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1.  Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion 
Nucleosomes are the fundamental unit of chromatin, but the analysis of transcription-independent nucleosome functions has been thwarted by the confounding gene expression changes resultant of histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation, and analyze the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that while nucleosome-free DNA can recruit nuclear envelope membranes, nucleosomes are required for spindle assembly, lamina and nuclear pore complex (NPC) formation. In addition to RCC1, we reveal that ELYS, the initiator of NPC formation, fails to associate with naked DNA, but directly binds histones H2A–H2B and nucleosomes. Tethering ELYS and RCC1 to DNA bypassed the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS.
PMCID: PMC4082469  PMID: 24952593
2.  Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging 
PLoS ONE  2014;9(9):e106271.
To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye∶protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).
PMCID: PMC4153647  PMID: 25184362
3.  Hierarchical Molecular Events Driven by Oocyte-Specific Factors Lead to Rapid and Extensive Reprogramming 
Molecular Cell  2014;55(4):524-536.
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.
Graphical Abstract
•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.
PMCID: PMC4156308  PMID: 25066233
4.  DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution 
Methyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues.
Using immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2-/y ) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2-/y and Mecp2 wt mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2-/y mice.
MECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.
PMCID: PMC4148084  PMID: 25170345
MECP2; MBD; Histone modifications; Nuclear architecture; Mouse retina; Retina development; Mouse tissues
5.  Role of latent membrane protein 1 in chronic active Epstein–Barr virus infection-derived T/NK-cell proliferation 
Cancer Medicine  2014;3(4):787-795.
Epstein–Barr virus (EBV) predominantly infects B cells and causes B-cell lymphomas, such as Burkitt lymphoma and Hodgkin lymphoma. However, it also infects other types of cells, including T and natural killer (NK) cells, and causes disorders, such as chronic active EBV infection (CAEBV) and T/NK-cell lymphoma. The CAEBV is a lymphoproliferative disease with poor prognosis, where EBV-positive T or NK cells grow rapidly, although the molecular mechanisms that cause the cell expansion still remain to be elucidated. EBV-encoded latent membrane protein 1 (LMP1) is an oncogene that can transform some cell types, such as B cells and mouse fibroblasts, and thus may stimulate cell proliferation in CAEBV. Here, we examined the effect of LMP1 on EBV-negative cells using the cells conditionally expressing LMP1, and on CAEBV-derived EBV-positive cells by inhibiting the function of LMP1 using a dominant negative form of LMP1. We demonstrated that LMP1 was responsible for the increased cell proliferation in the cell lines derived from CAEBV, while LMP1 did not give any proliferative advantage to the EBV-negative cell line.
PMCID: PMC4303147  PMID: 24799376
AKT; CAEBV; dominant negative; LMP1; NFκB
6.  Nuclear dynamics of topoisomerase IIβ reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain 
Nucleic Acids Research  2014;42(14):9005-9020.
DNA topoisomerase II (topo II) changes DNA topology by cleavage/re-ligation cycle(s) and thus contributes to various nuclear DNA transactions. It is largely unknown how the enzyme is controlled in a nuclear context. Several studies have suggested that its C-terminal domain (CTD), which is dispensable for basal relaxation activity, has some regulatory influence. In this work, we examined the impact of nuclear localization on regulation of activity in nuclei. Specifically, human cells were transfected with wild-type and mutant topo IIβ tagged with EGFP. Activity attenuation experiments and nuclear localization data reveal that the endogenous activity of topo IIβ is correlated with its subnuclear distribution. The enzyme shuttles between an active form in the nucleoplasm and a quiescent form in the nucleolus in a dynamic equilibrium. Mechanistically, the process involves a tethering event with RNA. Isolated RNA inhibits the catalytic activity of topo IIβ in vitro through the interaction with a specific 50-residue region of the CTD (termed the CRD). Taken together, these results suggest that both the subnuclear distribution and activity regulation of topo IIβ are mediated by the interplay between cellular RNA and the CRD.
PMCID: PMC4132749  PMID: 25034690
8.  Histone H4 Lys 20 Monomethylation of the CENP-A Nucleosome Is Essential for Kinetochore Assembly 
Developmental Cell  2014;29(6):740-749.
In vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A. Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. Here, we used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. We found that H4K20 monomethylation (H4K20me1) is enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. We conclude that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly.
