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1.
组蛋白H3在氨基末端Ser10、Ser28、Thr11和Thr3等氨基酸残基的磷酸化修饰是一类在时间上和空间上与细胞有丝分裂相关的翻译后修饰事件。为了研究Thr11位点磷酸化作用的功能,利用SDS-PAGE、Western Blot、间接免疫荧光标记技术和激光共聚焦显微技术检测分析了人乳腺癌细胞(MCF-7)中Thr11磷酸化组蛋白H3在有丝分裂过程中的动态分布,以研究其在有丝分裂过程中的功能。结果显示:在MCF-7细胞中,组蛋白H3 Thr11的磷酸化发生在早前期细胞染色体的着丝粒处,成点状分布,继而在早中期达到最高水平,并以点状集中在赤道板上,在有丝分裂后期开始脱磷酸化,并于末期完成脱磷酸化。事实表明,H3 Thr11的磷酸化与细胞有丝分裂过程存在着时间和空间上的相关性。Thr11磷酸化H3只存在于着丝粒表明它可能参与有丝分裂期间功能性动原体的组成。这与Ser10磷酸化H3的分布及可能的功能截然相反。  相似文献   

2.
In this study, indirect immunofluorescence labeling was used to examine the cellular dynamic distribution of Thr11 phosphorylated H3 at mitosis in MCF-7 cells. The Thr11 phosphorylation was observed beginning at prophase at centromeres. Upon progression of mitosis, fluorescence signal was enhanced in the central region of the metaphase plate and maintained till anaphase at centromeres. During telophase, the fluorescent signal of Thr11 phosphorylated H3 disappears from centromeres, but the signal appears again at the midbody during cytokinesis, which suggests that the modified histones may take part in the formation of the midbody and play a crucial role in cytokinesis. Chromatin immunoprecipitation (ChIP) was used to confirm that Thr11 phosphorylated H3 is specifically associated with centromere DNA at prophase to metaphase, which is coincident with the results observed by immunofluorescence. In conclusion, there was a precise spatial and temporal correlation between H3 phosphorylation of Thr11 and stages of chromatin condensation. The timing of Thr11 phosphorylation and dephosphorylation in mitosis were similar to that reported for Ser10 phosphorylation of H3. The Thr11 phosphorylated H3 localized at centromeres during mitosis, which was different from the Ser10 phosphorylated H3 localized at telomere regions and Thr3 phosphorylated H3 localized along the chromosome arms. The results suggest that the Thr11 phosphorylation of histone H3 may play a specific role which was different from Ser10 and Thr3 phosphorylation in mitosis.  相似文献   

3.
A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.  相似文献   

4.
H1 histones are progressively phosphorylated during the cell cycle. The number of phosphorylated sites is zero to three in late S phase and increases to five or six in late G2 phase and M phase. It is assumed that this phosphorylation modulates chromatin condensation and decondensation, but its specific role remains unclear. Recently, it was shown that the somatic H1 histone subtype H1.5 becomes pentaphosphorylated during mitosis, with phosphorylated threonine 10 being the last site to be phosphorylated. We have generated an antiserum specific for human H1.5 phosphorylated at threonine 10. Immunofluorescence labeling of HeLa cells with this antiserum revealed that the phosphorylation at this site appears in prometaphase and disappears in telophase, and that this hyperphosphorylated form of H1.5 is mainly chromatin-bound in metaphase when chromatin condensation is maximal. In search of the kinase responsible for the phosphorylation at this site, we found that threonine 10 of H1.5 can be phosphorylated by glycogen synthase kinase-3 in vitro, but not by cyclin-dependent kinase 1/cyclin B and cyclin-dependent kinase 5/p35, respectively. Furthermore, addition of specific glycogen synthase kinase-3 inhibitors led to a reduction in phosphorylation at this site both in vivo and in vitro.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
Haspin phosphorylates histone H3 at Thr3 (H3T3ph) during mitosis [1, 2], providing a chromatin binding site for the chromosomal passenger complex (CPC) at centromeres to regulate chromosome segregation [3-5]. H3T3ph becomes increasingly focused at inner centromeres during prometaphase [1, 2], but little is known about how its level or location and the consequent chromosomal localization of the CPC are regulated. In addition, CPC binding to shugoshin proteins contributes to centromeric Aurora B localization [5, 6]. Recruitment of the shugoshins to centromeres requires the phosphorylation of histone H2A at Thr120 (H2AT120ph) by the kinetochore kinase Bub1 [7], but the molecular basis for the collaboration of this pathway with H3T3ph has been unclear. Here, we show that Aurora B phosphorylates Haspin to promote generation of H3T3ph and that Aurora B kinase activity is required for normal chromosomal localization of the CPC, indicating an intimate linkage between Aurora B and Haspin functions in mitosis. We propose that Aurora B activity triggers a CPC-Haspin-H3T3ph feedback loop that promotes generation of H3T3ph on chromatin. We also provide evidence that the Bub1-shugoshin-CPC pathway supplies a signal that boosts the CPC-Haspin-H3T3ph feedback loop specifically at centromeres to produce the well-known accumulation of the CPC in these regions.  相似文献   

