Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells.

The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.

Detection and management of cardiomyopathy in female dystrophinopathy carriers.

Regular health checkups for mothers of patients with Duchenne muscular dystrophy have been performed at National Hospital Organization Tokushima Hospital since 1994. Among 43 mothers participated in this study, 28 dystrophinopathy carriers were identified. Skeletal and cardiac muscle functions of these subjects were examined. High serum creatine kinase was found in 23 subjects (82.1%). Obvious muscle weakness was present in 5 (17.8%) and had progressed from 1994 to 2015. Cardiomyopathy was observed in 15 subjects (60.0%), including dilated cardiomyopathy-like damage that was more common in the left ventricular (LV) posterior wall. Late gadolinium enhancement on cardiac MRI was found in 5 of 6 subjects, suggesting fibrotic cardiac muscle. In speckle tracking echocardiography performed seven years later, global longitudinal strain was decreased in these subjects, indicating LV myocardial contractile abnormality. These results suggest that female dystrophinopathy carriers should receive regular checkups for detection and treatment of cardiomyopathy, even if they have no cardiac symptoms.

Zfp296 negatively regulates H3K9 methylation in embryonic development as a component of heterochromatin.

The Cys2/His2-type zinc finger protein Zfp296 has been implicated in stem cell pluripotency and tumor pathogenesis. However, its mechanisms remain elusive. Here, we demonstrated that a Zfp296 deficiency in mice impairs germ-cell development and embryonic growth. Zfp296 was intracellularly localized to heterochromatin in embryos. A GST-Zfp296 pull-down experiment using ES cell nuclear extract followed by LC-MS/MS showed that Zfp296 interacts with component proteins of heterochromatin (such as HP1, Dnmt1, Dnmt3b, and ATRX) and the NuRD complex. We focused on H3K9 methylation as a hallmark of heterochromatin, and found that Zfp296 overexpression in cultured cells reduces the Suv39h1-mediated H3K9 methylation. Consistent with this finding, in Zfp296 -/- mouse embryos, we observed a global increase in H3K9 methylation in a developmental stage-dependent manner, and showed, by ChIP-qPCR, that the H3K9me3 levels at major satellite repeats were elevated in Zfp296 -/- embryos. Our results demonstrate that Zfp296 is a component of heterochromatin that affects embryonic development by negatively regulating H3K9 methylation.

Mumps encephalitis with akinesia and mutism.

Measles-rubella-mumps vaccination is routine in many countries, but the mumps vaccine remains voluntary and is not covered by insurance in Japan. A 5-year-old Japanese boy who had not received the mumps vaccine was affected by mumps parotitis. Several days later, he presented with various neurological abnormalities, including akinesia, mutism, dysphagia, and uncontrolled respiratory disorder. Mumps encephalitis was diagnosed. Despite steroid pulse and immunoglobulin treatment, the disease progressed. Magnetic resonance imaging showed necrotic changes in bilateral basal ganglia, midbrain, and hypothalamus. At 1 year follow up, he was bedridden and required enteral feeding through a gastric fistula and tracheostomy. Mumps vaccination should be made routine as soon as possible in Japan, because mumps encephalitis carries the risk of severe sequelae.

Functional analysis of Tcl1 using Tcl1-deficient mouse embryonic stem cells.

Tcl1 is highly expressed in embryonic stem (ES) cells, but its expression rapidly decreases following differentiation. To assess Tcl1's roles in ES cells, we generated Tcl1-deficient and -overexpressing mouse ES cell lines. We found that Tcl1 was neither essential nor sufficient for maintaining the undifferentiated state. Tcl1 is reported to activate Akt and to enhance cell proliferation. We found that Tcl1 expression levels correlated positively with the proliferation rate and negatively with the apoptosis of ES cells, but did not affect Akt phosphorylation. On the other hand, the phosphorylation level of ß-catenin decreased in response to Tcl1 overexpression. We measured the ß-catenin activity using the TOPflash reporter assay, and found that wild-type ES cells had low activity, which Tcl1 overexpression enhanced 1.8-fold. When the canonical Wnt signaling is activated by ß-catenin stabilization, it reportedly helps maintain ES cells in the undifferentiated state. We then performed DNA microarray analyses between the Tcl1-deficient and -expressing ES cells. The results revealed that Tcl1 expression downregulated a distinct group of genes, including Ndp52, whose expression is very high in blastocysts but reduced in the primitive ectoderm. Based on these results, we discuss the possible roles of Tcl1 in ES cells.

Decreased resting energy expenditure in patients with Duchenne muscular dystrophy.