Graphical Abstract
•H4K20 monomethylation is enriched at both chicken and human centromeres•H4K20 monomethylation occurs primarily at the CENP-A-containing nucleosomes•H4K20 monomethylation is essential for kinetochore assembly in chicken cells
Deposition of histone CENP-A characterizes vertebrate centromeres, but the full epigenetic landscape of centromeres remains undefined. Hori et al. show that monomethylation of histone H4K20 is associated with CENP-A nucleosomes and is enriched at human and chicken centromeres. Importantly, they show that this chromatin modification is essential for kinetochore assembly.
PMCID: PMC4081567  PMID: 24960696
9.  Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity 
Loss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions.
Levels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro.
H4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro.
H4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner.
PMCID: PMC4229880  PMID: 24953066
10.  Heterochromatin Dynamics during the Differentiation Process Revealed by the DNA Methylation Reporter Mouse, MethylRO 
Stem Cell Reports  2014;2(6):910-924.
In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.
Graphical Abstract
•Changes in DNA methylation are tracked in living mice•Heterochromatin structure changes dynamically during development and differentiation•Heterochromatin of preimplantation embryonic cells is highly dynamic than ESCs•Heterochromatin pattern in nucleus can be a marker for cell differentiation states
DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. To track these dynamics, DNA methylation reporter mouse was generated and named MethylRO (methylation probe in ROSA26 locus). Using this mouse, Yamagata and colleagues discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status.
PMCID: PMC4050349  PMID: 24936475
11.  Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the KLF4 Gene in Endothelial Cells 
PLoS ONE  2014;9(5):e96005.
Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
PMCID: PMC4010393  PMID: 24797675
13.  NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies 
Molecular Biology of the Cell  2014;25(1):169-183.
Paraspeckles are subnuclear structures formed around NEAT1 lncRNA. Paraspeckles became enlarged after proteasome inhibition caused by NEAT1 transcriptional activation, leading to protein sequestration into paraspeckles. The NEAT1-dependent sequestration affects the transcription of several genes, arguing for a novel role for lncRNA in gene regulation.
Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENε/β long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno–electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1−/− fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.
PMCID: PMC3873887  PMID: 24173718
14.  Concurrent Activation of Acetylation and Tri-Methylation of H3K27 in a Subset of Hepatocellular Carcinoma with Aggressive Behavior 
PLoS ONE  2014;9(3):e91330.
Analysis of acetylation and tri-methylation of the same residue of histone molecules might identify a subset of hepatocellular carcinoma (HCC) with aggressive behavior. In the present study, we examined acetylation and tri-methylation of lysine 27 on histone H3 (H3K27ac and H3K27me3, respectively) because these two modifications are known to exhibit opposite effects (enhancing and silencing) on gene expression. Neoplastic and non-neoplastic tissues from 198 HCC cases were immunostained with specific monoclonal antibodies against H3K27ac and H3K27me3. The stained tissues were evaluated by an image analyzing program to generate histological scores (H-scores, range 0–300), which were determined by multiplying the percentage of positive-stained cells with the classified immunohistochemical marker intensity (0–3). HCC tissues showed significantly higher H3K27ac (156.7±86.8) and H3K27me3 H-scores (151.8±78.1) compared with the background liver (40.3±33.0 and 64.7±45.6, respectively) (both P<0.001). The cases with H-scores of high-H3K27ac/high-H3K27me3 (n = 54) showed significant correlation with poor differentiation of morphology (P<0.01) and p53-positive staining (P<0.05), and poor prognosis (P<0.01). Confocal microscopy revealed segregated intranuclear localization of both modifications in the individual cancer cells: H3K27ac localization in central euchromatin regions and H3K27me3 in peripheral heterochromatin regions. Concurrent acetylation and methylation at H3K27 occurs in HCC cells in association with p53 abnormalities. These findings demonstrate that image analyzer-assisted H-scores of H3K27ac and H3K27me3 identified an aggressive subgroup of HCC, and could serve as a prognostic marker for HCC.
PMCID: PMC3948868  PMID: 24614346
15.  Haematopoietic cells produce BDNF and regulate appetite upon migration to the hypothalamus 
Nature communications  2013;4:1526.
Brain-derived neurotrophic factor (BDNF) suppresses food intake by acting on neurons in the hypothalamus. Here we show that BDNF-producing haematopoietic cells control appetite and energy balance by migrating to the hypothalamic paraventricular nucleus. These haematopoietic-derived paraventricular nucleus cells produce microglial markers and make direct contacts with neurons in response to feeding status. Mice with congenital BDNF deficiency, specifically in haematopoietic cells, develop hyperphagia, obesity and insulin resistance. These abnormalities are ameliorated by bone marrow transplantation with wild-type bone marrow cells. Furthermore, when injected into the third ventricle, wild-type bone marrow mononuclear cells home to the paraventricular nucleus and reverse the hyperphagia of BDNF-deficient mice. Our results suggest a novel mechanism of feeding control based on the production of BDNF by haematopoietic cells and highlight a potential new therapeutic route for the treatment of obesity.
