In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin ...tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.
The Xenopus oocyte is known to accumulate filamentous or F‐actin in the nucleus, but it is currently unknown whether F‐actin also accumulates in embryo nuclei. Using fluorescence‐labeled actin ...reporters, we examined the actin distribution in Xenopus embryonic cells and found that F‐actin accumulates in nuclei during the blastula stage but not during the gastrula stage. To further investigate nuclear F‐actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F‐actin accumulates. Using this extract, we found that F‐actin accumulates primarily at the subnuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well‐developed nuclei. We also provide evidence that nuclear F‐actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F‐actin accumulation in rapidly dividing large Xenopus blastula cells.
While nuclear F‐actin accumulates in Xenopus oocytes, it is unknown whether it does so in embryos. Using fluorescence‐labeled actin reporters and Xenopus egg extracts, we demonstrate that F‐actin accumulates in blastula nuclei, promoting chromatin–nuclear envelope binding, nuclear structural stability and M‐phase mitotic chromosome alignment.
Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not ...constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or "intranuclear DNA density." Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.
Histone chaperones are a group of histone-binding proteins that facilitate the assembly of nucleosomes, the fundamental structural units of chromatin in eukaryotes. In nucleosome assembly, deposition ...of a histone H3-H4 tetramer onto DNA is the first and critical step, which is mediated by the histone chaperones HIRA and CAF-1. HIRA and CAF-1 are reportedly involved in DNA replication independent (RI) and replication coupled nucleosome assembly, respectively. However, the mechanisms by which they mediate histone deposition remain unclear. In this study, we focused on the mechanism by which HIRA induces RI-nucleosome assembly. We looked for HIRA domains that are required for nucleosome assembly and its localization to chromatin. We used cell-free extracts from Xenopus eggs that carry out RI-nucleosome assembly of plasmid DNA. We confirmed that HIRA formed stable complexes with Asf1, another histone H3-H4 chaperone, and the HIRA-Asf1 complex was solely responsible for RI-nucleosome assembly in egg extracts. We further demonstrated that the HIRA N-terminus containing the WD40 domain, which comprises seven WD40 repeats, and the B domain, to which Asf1 binds, were essential for RI-nucleosome assembly; the three WD40 repeats from the N-terminus were especially critical. Using egg extracts that reproduce nuclear formation accompanying the duplication of chromatin, we also demonstrated that the Hir domain was indispensable for the binding of HIRA to chromatin. Thus, the WD40 and B domains are the core elements for inducing RI-nucleosome assembly. Hir domain regulates the binding to chromatin. Based on these findings, similarities and differences between HIRA and CAF-1 are discussed.
Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the ...anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.
Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid ...transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic- for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.
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•Xenopus oocytes induce an oocyte transcription pattern in mouse nuclei in 2 days•Reprogramming requires a switch from somatic to oocyte transcriptional components•Unusually high amounts of oocyte-derived RNA polymerase II drive reprogramming•The pattern of oocyte linker histone binding to somatic chromatin is revealed
Jullien et al. identify a sequence of events leading to rapid transcriptional reprogramming of somatic nuclei after their transplantation to Xenopus oocytes. Transcription is switched selectively to an oocyte-type program in a process involving stepwise loading of oocyte-specific factors onto transplanted chromatin.
At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II ...(MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.
Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, ...comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency.
In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are ...known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA. Here we report that, in Xenopus eggs, the linker histone is associated with the Xenopus homologue of nucleosome assembly protein-1 (NAP-1), which is known to be a chaperone for the core histones H2A and H2B in Drosophila and mammalian cells Ito, T., Bulger, M., Kobayashi, R. & Kadonaga, J. T. (1996) Mol. Cell Biol. 16, 3112-3124; Chang, L., Loranger, S. S., Mizzen, C., Ernst, S. G., Allis, C. D. & Annunziato, A. T. (1997) Biochemistry 36, 469-480. We show that NAP-1 acts as the chaperone for the linker histone in both sperm chromatin remodeling into nucleosomes and linker histone binding to nucleosome core dimers. In the presence of NAP-1, the linker histone is properly deposited onto linker DNA at physiological ionic strength, without formation of nonspecific aggregates. These results strongly that NAP-1 functions as a chaperone for the linker histone in Xenopus eggs.
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