosomes when the oocytes reached the M-I to M-II stages. The results of the present study indicate that MAD1 is present in mouse oocytes at all stages during the first meiosis and that it participates in spindle checkpoint during meiosis. However, MAD1 could not check misaligned chromosomes during spindle recovery after the spindles were destroyed by drug or cooling, which caused some chromosomes to scatter in the oocytes.     

Histone modifications related to chromosome silencing and elimination during male meiosis in Bengalese finch

We report here that a germline-restricted chromosome (GRC) is regularly present in males and females of the Bengalese finch (Lonchura domestica). While the GRC is euchromatic in oocytes, in spermatocytes this chromosome is cytologically seen as entirely heterochromatic and presumably inactive. The GRC is observed in the cytoplasm of secondary spermatocytes, indicating that its elimination from the nucleus occurs during the first meiotic division. By immunofluorescence on microspreads, we investigated the presence of histone H3 modifications throughout male meiosis, as well as in postmeiotic stages. We found that the GRC is highly enriched in di- and trimethylated histone H3 at lysine 9 during prophase I, in agreement with the presumed inactive state of this chromosome. At metaphase I, dimethylated histone H3 is no longer detectable on the GRC and its chromatin is more faintly stained with DAPI. The condensed GRC is underphosphorylated at serine 10 compared to the regular chromosomes during metaphase I, being phosphorylated later at this site after the first meiotic division. From these results, we proposed that trimethylation of histone H3 at lysine 9 on the GRC chromatin increases during metaphase I. This hypermethylated state at lysine 9 may preclude the phosphorylation of the adjacent serine 10 residue, providing an example of cross-talk of histone H3 modifications as described in experimental systems. The differential underphosphorylation of the GRC chromatin before elimination is interpreted as a cytologically detectable byproduct of deficient activity of Aurora B kinase, which is responsible for the phosphorylation of H3 at serine 10 during mitosis and meiosis.     

The centromeric heterochromatin of Costus spiralis: poorly methylated and transiently acetylated during meiosis

The centromeric region of Costus spiralis is characteristically composed of a small heterochromatic DAPI(+) band flanked by a discrete decondensed region. High concentrations of serine 10 of histone H3 (H3S10ph) around the DAPI(+) band in pachytene chromosomes and the location of this heterochromatin at the chromosome region directed towards the poles during metaphase-anaphase I confirm its integration into the centromeric region. Antibodies against both typical components of euchromatin histones (histone H4 acetylated at lysine 5 (H4K5ac) and histone H3 dimethylated at lysine 4 (H3K4me2)) and heterochromatin (dimethylated lysine 9 of H3 (H3K9me2) and anti-5-methylcytosine (5-mC)) were used to characterize the centromeric chromatin of this species during meiosis. In pachytene chromosomes, the decondensed terminal euchromatin of the chromosome arms were seen to be richer in H4K5ac and H3K4me2 histones, while the more condensed proximal region was relatively stronger labeled with anti-H3K9me2 and anti-5-methylcytosine (5-mC). The centromeric region itself, including the DAPI(+) band, was poor in all of these chromatin modifications, but it was highly enriched in H4K5ac at pachytene. Before and after this stage, the centromeric region was poorly labeled with anti-H4K5ac. Hypomethylation and hyperacetylation of any kind of heterochromatin has rarely been reported, and it may be related to the dominant role of the centromere domain over the heterochromatin repeats.     

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.