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RNA-directed transposon silencing operates in the mammalian soma and germline to safeguard genomic integrity. The piRNA pathway and the HUSH complex identify active transposons through recognition of their nascent transcripts, but mechanistic understanding of how these distinct pathways evolved is lacking. TASOR is an essential component of the HUSH complex. TASOR's DUF3715 domain adopts a pseudo-PARP structure and is required for transposon silencing in a manner independent of complex assembly. TEX15, an essential piRNA pathway factor, also contains the DUF3715 domain. Here, we show that TASOR's and TEX15's DUF3715 domain share extensive structural homology. We found that the DUF3715 domain arose in early eukaryotes and that in vertebrates it is restricted to TEX15, TASOR, and TASORB orthologs. While TASOR-like proteins are found throughout metazoa, TEX15 is vertebrate-specific. The branching of TEX15 and the TASOR-like DUF3715 domain likely occurred in early metazoan evolution. Remarkably, despite this vast evolutionary distance, the DUF3715 domain from divergent TEX15 sequences can functionally substitute the DUF3715 domain of TASOR and mediates transposon silencing. We have thus termed this domain of unknown function as the RNA-directed pseudo-PARP transposon silencing (RDTS) domain. In summary, we show an unexpected functional link between these critical transposon silencing pathways.

The identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein–DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps, or chemical protein–DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical limitations. Here we describe an optimized DamID method coupled with next-generation sequencing (DamID-seq) in mouse cells and demonstrate the identification of the binding sites of two TFs, POU5F1 (also known as OCT4) and SOX2, in as few as 1000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. POU5F1 DamID-seq in the gastrulating mouse embryo at 7.5 d post coitum (dpc) successfully identified multiple POU5F1 binding sites proximal to genes involved in embryo development, neural tube formation, and mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigation of TF–DNA interactions and GRNs in specific cell types of limited availability in mammals, including in vivo samples.

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self‐renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self‐renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. StemCells2015;33:699–712

The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for embryonic development and pluripotent stem cell differentiation. In this study, we investigated whether NuRD is also involved in the reverse biological process of induction of pluripotency in neural stem cells. By knocking out MBD3, an essential scaffold subunit of the NuRD complex, at different time points in reprogramming, we found that efficient formation of reprogramming intermediates and induced pluripotent stem cells from neural stem cells requires NuRD activity. We also show that reprogramming of epiblast-derived stem cells to naive pluripotency requires NuRD complex function and that increased MBD3/NuRD levels can enhance reprogramming efficiency when coexpressed with the reprogramming factor NANOG. Our results therefore show that the MBD3/NuRD complex plays a key role in reprogramming in certain contexts and that a chromatin complex required for cell differentiation can also promote reversion back to a naive pluripotent cell state.

Oct4 is an essential regulator of pluripotency in vivo and in vitro in embryonic stem cells, as well as a key mediator of the reprogramming of somatic cells into induced pluripotent stem cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here, we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopushomolog of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported embryonic stem cell self-renewal in vitro at lower concentrations than that required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes.

We reviewed our structure studies on poly(vinyl alcohol) (PVA) gels formed in the mixtures of dimethyl sulfoxide (DMSO) and water using various scattering methods, including wide-, small- and ultra-small-angle neutron scattering and light scattering. These studies revealed the hierarchic structure of PVA gels formed in DMSO/water (60/40 v/v) in a very wide spatial scale from 1 Å to several micrometers. The crosslinking points are crystallites with radii of ~70 Å, the distance between the neighboring crystallites is 180–200 Å, and the bicontinuous structure due to liquid–liquid phase separation is in µm scale. We also review our dynamic studies on three kinds of PVA gels at the nanometer scale using neutron spin echo techniques to separate the concentration fluctuations observed by small-angle neutron scattering into the static and dynamics contributions.

In situ observation of the formation process of a mesophase of isotactic polypropylene is reported from a structural point of view. A very rapid transformation from the molten state to the mesophase has, for many years, made in situ observation difficult. In the present study, a rapid temperature jump and high-flux synchrotron radiation X-ray scattering techniques were combined effectively to observe this transformation. Fast time-resolved wide-angle X-ray diffraction during rapid cooling clearly shows the time evolution of the transformation from the molten state to the mesophase. The transformation proceeded very quickly in a narrow temperature range below ca 35?°C. Furthermore, the transformation was accompanied by instantaneous density fluctuations throughout the system, as visualized by microscopic observations. These observations suggest that the mesophase formation proceeds similarly to spinodal decomposition.