8.
The chromosomal location of centromere-specific histone H3 (CENH3) is the assembly site for the kinetochore complex of active centromeres. Chromatin immunoprecipitation data indicated that CENH3 interacts in barley with cereba, a centromeric retroelement (CR)-like element conserved among cereal centromeres and barley-specific GC-rich centromeric satellite sequences. Anti-CENH3 signals on extended chromatin fibers always colocalized with the centromeric sequences but did not encompass the entire area covered by such centromeric repeats. This indicates that the CENH3 protein is bound only to a fraction of the centromeric repeats. At mitotic metaphase, CENH3, histone H3, and serine 10 phosphorylated histone H3 predominated within distinct structural subdomains of the centromere, as demonstrated by immunogold labeling for high resolution scanning electron microscopy.  相似文献   

9.
10.
在时间上与细胞周期相关并且在功能上又与染色质凝集偶联的一类组蛋白翻译后修饰就是组蛋白H3磷酸化。运用一个针对H3 Ser10磷酸化的特异性抗体 ,通过SDS PAGE、免疫印迹和免疫荧光标记检测了磷酸化H3在MCF 7细胞周期中的分布。共聚焦显微结果显示 :H3磷酸化在早前期细胞核膜附近以斑点状起始 ,之后扩展到整个凝集的染色质上 ,然后在早中期达到最高水平。H3去磷酸化开始于有丝分裂后期 ,很快在末期完成 ,而此时末期细胞凝集的染色质并未完全解凝集。H3磷酸化与染色质初期凝集之间存在着精确的时间和空间上的相关性。另外 ,对H3磷酸化可能的作用进行了讨论。  相似文献   

11.
Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.  相似文献   

12.
The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.  相似文献   

13.
In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.  相似文献   

14.
《Epigenetics》2013,8(7):672-675
The kinetochore is formed on centromeric DNA as a key interface with microtubules from the mitotic spindle to achieve accurate chromosome segregation during mitosis. However, in contrast to other regions of the chromosome, the position of the kinetochore is specified by sequence-independent epigenetic mechanisms. Most recent work on kinetochore specification has focused on the centromere-specific histone H3-variant CENP-A. Whereas CENP-A is an important epigenetic marker for the kinetochore specification, it is unclear how centromeric chromatin structure is organized. To understand centromeric chromatin structure, we focused on additional centromere proteins that have an intrinsic DNA binding activity and identified the DNA binding CENP-T-W-S-X complex. Tetramer formation of CENP-T-W-S-X is essential for functional kinetochore assembly in vertebrate cells. Our structural and biochemical analysis reveals that the CENP-T-W-S-X complex is composed of four histone-fold domains with structural similarity to nucleosomes and displays DNA supercoiling activity. These results suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure at centromeric chromatin. In addition, CENP-S and CENP-X function at non-centromeric sites. The intriguing histone-like properties of these proteins suggest that they may form nucleosome-like structures at various genome loci, extending the chromatin code beyond classical histone variants.  相似文献   

15.
Cobb J  Miyaike M  Kikuchi A  Handel MA 《Chromosoma》1999,108(7):412-425
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.  相似文献   

16.
The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere‐specific histone H3 variant CenH3, originally called CENP‐A, which replaces canonical histone H3 at centromeres. CenH3‐chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3‐chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly.  相似文献   

17.
The kinetochore is a complex multiprotein structure located at centromeres that is essential for proper chromosome segregation. Budding-yeast Cse4 is an essential evolutionarily conserved histone H3 variant recruited to the centromere by an unknown mechanism. We have identified Scm3, an inner kinetochore protein that immunopurifies with Cse4. Scm3 is essential for viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin after DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. We propose a model in which Ndc10/Scm3 binds to centromeric DNA, which is in turn essential for targeting Cse4 to centromeres.  相似文献   

18.
Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.  相似文献   

19.
CENP-A locates at nucleosome as histone H3-like proteins, and is phosphorylated during mitosis. We investigated the dynamic distribution of p-CENP-A to explore the details of its function. We found that p-CENP-A was phosphorylated at late prophase, and the signal of p-CENP-A arranged at equatorial plate along with nucleosomes at metaphase, but moved to midbody at later phase of mitosis. The phosphorylation modification of CENP-A shares some characters of H3, but has different temporal patterns during mitosis. Our results suggested that the CENP-A might have similar functions as H3, but with different patterns for their different binging materials. Dengwen Li and Ruming Liu had contributed equally to this paper.  相似文献   

20.
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