Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1 and TIF1 from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.Epigenetic regulation of cell differentiation is surprisingly reversible, as demonstrated in many vertebrate species by somatic cell nuclear cloning, a procedure to create genetically identical animals by replacing egg nuclei with somatic cell nuclei (29, 38, 52). The most striking evidence of this reversibility is the establishment of fertile mouse clones by using nuclei isolated from terminally differentiated lymphocytes and olfactory sensory neurons (24,33). Whereas the success rate of mouse cloning is less than 5% (72), some of the surviving mouse clones have unexpectedly normal gene expression profiles, as shown by proper expression of over 11,000 genes in the placentae and livers of newborn mouse clones (36, 69). Because no other experimental models with a comparable degree of genomic reversibility exist, with the exception of cell fusion between somatic cells and embryonic stem cells (20), nuclear cloning provides a valuable opportunity for us to investigate the mechanisms of genome-wide epigenetic reprogramming activities that are important for the future of regeneration medicine. One of the key questions in nuclear cloning is whether a few general reprogramming factors exist that can nonspecifically affect multiple genes in addition to the obviously necessary gene-specific activators and suppressors. Currently, there is no evidence to support the existence of such general reprogramming factors in egg cytoplasm.Massive nuclear swelling accompanied by global chromatin decondensation is one of the ha...
In Xenopus somatic cell nuclear cloning, the nucleoli of donor nuclei rapidly and almost completely disappear in egg cytoplasm. We previously showed that the germ cell-specific proteins FRGY2a and FRGY2b were responsible for this unusually drastic nucleolar disassembly. The nucleolar disassembly occurs without inhibition of pre-rRNA transcription, a well known trigger for nucleolar segregation, and the mechanism for the nucleolar disassembly by FRGY2a and FRGY2b remains largely unknown. In this study, we searched for FRGY2a-interacting proteins and investigated the functional consequences of their interactions through a series of experiments. We showed that during the nucleolar disassembly, FRGY2a localized to the nucleoli of isolated nuclei and was capable of disassembling purified nucleoli, suggesting a direct interaction between FRGY2a and nucleolar components. Using a His tag pulldown approach, we identified the abundant and multifunctional nucleolar protein B23 as a potential target of FRGY2a and its related human protein YB1. A specific interaction between FRGY2a/YB1 and B23 was confirmed by co-immunoprecipitation. Finally, B23 knockdown using short interfering RNA and a subsequent add-back experiment confirmed that B23 was necessary for nucleolar disassembly by YB1. We propose that FRGY2a and YB1 disassemble nucleoli by sequestering B23, which is associated with pre-ribosomes and other structurally important nucleolar components.Reversible disassembly of nucleoli is one of the most striking phenomena that donor nuclei exhibit in Xenopus somatic cell nuclear cloning. Physiologically, Xenopus eggs and early embryos do not contain nucleoli or transcribe rRNA until the midblastula transition, when zygotic transcription initiates and nucleoli are assembled (1). When somatic nuclei are injected into eggs, the nucleoli of the donor nuclei disappear within 40 min and reappear during the midblastula transition, exactly recapitulating physiological nucleolar disassembly and reassembly (2). To understand the molecular mechanism of nucleolar disassembly in the donor nuclei and gain new insight into the physiological dynamics of nucleoli in early Xenopus embryos, we established an in vitro nucleolar disassembly assay by combining Xenopus egg extract and somatic nuclei. Using this assay, we have found that the Xenopus germ cell-specific proteins FRGY2a and FRGY2b (collectively called FRGY2a/b) are responsible for the reversible disassembly of somatic nucleoli in egg cytoplasm (3).FRGY2a/b were initially isolated as key proteins responsible for masking maternally derived mRNA within eggs and early embryos to prevent premature translation of mRNA (4). Later, they were additionally discovered to be transcription factors for several germ cell-specific genes (5). FRGY2a (336 amino acids) and FRGY2b (324 amino acids) share 83.0% identity at the amino acid level, and each recombinant protein can disassemble nucleoli on its own (3). The N-terminal domains of these proteins contain the evolutionarily conserved cold shock domain...
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