SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones.

The histone chaperone complex FACT comprises SPT16 and SSRP1 and contributes to DNA replication, transcription, and repair, but how it plays such various roles is unclear. Here, we show that human SPT16 is ubiquitylated at lysine-674 (K674) by the DCAF14-CRL4 ubiquitin ligase. K674 is located in the middle domain of SPT16, and the corresponding residue of the yeast ortholog is critical for binding to histone H3.1-H4. We show that the middle domain of human SPT16 binds to histone H3.1-H4 and that this binding is inhibited by K674 ubiquitylation. Cells with heterozygous knockin of a K674R mutant of SPT16 manifest reduction of both SPT16 ubiquitylation and H3.1 in chromatin, a reduced population in mid S phase, impaired proliferation, and increased susceptibility to S phase stress. Our data thus indicate that SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones and may thereby control DNA replication-coupled histone incorporation into chromatin.

Late testicular relapse two decades after primary extragonadal germ cell tumor with uncommon metastases: a case report.

BACKGROUND: Extragonadal germ cell tumor (EGCT) is a relatively rare condition, reportedly representing 3-7% of all germ cell tumors. We report a patient who had metachronous testicular tumor with uncommon metastases 20 years after primary retroperitoneal EGCT treatment, along with a corresponding literature review. CASE PRESENTATION: A 49-year-old Japanese man visited our department in November 2017 with chief complaints of indolent right scrotum enlargement and a right inguinal mass. History showed that the patient visited our department of gastroenterology with chief complaints of blackish feces and ill complexion in February 1997. Computed tomography (CT) showed a right retroperitoneal tumor, which was removed in the same month. Histopathological examination showed a teratoma and yolk sac tumor. He was diagnosed with primary retroperitoneal EGCT and received three courses of chemotherapy (bleomycin/etoposide/cisplatin; BEP). Periodic imaging and the determination of tumor markers (alpha-fetoprotein [AFP], human chorionic gonadotropin [HCG], and lactate dehydrogenase [LDH]) showed no recurrence or metastasis during the 5 years postoperatively. Subsequently, he did not visit the outpatient ward. In August 1999, he underwent surgery of right hydrocele. Contrast-enhanced CT showed a 35-mm contrast effect with uneven content in the right testicle and enlarged nodes that raised suspicion for metastases in the right inguinal and right external iliac lymph nodes. All tumor markers were within normal ranges. He underwent right high orchiectomy and resection of the right inguinal lymph nodes in the same month. Histopathological findings revealed seminoma (pT1, pN2, M0, S0, and TNM stage IIB). He received postoperative chemotherapy, one course of BEP therapy, and three courses of etoposide and cisplatin therapy. Post-chemotherapy CT confirmed a complete clinical response at the right external iliac lymph nodes, and this response continued 12 months later. No recurrence or metastasis has been found so far. CONCLUSIONS: We report a patient in whom a testicular tumor with uncommon metastases occurred 20 years after primary retroperitoneal EGCT treatment. After EGCT treatment, testicular relapses tend to occur after relatively long-term follow-up. After EGCT treatment, such patients must be closely monitored for testicular recurrences and onset of testicular tumor.

Skin grafting utilizing a skin of lateral thoracic area for chest wall reconstruction in patient who underwent mastectomy for locally advanced breast cancer.

Locally advanced breast cancer sometimes results in a large chest wall defect at mastectomy. When closing the wound horizontally, the skin tension is usually severe in the middle of the wound, while the skin of the lateral area tends to make a dog-ear deformity. Triangle technique is a procedure to prevent the dog ear in which the skin and subcutaneous fat of the axilla are cut into an equilateral triangle. Herein, we present a case of breast cancer who underwent a mastectomy and closed the wound with a skin graft by utilizing the skin removed from lateral thoracic area using triangle technique. An 85-year-old female visited our institution complaining about the mass on her right breast. Preoperative images showed a 10 cm-sized mass with suspicious axillary and mediastinal lymph nodes swelling. A biopsy revealed a hormone receptor-negative, HER2-positive invasive ductal carcinoma. A mastectomy and axillary lymph node sampling were performed for a local control as the tumor did not respond to four cycles of triweekly trastuzumab combined with S-1. After a transverse elliptical incision, a skin of the lateral thoracic area was harvested using triangle technique. As the middle of the wound had excessive closing tension, the skin was grafted on the defect. After 10 day fixation by a tie-over dressing, the wound healed without complications. This procedure is a simple method for closing a large defect after mastectomy preventing both the dog-ear deformity and a new wound scarring of a donor site.