Small‐angle neutron scattering (SANS) measurements were performed on poly(vinyl alcohol) (PVA) gels in a mixture of deuterated dimethyl sulfoxide (DMSO‐d6) and D2O with volume ratio of 60/40 to see the structure changes of the crosslinking points, which are crystallites, and of the gel network during the melting process. The observed SANS intensities were fitted to the Ornstein–Zernike (OZ) formula and a power law in scattering vector Q ranges from 0.01 to 0.035 and 0.05 to 0.1 Å−1, respectively, to evaluate the correlation length ξ and the power law exponent n. It was found that the exponent n is 4 and the correlation length ξ is ∼ 150 Å below ≈70°C, suggesting that the crystallite surface is smooth and the average distance between the neighboring crystallites is ≈150Å. On the other hand, they begin to decrease >70°C. The decrease of n suggests that the surface of the crystallites becomes rougher with increasing temperature. As for the correlation length ξ, analyses in terms of distance distribution function suggested that the decrease of ξ is apparent, and the intercrystallite distance increases with temperature >70°C because the crystallites decrease in number because of melt. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 95: 157–160, 2005

It has been considered that crystallization of polymer from melt proceeds via the coexistence of molten matrix and growing crystals that have once overcome a nucleation barrier to a critical size. The nucleation process has often been explained analogously with so-called nucleation and growth (NG) behavior of the phase separation of a binary mixture in metastable conditions, although the crystallization in one-component polymer is not a real component separation but a phase transition. Among the mechanisms of polymer crystallization, the topic is whether a liquid–liquid transition between states of different densities within one-component polymers takes place before the aforementioned nucleation process. The liquid–liquid transition between states, which is probably driven by chain orientation, is also categorized into NG and the controversial spinodal decomposition (SD) type processes depending on the quenching depth. This article provides the optical microscopic observations that favor the occurrence of the SD-like process when a one-component polymer melt is very rapidly quenched below a stability limit, including a drastic morphological change from a spherulitic to a spinodal pattern at the critical (or spinodal) temperature. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 1817–1822, 2004

The origin of the variety of characteristics of the reduced viscosity of aqueous suspensions of charged spherical particles has been an unsolved problem. To solve the problem, the reduced viscosity due to interparticle electrostatic interactions between charged spherical particles are calculated as a function of particle concentration with scanning various parameters, such as diameter of particle, number of charges per particle, and added-salt concentration. The result successfully reproduced the variety of characteristics. Of all the scanned parameters, the diameter of the particle has a significant role to display the variety of characteristics when other parameters are fixed. When the diameter is very small (~0 Å), the calculated reduced viscosity of aqueous suspensions of charged spherical particles increases with decreasing particle concentration and it shows a maximum. This behavior is very similar to the reduced viscosity of linear chain polyelectrolyte solutions. Whereas, when the diameter is large (>2000 Å), the calculated reduced viscosity decreases with decreasing particle concentration and it does not show a maximum. When the diameter is <1000 Å, the calculated reduced viscosity shows both the maximum and minimum. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 1068–1074, 2004

Intermolecular electrostatic interactions of polyions contributing to the reduced viscosity are calculated for two molecular models of charged points and skewed rods. These simplified models approximately describe inter-polyion configurations in the actual polyelectrolyte solutions to qualitatively reproduce the characteristic peak of the reduced viscosity appearing at lower concentrations. The long-standing theoretical equation by Rice and Kirkwood was adopted for this calculation because of its simplicity. For the potential term of this equation, we assumed a Debye type screening potential as a function of ionic strength. The results show that the skewed rods model well fits with the experimental data. Furthermore, it should be pointed out that the configurations of this model correspond to the basic constituents of the so-called isotropic model proposed by de Gennes et al.

Although tumor necrosis factor receptor‐associated factor 6 (TRAF6) is required in receptor activator of NF‐κB‐receptor activator of NF‐κB ligand (RANK‐RANKL) signaling for osteoclastogenesis, it has remained unclear whether TRAF6 is crucial in tumor necrosis factor α (TNF‐α)‐induced osteoclastogenesis. We examined TRAF6 function in the TNF‐α‐induced osteoclastogenesis by using osteoclast progenitor cells from TRAF6‐deficient mice. The results indicated that TNF‐α did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c‐junN‐terminal kinase (JNK) and TNF‐α activation was observed in osteoclast progenitor cells. Thus, we have provided the first evidence showing that TRAF6 is involved in TNF‐α‐induced osteoclastogenesis.

Although tumor necrosis factor receptor‐associated factor 6 (TRAF6) is required in receptor activator of NF‐κB‐receptor activator of NF‐κB ligand (RANK‐RANKL) signaling for osteoclastogenesis, it has remained unclear whether TRAF6 is crucial in tumor necrosis factor α (TNF‐α)‐induced osteoclastogenesis. We examined TRAF6 function in the TNF‐α‐induced osteoclastogenesis by using osteoclast progenitor cells from TRAF6‐deficient mice. The results indicated that TNF‐α did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c‐junN‐terminal kinase (JNK) and TNF‐α activation was observed in osteoclast progenitor cells. Thus, we have provided the first evidence showing that TRAF6 is involved in TNF‐α‐induced osteoclastogenesis.

