Nobuaki Kikyo

Regulation of embryonic stem cell self-renewal and pluripotency by leukaemia inhibitory factor

LIF (leukaemia inhibitory factor) is a key cytokine for maintaining self-renewal and pluripotency of mESCs (mouse embryonic stem cells). Upon binding to the LIF receptor, LIF activates three major intracellular signalling pathways: the JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3), PI3K (phosphoinositide 3-kinase)/AKT and SHP2 [SH2 (Src homology 2) domain-containing tyrosine phosphatase 2]/MAPK (mitogen-activated protein kinase) pathways. These pathways converge to orchestrate the gene expression pattern specific to mESCs. Among the many signalling events downstream of the LIF receptor, activation and DNA binding of the transcription factor STAT3 plays a central role in transducing LIFs functions. The fundamental role of LIF for pluripotency was highlighted further by the discovery that LIF accelerates the conversion of epiblast-derived stem cells into a more fully pluripotent state. In the present review, we provide an overview of the three major LIF signalling pathways, the molecules that interact with STAT3 and the current interpretations of the roles of LIF in pluripotency.

Chromatin Decondensation and Nuclear Reprogramming by Nucleoplasmin

ABSTRACT 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 HP1β and TIF1β 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.

Reversible disassembly of somatic nucleoli by the germ cell proteins FRGY2a and FRGY2b

Egg cytoplasm has the capability to reprogramme differentiated somatic nuclei, as shown by nuclear transplantation in animal cloning. The nucleoli of donor nuclei are rapidly disassembled on injection into interphase eggs and are correctly reassembled when donor transcription initiates in the early embryos of frogs and mammals, recapitulating the physiological nucleolar dynamics of early embryogenesis. This is one of the most remarkable structural reorganizations of somatic nuclei in nuclear cloning. Despite the long history of nuclear cloning, almost nothing is known about the molecular mechanism of nucleolar disassembly in egg cytoplasm. Here we show that the Xenopus germ cell proteins FRGY2a and FRGY2b reversibly disassemble somatic nucleoli in egg cytoplasm, independently of continuing ribosomal RNA transcription. The carboxy-terminal domain of FRGY2a, which localizes to the nucleoli, is sufficient for nucleolar disassembly in transfected cells. Our results show that a single protein fragment can trigger reversible disassembly of the complex nucleolar structure.

Critical role of nucleostemin in pre-rRNA processing.

Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation.

Multiple ISWI ATPase complexes from Xenopus laevis: Functional conservation of an ACF/CHRAC homolog

The nucleosomal ATPase ISWI is the catalytic subunit of several protein complexes that either organize or perturb chromatin structure in vitro. This work reports the cloning and biochemical characterization of a Xenopus ISWI homolog. Surprisingly, whereas we find four complex forms of ISWI in egg extracts, we find no functional homolog of NURF. One of these complexes, xACF, consists of ISWI, Acf1, and a previously uncharacterized protein of 175 kDa. Like both ACF and CHRAC, this complex organizes randomly deposited histones into a regularly spaced array. The remaining three forms include two novel ISWI complexes distinct from known ISWI complexes plus a histone-dependent ATPase complex. This comprehensive biochemical characterization of ISWI underscores the evolutionary conservation of the ACF/CHRAC family.

Radical Acceleration of Nuclear Reprogramming by Chromatin Remodeling with the Transactivation Domain of MyoD

Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c‐Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M3O). Transduction of M3O as well as Sox2, Klf4, and c‐Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50‐fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far‐reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology. STEM CELLS 2011; 29:1349–1361

Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain

AIMS Fibroblasts can be directly reprogrammed to cardiomyocyte-like cells by introducing defined genes. However, the reprogramming efficiency remains low, delaying the clinical application of this strategy to regenerative cardiology. We previously showed that fusion of the MyoD transactivation domain to the pluripotency transcription factor Oct4 facilitated the transcriptional activity of Oct4, resulting in highly efficient production of induced pluripotent stem cells. We examined whether the same approach can be applied to cardiac transcription factors to facilitate cardiac reprogramming. METHODS AND RESULTS We fused the MyoD domain to Mef2c, Gata4, Hand2, and Tbx5 and transduced these genes in various combinations into mouse non-cardiac fibroblasts. Transduction of the chimeric Mef2c with the wild-types of the other three genes produced much larger beating clusters of cardiomyocyte-like cells faster than the combination of the four wild-type genes, with an efficiency of 3.5%, >15-fold greater than the wild-type genes. CONCLUSION Fusion of a powerful transactivation domain to heterologous factors can increase the efficiency of direct reprogramming of fibroblasts to cardiomyocytes.

Nuclear reprogramming in mammalian somatic cell nuclear cloning

Nuclear cloning is still a developing technique used to create genetically identical animals by somatic cell nuclear transfer into unfertilized eggs. Despite an intensive effort in a number of laboratories, the success rate of obtaining viable offspring from this technique remains less than 5%. In the past few years many investigators reported the reprogramming of specific nuclear activities in cloned animals, such as genome-wide gene expression patterns, DNA methylation, genetic imprinting, histone modifications and telomere length regulation. The results highlight the tremendous difficulty the clones face to reprogram the original differentiation status of the donor nuclei. Nevertheless, nuclei prepared from terminally differentiated lymphocytes can overcome this barrier and produce apparently normal mice. Study of this striking nuclear reprogramming activity should significantly contribute to our understanding of cell differentiation in more physiological settings.

Microvesicles as mediators of tissue regeneration

The use of stem cells in the treatment of various diseases and injuries has received increasing interest during the past decade. Injected stem cells, such as mesenchymal stem cells, stimulate tissue repair largely through the secretion of soluble factors that regulate various processes of tissue regeneration, including inflammatory responses, apoptosis, host cell proliferation, and angiogenesis. Recently, it has become apparent that stem cells also use membranous small vesicles, collectively called microvesicles, to repair damaged tissues. Microvesicles are released by many types of cells and exist in almost all types of body fluids. They serve as a vehicle to transfer protein, messenger RNA, and micro RNA to distant cells, altering the gene expression, proliferation, and differentiation of the recipient cells. Although animal models and in vitro studies have suggested promising applications for microvesicles-based regeneration therapy, its effectiveness and feasibility in clinical medicine remain to be established. Further studies of the basic mechanisms responsible for microvesicle-mediated tissue regeneration could lead to novel approaches in regenerative medicine.

Novel role of nucleostemin in the maintenance of nucleolar architecture and integrity of small nucleolar ribonucleoproteins and the telomerase complex.

Nucleostemin (NS) is a nucleolar protein involved in the regulation of cell proliferation. Both overexpression and knockdown of NS increase the activity of the tumor suppressor protein p53, resulting in cell cycle arrest. In addition, NS regulates processing of pre-rRNA and consequently the level of total protein synthesis. Here, we describe a previously uncharacterized function of NS in the maintenance of the tripartite nucleolar structure as well as the integrity of small nucleolar ribonucleoproteins (snoRNPs). NS is also necessary to maintain the telomerase complex which shares common protein subunits with the H/ACA box snoRNPs. First, immunofluorescence microscopy and electron microscopy demonstrated that knockdown of NS disorganized the nucleolar architecture, in particular, the dense fibrillar component where snoRNPs are localized. Second, gel filtration chromatography and immunoprecipitation indicated that NS depletion leads to dissociation of the components of snoRNPs and the telomerase complex. Third, NS depletion reduced both telomerase activity and the cellular level of pseudouridine, an H/ACA snoRNP-mediated modification of rRNA and other RNAs that are important for their folding and stability. These morphological, biochemical and functional studies demonstrate that NS plays an important role to maintain nucleolar structure and function on a more fundamental level than previously thought.