BACKGROUND: Skeletal muscle metabolism is a major determinant of resting energy expenditure (REE). Although the severe muscle loss that characterizes Duchenne muscular dystrophy (DMD) may alter REE, this has not been extensively investigated. METHODS: We studied REE in 77 patients with DMD ranging in age from 10 to 37 years using a portable indirect calorimeter, together with several clinical parameters (age, height, body weight (BW), body mass index (BMI), vital capacity (VC), creatine kinase, creatinine, albumin, cholinesterase, prealbumin), and assessed their influence on REE. In addition, in 12 patients maintaining a stable body weight, the ratio of energy intake to REE was calculated and defined as an alternative index for the physical activity level (aPAL). RESULTS: REE (kcal/day, mean±SD) in DMD patients was 1123 (10-11 years), 1186±188 (12-14 years), 1146±214 (15-17 years), 1006±136 (18-29 years) and 1023±97 (≥30 years), each of these values being significantly lower than the corresponding control (p<0.0001). VC (p<0.001) was the parameter most strongly associated with REE, followed by BMI (p<0.01) and BW (p<0.05). The calculated aPAL values were 1.61 (10-11 years), 1.19 (12-14 years), 1.16 (15-17 years), and 1.57 (18-29 years). CONCLUSION: The REE in DMD patients was significantly lower than the normal value in every age group, and strongly associated with VC. Both the low REE and PAL values during the early teens, resulting in a low energy requirement, might be related to the obesity that frequently occurs in this age group. In contrast, the high PAL value in the late stage of the disease, possibly due to the presence of respiratory failure, may lead to a high energy requirement, and thus become one of the risk factors for development of malnutrition.

Maternal-effect gene Ces5/Ooep/Moep19/Floped is essential for oocyte cytoplasmic lattice formation and embryonic development at the maternal-zygotic stage transition.

In a search for genes specifically expressed in mouse embryonic stem cells, we identified one we called Ces5. We found that it corresponded to the Ooep gene, which was recently reported to be expressed specifically in oocytes. Mouse Ces5/Ooep, also called Moep19 or Floped, encoded a 164-amino acid protein, which was detected in the cytoplasm of developing and mature oocytes and in embryos throughout the preimplantation period. To examine its function, we carried out targeted disruption of this gene. The Ces5/Ooep-null mice were grossly normal, but the females were infertile. Although the ovaries and ovulation appeared normal, the embryos from Ces5/Ooep-null females mated with wild-type males showed developmental arrest at the two- or four-cell stage. In addition, their first cleavage was considerably delayed and often asymmetrical. Thus, Ces5/Ooep is a maternal-effect gene. By electron microscopy, we found that the eggs from Ces5/Ooep-null females lacked oocyte cytoplasmic lattices (CPLs), which have long been predicted to function as a storage form for components that are maternally contributed to the early embryo. Further analysis showed that CES5/OOEP was directly associated with the CPLs. These results indicate that CES5/OOEP is an essential component of the CPLs and is required for embryonic development at the maternal-zygotic stage transition.

Increased mid-left ventricular rotation in patients with Duchenne muscular dystrophy using two-dimensional speckle tracking echocardiography.

BACKGROUND: In patients with Duchenne muscular dystrophy (DMD), cardiomyopathy initially occurs during adolescence. In routine echocardiographic examination, we often recognize increased rotational movement of the left ventricle in DMD patients even if their conventional echocardiographic finding is normal. Two-dimensional speckle tracking echocardiography can assess left ventricular (LV) rotation. The aim of this study was to analyze the mid-LV rotation and to investigate the clinical implication of this abnormal movement. METHODS: Nineteen DMD patients (age 15.5 ± 3.1 years) and 22 age-matched healthy subjects were recruited. The two-dimensional speckle tracking method was used to determine the mid-LV rotation at the papillary muscle level. The mid-LV rotation and rotational velocity were calculated and were compared with other echocardiographic data and indices of autonomic function. RESULTS: Total rotation was greater in the DMD group than in the normal group (7.3 ± 1.4° versus 5.2° ± 1.3°, p < 0.05). Both peak diastolic clockwise and counterclockwise rotational velocity were greater in the DMD group (p < 0.005 and p < 0.05, respectively). Time from the second heart sound to peak diastolic clockwise rotation (% diastolic duration) was greater in the DMD group (p < 0.005). Total rotation and percentage of adjacent normal R-R intervals more than 50 ms different showed a negative correlation (r = -0.72) in the DMD group. CONCLUSION: In DMD patients, cases diagnosed with normal LV fractional shortening showed an increase in mid-LV rotation that might be due to relative increase of sympathetic nervous function before global cardiac function decreases.

Gtsf1/Cue110, a gene encoding a protein with two copies of a CHHC Zn-finger motif, is involved in spermatogenesis and retrotransposon suppression in murine testes.

We recently reported that the Gtsf1/Cue110 gene, a member of the evolutionarily conserved UPF0224 family, is expressed predominantly in male germ cells, and that the GTSF1/CUE110 protein is localized to the cytoplasm of these cells in the adult testis. Here, to analyze the roles of the Gtsf1/Cue110 gene in spermatogenesis, we produced Gtsf1/Cue110-null mice by gene targeting. The Gtsf1/Cue110-null mice grew normally and appeared healthy; however, the males were sterile due to massive apoptotic death of their germ cells after postnatal day 14. In contrast, the null females were fertile. Detailed analyses revealed that the Gtsf1/Cue110-null male meiocytes ceased meiotic progression before the zygotene stage. Thus, the Gtsf1/Cue110 gene is essential for spermatogenesis beyond the early meiotic phase. Furthermore, the loss of the Gtsf1/Cue110 gene caused increased transcription of the long interspersed nucleotide element (Line-1) and the intracisternal A-particle (IAP) retrotransposons, accompanied by demethylation of their promoter regions. These observations indicate that Gtsf1/Cue110 is required for spermatogenesis and involved in retrotransposon suppression in male germ cells.