PMCID: PMC3640364  PMID: 23443554
16.  Tipepidine in children with attention deficit/hyperactivity disorder: a 4-week, open-label, preliminary study 
Tipepidine (3-[di-2-thienylmethylene]-1-methylpiperidine) has been used solely as a nonnarcotic antitussive in Japan since 1959. The safety of tipepidine in children and adults has already been established. It is reported that tipepidine inhibits G-protein-coupled inwardly rectifying potassium (GIRK)-channel currents. The inhibition of GIRK channels by tipepidine is expected to modulate the level of monoamines in the brain. We put forward the hypothesis that tipepidine can improve attention deficit/hyperactivity disorder (ADHD) symptoms by modulating monoaminergic neurotransmission through the inhibition of GIRK channels. The purpose of this open-label trial was to confirm whether treatment with tipepidine can improve symptoms in pediatric patients with ADHD.
Subjects and methods
This was a 4-week, open-label, proof-of-efficacy pilot study for pediatric subjects with ADHD. Ten pediatric ADHD subjects (70% male; mean age, 9.9 years; combined [inattentive and hyperactive/impulsive] subtype, n=7; inattentive subtype, n=3; hyperimpulsive subtype, n=0) received tipepidine hibenzate taken orally at 30 mg/day for 4 weeks. All subjects were assessed using the ADHD Rating Scale IV (ADHD-RS), Japanese version, and the Das–Naglieri Cognitive Assessment System (DN-CAS), Japanese version.
A comparison of baseline scores and 4-week end-point scores showed that all the ADHD-RS scores (total scores, hyperimpulsive subscores, and inattentive subscores) improved significantly (P<0.001). Furthermore, a comparison of baseline DN-CAS total scores and 4-week end-point scores showed a mild trend of improvement (P=0.093). Tipepidine was well tolerated, with no patients discontinuing medication because of side effects.
Our pilot study suggests that tipepidine therapy may prove to be an effective alternative treatment for pediatric patients with ADHD. Nonetheless, more detailed randomized, double-blind trials are needed to confirm tipepidine’s efficacy.
PMCID: PMC3908907  PMID: 24493927
attention deficit/hyperactivity disorder; tipepidine; GIRK channel; pediatric; antitussive; nucleus accumbens
17.  Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin 
Scientific Reports  2013;3:3510.
Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.
PMCID: PMC3863819  PMID: 24336483
18.  A probable identical Epstein-Barr virus clone-positive composite lymphoma with aggressive natural killer-cell leukemia and cytotoxic T-cell lymphoma 
The patient was a 52-year old woman with a history of mosquito-bite hypersensitivity since childhood. In July 2011, she developed pyrexia, headaches, and nausea, and Epstein-Barr virus (EBV)-positive aggressive natural killer leukemia (ANKL) was diagnosed on the basis of both a peripheral blood and bone marrow examination. An inguinal lymph node biopsy, on the other hand, revealed EBV-positive cytotoxic T-cell lymphoma plus the presence of a small number of EBV-positive ANKL cells, and a diagnosis of EBV-positive composite lymphoma was made. Both the cytotoxic T-cell lymphoma and ANKL exhibited EBV terminal repeat (Southern blot analysis) monoclonal patterns, and they were almost the same size, approximately 9.0 kb. If it was the identical EBV clone, it is possible that EBV infected progenitor cells common to both NK cells and T cells, that the progenitor cells then differentiated into NK cells and T cells, a chronic active Epstein-Barr virus infection developed, and neoplastic transformation occurred. If it was not the identical EBV clone, fairly similar EBVs must have infected NK cells and T cells separately, and they then underwent neoplastic transformation. Because the mechanism by which EBV infects NK cells or T cells is still unknown, we concluded that this case is also important from the standpoint of elucidating it. We are currently in the process of conducting gene analyses to determine whether the fairly similar EBVs that infected the ANKL and cytotoxic T-cell lymphoma are the identical clone.
PMCID: PMC3885499  PMID: 24427365
Aggressive NK leukemia; Epstein-Barr virus; cytotoxic T-cell lymphoma; composite lymphoma; large granular lymphocytes
19.  Skin Autofluorescence Is Associated with the Progression of Chronic Kidney Disease: A Prospective Observational Study 
PLoS ONE  2013;8(12):e83799.