Osteoclasts are thought to belong to a macrophage lineage. However, the nature of common precursors of osteoclasts and macrophages remains to be investigated. We have characterized the differentiation potential of mouse bone marrow macrophages into mature osteoclasts. Monocyte macrophage‐colony‐stimulating factor (M‐CSF) stimulated the proliferation of bone marrow macrophages in a dose‐dependent manner and these M‐CSF‐dependent bone marrow macrophage (MDBM) cells efficiently differentiated into the tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in the presence of soluble RANKL (sRANKL) and M‐CSF in the in vitro culture. The macrophage‐like cell line TMC16 was established from tsA58 (temperature‐sensitive SV40 large T‐antigen) transgenic mice in the same manner to the preparation of MDBM cells and also differentiated into mature osteoclasts. During this differentiation in vitro, the morphology of the cells changed from spindle to round and smaller (termed pOC) on day 2 and to multinuclear (termed multinucleated cells [MNCs]) on day 4. The surface expression of macrophage marker CD14 was down‐regulated and that of CD43 was up‐regulated on pOC, analyzed by flow cytometry. RNA analysis revealed that osteoclast marker genes such as calcitonin receptor (CTR), carbonic anhydrase II (CAII), cathepsin K (cath K), MMP9, and TRAP were strongly expressed in MNCs and weakly in pOC whereas MDBM cells did not express these genes. However, the osteopontin (OPN) gene was strongly expressed in MDBM cells and this expression became weakened after differentiation into pOC. The TMC16 cell line weakly expressed cath K, TRAP, and OPN, suggesting that the TMC16 cell line is immortalized at a stage slightly differentiated from MDBM cells. Furthermore, cell sorting analysis revealed that osteoclast early progenitors in bone marrow cells are preferentially present in the Mac‐1−F4/80dullpopulation, which differentiated into MDBM cells (the osteoclast progenitor) expressing Mac‐1+F4/80int, suggesting that M‐CSF plays roles of a differentiation factor as well as a growth factor for osteoclast early progenitors. These results showed the transition of morphology, surface markers, and gene expression from the early to mature stage in osteoclast differentiation. We propose three differentiation stages in the osteoclast lineage: the pro‐osteoclast (spindle‐shaped macrophage cells), the pre‐osteoclast (small round mononucleated TRAP‐positive cells), and the mature osteoclast (multinucleated TRAP‐positive cells) stage.

Osteoclasts are thought to belong to a macrophage lineage. However, the nature of common precursors of osteoclasts and macrophages remains to be investigated. We have characterized the differentiation potential of mouse bone marrow macrophages into mature osteoclasts. Monocyte macrophage‐colony‐stimulating factor (M‐CSF) stimulated the proliferation of bone marrow macrophages in a dose‐dependent manner and these M‐CSF‐dependent bone marrow macrophage (MDBM) cells efficiently differentiated into the tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in the presence of soluble RANKL (sRANKL) and M‐CSF in the in vitro culture. The macrophage‐like cell line TMC16 was established from tsA58 (temperature‐sensitive SV40 large T‐antigen) transgenic mice in the same manner to the preparation of MDBM cells and also differentiated into mature osteoclasts. During this differentiation in vitro, the morphology of the cells changed from spindle to round and smaller (termed pOC) on day 2 and to multinuclear (termed multinucleated cells [MNCs]) on day 4. The surface expression of macrophage marker CD14 was down‐regulated and that of CD43 was up‐regulated on pOC, analyzed by flow cytometry. RNA analysis revealed that osteoclast marker genes such as calcitonin receptor (CTR), carbonic anhydrase II (CAII), cathepsin K (cath K), MMP9, and TRAP were strongly expressed in MNCs and weakly in pOC whereas MDBM cells did not express these genes. However, the osteopontin (OPN) gene was strongly expressed in MDBM cells and this expression became weakened after differentiation into pOC. The TMC16 cell line weakly expressed cath K, TRAP, and OPN, suggesting that the TMC16 cell line is immortalized at a stage slightly differentiated from MDBM cells. Furthermore, cell sorting analysis revealed that osteoclast early progenitors in bone marrow cells are preferentially present in the Mac‐1−F4/80dullpopulation, which differentiated into MDBM cells (the osteoclast progenitor) expressing Mac‐1+F4/80int, suggesting that M‐CSF plays roles of a differentiation factor as well as a growth factor for osteoclast early progenitors. These results showed the transition of morphology, surface markers, and gene expression from the early to mature stage in osteoclast differentiation. We propose three differentiation stages in the osteoclast lineage: the pro‐osteoclast (spindle‐shaped macrophage cells), the pre‐osteoclast (small round mononucleated TRAP‐positive cells), and the mature osteoclast (multinucleated TRAP‐positive cells) stage.