Sohlh2 affects differentiation of KIT positive oocytes and spermatogonia.

The differentiation programs of spermatogenesis and oogenesis are largely independent. In the early stages, however, the mechanisms partly overlap. Here we demonstrated that a germ-cell-specific basic helix-loop-helix (bHLH) transcription factor gene, Sohlh2, is required for early spermatogenesis and oogenesis. SOHLH2 was expressed in mouse spermatogonia from the undifferentiated stage through differentiation and in primordial-to-primary oocytes. Sohlh2-null mice, produced by gene targeting, showed both male and female sterility, owing to the disrupted differentiation of mature (KIT(+)) spermatogonia and oocytes. The Sohlh2-null mice also showed the downregulation of genes involved in spermatogenesis and oogenesis, including the Sohlh1 gene, which is essential for these processes. Furthermore, we showed that SOHLH2 and SOHLH1 could form heterodimers. These observations suggested that SOHLH2 might coordinate with SOHLH1 to control spermatogonial and oocyte genes, including Sohlh1, to promote the differentiation of KIT(+) germ cells in vivo. This study lays the foundation for further dissection of the bHLH network that regulates early spermatogenesis and oogenesis.

Gene expression pattern of Cue110: a member of the uncharacterized UPF0224 gene family preferentially expressed in germ cells.

The large number of expressed sequence tags (ESTs) now available in databases has enabled the analysis of gene expression profiles in silico. We searched public databases for uncharacterized transcripts specifically expressed in germ cells, in an attempt to identify genes involved in gametogenesis. We found a transcript that is expressed in unfertilized eggs, ovaries, and testes of the mouse. It has an open reading frame (ORF) encoding a 167-amino acid protein belonging to the UPF0224 (unknown protein family 0224) family. We called the novel gene Cue110. We examined the Pfam database for other members of the UPF0224 family, and found a conserved N-terminal portion among members of various species. To study the cellular localization of the Cue110 transcript and protein, we performed in situ hybridization and immunohistochemical analysis of the adult mouse ovary and testis. In the testis, specific hybridization signals were observed weakly in preleptotene spermatocytes but maximally in late round spermatids. Immunostaining showed that Cue110 protein was present predominantly in the cytoplasm of pachytene spermatocytes and round spermatids. In the ovary, weak hybridization signals were observed in primary oocytes in the primordial, primary, and secondary follicles, but Cue110 protein was not detected in oocytes by immunostaining. We next examined the developmental expression pattern of the Cue110 gene using RT-PCR and western blotting, and found its increasing expression coincided with the appearance of spermatocytes. Thus, the Cue110 gene is expressed predominantly in male germ cells at stages from the pachytene spermatocytes to round spermatids.

Development of a single-cassette system for spatiotemporal gene regulation in mice.

The tetracycline-regulated gene expression system has been widely used in mice to turn a transgene on and off in a target organ, but with only limited success. We developed an advanced system in which a Tet-off regulation unit was integrated into the ROSA26 locus and became active after Cre-mediated excision of the neo(r) gene. We examined the utility of this system through regulable expression of the homeodomain transcription factor pdx-1 and enhanced green fluorescent protein. The resulting mice showed strict tetracycline-regulable gene expression in all the organs where the neo(r) gene had been removed. When combined with organ-specific Cre recombinase transgenic mice, our system allows us to manipulate the gene expression in an organ-specific and temporal manner. This Tet-off system should serve as an efficient tool to analyze the roles of genes in complex biological systems, such as embryogenesis, metabolism, immune system, etc.

Systemic administration of IL-18 promotes diabetes development in young nonobese diabetic mice.

IL-18 is now identified as a pleiotropic cytokine that acts as a cofactor for both Th1 and Th2 cell development. Type 1 diabetes is considered a Th1-type autoimmune disease, and to date, the suppressive effect of exogenous IL-18 on the development of diabetes has been reported in 10-wk-old nonobese diabetic (NOD) mice. In the present study we administered exogenous IL-18 systemically in 4-wk-old NOD mice using i.m. injection of the IL-18 expression plasmid DNA (pCAGGS-IL-18) with electroporation. Contrary to previous reports, the incidence of diabetes development was significantly increased in NOD mice injected with pCAGGS-IL-18 compared with that in control mice. Systemic and pancreatic cytokine profiles deviated to a Th1-dominant state, and the the frequency of glutamic acid decarboxylase-reactive IFN-gamma-producing CD4(+) cells was also high in the IL-18 group. Moreover, it was suggested that the promoting effect of IL-18 might be associated with increased peripheral IL-12, CD86, and pancreatic IFN-inducible protein-10 mRNA expression levels. In conclusion, we demonstrate here that IL-18 plays a promoting role as an enhancer of Th1-type immune responses in diabetes development early in the spontaneous disease process, which may contribute to elucidating the pathogenesis of type 1 diabetes.