Advanced glycation end product (AGE) accumulation is thought to be a measure of cumulative metabolic stress that has been reported to independently predict cardiovascular disease in diabetes and renal failure. The aim of this study was to evaluate the association between AGE accumulation, measured as skin autofluorescence, and the progression of renal disease in pre-dialysis patients with chronic kidney disease (CKD).
Skin autofluorescence was measured noninvasively with an autofluorescence reader at baseline in 449 pre-dialysis patients with CKD. The primary end point was defined as a doubling of serum creatinine and/or need for dialysis.
Thirty-three patients were lost to follow-up. Forty six patients reached the primary end point during the follow-up period (Median 39 months). Kaplan-Meier analysis showed a significantly higher risk of development of the primary end points in patients with skin autofluorescence levels above the optimal cut-off level of 2.31 arbitrary units, derived by receiver operator curve analysis. Cox regression analysis revealed that skin autofluorescence was an independent predictor of the primary end point, even after adjustment for age, gender, smoking history, diabetes, estimated glomerular filtration rate and proteinuria (adjusted hazard ratio 2.58, P = 0.004).
Tissue accumulation of AGEs, measured as skin autofluorescence, is a strong and independent predictor of progression of CKD. Skin autofluorescence may be useful for risk stratification in this group of patients; further studies should clarify whether AGE accumulation could be one of the therapeutic targets to improve the prognosis of CKD.
PMCID: PMC3861518  PMID: 24349550
20.  Different Distributions of Epstein-Barr Virus Early and Late Gene Transcripts within Viral Replication Compartments 
Journal of Virology  2013;87(12):6693-6699.
Productive replication of the Epstein-Barr virus (EBV) occurs in discrete sites in nuclei, called replication compartments, where viral genome DNA synthesis and transcription take place. The replication compartments include subnuclear domains, designated BMRF1 cores, which are highly enriched in the BMRF1 protein. During viral lytic replication, newly synthesized viral DNA genomes are organized around and then stored inside BMRF1 cores. Here, we examined spatial distribution of viral early and late gene mRNAs within replication compartments using confocal laser scanning microscopy and three-dimensional surface reconstruction imaging. EBV early mRNAs were mainly located outside the BMRF1 cores, while viral late mRNAs were identified inside, corresponding well with the fact that late gene transcription is dependent on viral DNA replication. From these results, we speculate that sites for viral early and late gene transcription are separated with reference to BMRF1 cores.
PMCID: PMC3676136  PMID: 23552415
21.  Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2 
The crystal structures of human nucleosomes containing H2A.Z.1 and H2A.Z.2 have been determined. Structural polymorphisms were found in the L1 loop regions of H2A.Z.1 and H2A.Z.2 in the nucleosomes that are likely to be caused by their flexible nature.
The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.
PMCID: PMC3852653  PMID: 24311584
H2A.Z.1; H2A.Z.2; nucleosomes; chromatin; FRAP
22.  Distinct Localization of Peripheral and Central Types of Choline Acetyltransferase in the Rat Cochlea 
Acta Histochemica et Cytochemica  2013;46(5):145-152.
We previously discovered a splice variant of choline acetyltransferase (ChAT) mRNA, and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, we examined the immunohistochemical localization of pChAT in rat cochlea and compared the distribution pattern to those of common ChAT (cChAT) and acetylcholinesterase. Some neuronal cell bodies and fibers in the spiral ganglia showed immunoreactivity for pChAT, predominantly the small spiral ganglion cells, indicating outer hair cell type II neurons. In contrast, cChAT- and acetylcholinesterase-positive structures were localized to fibers and not apparent in ganglion cells. After ablation of the cochlear nuclei, many pChAT-positive cochlear nerve fibers became clearly visible, whereas fibers immunopositive for cChAT and acetylcholine esterase disappeared. These results suggested that pChAT and cChAT are localized in different systems of the rat cochlea; pChAT in the afferent and cChAT in the efferent structures.
PMCID: PMC3814435  PMID: 24194628
acetylcholine; cholinergic neurons; choline acetyltransferase; cochlear nervous system; spiral ganglion
23.  Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations  
The Journal of Clinical Investigation  2013;123(11):4627-4640.
Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal–truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2–mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT–induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.
PMCID: PMC3809801  PMID: 24216483
24.  Genetically encoded system to track histone modification in vivo 
Scientific Reports  2013;3:2436.
Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.
PMCID: PMC3743053  PMID: 23942372
25.  Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication 
Journal of Virology  2013;87(4):2120-2127.
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication.
PMCID: PMC3571458  PMID: 23221557

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