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-κB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitrowithout stromal cells. Tumor necrosis factor-α (TNF-α), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-α can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-α directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitroin the presence of M-CSF. The bone resorption activity of TNF-α-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1β stimulated this activity of TNF-α-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1β did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-α was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-α is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-α and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.

In order to clarify the preparation process of crystal nucleation of polymers, we have studied the conformational changes and orientation fluctuations of isotactic polystyrene (iPS) in the induction period of crystallization using time-resolved Fourier transform infrared (FT-IR) spectroscopy and depolarized light scattering (DPLS) techniques, respectively. Their measurements were carried out for the so-called cold crystallization, when it was crystallized somewhat above the glass transition temperature Tg=100°C from the glassy states. It is found from the time-resolved FT-IR measurements that the polymer chains begin to assume alternate conformations of trans (T) and gauche (G) in the induction period, which are characteristic to the 3/1 helix in the crystalline state. On the other hand, the time-resolved DPLS observations reveal that orientation fluctuations begin to grow in the induction period after a temperature jump to the crystallization temperature, which means that some rod-like segments of polymer chains actually start to orient parallel to one another prior to crystallization. These results are discussed in terms of the Doi’s theory on the isotropic-to-nematic transition of liquid crystals where we assumed that the 3/1 helical segments are stiff and have a persistence length being almost equal to the length of the heilx axis. Thus, the growth of the helices involves the increase in persistence length and hence in excluded volume, to cause the parallel ordering of the helix parts as stiff segments.

We have succeeded in growing Saccharomyces cerevisiae(baker's yeast) on media containing 2H2O and isolating the core histones highly deuterated in the non‐exchangeable positions. The deuterated histones obtained here are of great value for their possible widespread use for structural studies of chromatin.

We have succeeded in growing Saccharomyces cerevisiae(baker's yeast) on media containing 2H 2O and isolating the core histones highly deuterated in the non-exchangeable positions. The deuterated histones obtained here are of great value for their possible widespread use for structural studies of chromatin.

During somatic reprogramming, Yamanaka’s pioneer factors regulate a complex sequence of molecular events leading to the activation of a network of pluripotency factors, ultimately resulting in the acquisition and maintenance of a pluripotent state. Here, we show that, contrary to the pluripotency factors studied so far, overexpression of Mybl2 inhibits somatic reprogramming. Our results demonstrate that Mybl2 levels are crucial to the dynamics of the reprogramming process. Mybl2 overexpression changes chromatin conformation, affecting the accessibility of pioneer factors to the chromatin and promoting accessibility for early immediate response genes known to be reprogramming blockers. These changes in the chromatin landscape ultimately lead to a deregulation of key genes that are important for the mesenchymal-to-epithelial transition. This work defines Mybl2 level as a gatekeeper for the initiation of reprogramming, providing further insights into the tight regulation and required coordination of molecular events that are necessary for changes in cell fate identity during the reprogramming process.

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.

We have studied the low energy excitations in the glassy simple hydrocarbon molecules. For all of the samples studied, non-Debye heat capacity was observed below 30 K and a clear boson peak appeared at 2–3 meV in their inelastic neutron scattering spectra. These data provided the absolute value of the excess density of states GLEE(ω) associated with the low energy excitations. The GLEE(ω) was analyzed in terms of the soft potential model. The magnitude of the low energy excitations and the boson peak energy were both dependent on the molecular weight and molecular structure.

We report neutron scattering experiments on amorphous polymers below and above the glass transition temperature Tg in a wide Q (magnitude of scattering vector) range up to 6.4 Å−1. From the Q-dependence of the incoherent elastic scattering intensity, we have evaluated the non-Gaussian parameter A as functions of temperature and “fragility” of polymers. The results are discussed in terms of dynamical heterogeneity due to different local environments.

We have studied the low energy excitations in the glassy simple hydrocarbon molecules. For all of the samples studied, non-Debye heat capacity was observed below 30 K and a clear boson peak appeared at 2–3 meV in their inelastic neutron scattering spectra. These data provided the absolute value of the excess density of states GLEE(?) associated with the low energy excitations. The GLEE(?) was analyzed in terms of the soft potential model. The magnitude of the low energy excitations and the boson peak energy were both dependent on the molecular weight and molecular structure.

We report neutron scattering experiments on amorphous polymers below and above the glass transition temperature Tg in a wide Q (magnitude of scattering vector) range up to 6.4 Å-1. From the Q-dependence of the incoherent elastic scattering intensity, we have evaluated the non-Gaussian parameter A as functions of temperature and “fragility” of polymers. The results are discussed in terms of dynamical heterogeneity due to different local environments.