An Amyotrophic Lateral Sclerosis-Associated Mutant of C21ORF2 Is Stabilized by NEK1-Mediated Hyperphosphorylation and the Inability to Bind FBXO3.

C21ORF2 and NEK1 have been identified as amyotrophic lateral sclerosis (ALS)-associated genes. Both genes are also mutated in certain ciliopathies, suggesting that they might contribute to the same signaling pathways. Here we show that FBXO3, the substrate receptor of an SCF ubiquitin ligase complex, binds and ubiquitylates C21ORF2, thereby targeting it for proteasomal degradation. C21ORF2 stabilizes the kinase NEK1, with the result that loss of FBXO3 stabilizes not only C21ORF2 but also NEK1. Conversely, NEK1-mediated phosphorylation stabilizes C21ORF2 by attenuating its interaction with FBXO3. We found that the ALS-associated V58L mutant of C21ORF2 is more susceptible to phosphorylation by NEK1, with the result that it is not ubiquitylated by FBXO3 and therefore accumulates together with NEK1. Expression of C21ORF2(V58L) in motor neurons induced from mouse embryonic stem cells impaired neurite outgrowth. We suggest that inhibition of NEK1 activity is a potential therapeutic approach to ALS associated with C21ORF2 mutation.

The Autism-Related Protein SETD5 Controls Neural Cell Proliferation through Epigenetic Regulation of rDNA Expression.

Haploinsufficiency of SETD5 is implicated in syndromic autism spectrum disorder (ASD), but the molecular mechanism underlying the pathological role of this protein has remained unclear. We have now shown that Setd5+/- mice manifest ASD-related behavioral phenotypes and that the expression of ribosomal protein genes and rDNA is disturbed in the brain of these mice. SETD5 recruited the HDAC3 complex to the rDNA promoter, resulting in removal of the histone mark H4K16ac and its reader protein TIP5, a repressor of rDNA expression. Depletion of SETD5 attenuated rDNA expression, translational activity, and neural cell proliferation, whereas ablation of TIP5 in SETD5-deficient cells rescued these effects. Translation of cyclin D1 mRNA was specifically down-regulated in SETD5-insufficient cells. Our results thus suggest that SETD5 positively regulates rDNA expression via an HDAC3-mediated epigenetic mechanism and that such regulation is essential for translation of cyclin D1 mRNA and neural cell proliferation.

Pathogenic mutations in the ALS gene CCNF cause cytoplasmic mislocalization of Cyclin F and elevated VCP ATPase activity.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease characterized by a progressive decline in motor function. Genetic analyses have identified several genes mutated in ALS patients, and one of them is Cyclin F gene (CCNF), the product of which (Cyclin F) serves as the substrate-binding module of a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex. However, the role of Cyclin F in ALS pathogenesis has remained unclear. Here, we show that Cyclin F binds to valosin-containing protein (VCP), which is also reported to be mutated in ALS, and that the two proteins colocalize in the nucleus. VCP was found to bind to the NH2-terminal region of Cyclin F and was not ubiquitylated by SCFCyclin F in transfected cells. Instead, the ATPase activity of VCP was enhanced by Cyclin F in vitro. Furthermore, whereas ALS-associated mutations of CCNF did not affect the stability of Cyclin F or disrupt formation of the SCFCyclin F complex, amino acid substitutions in the VCP binding region increased the binding ability of Cyclin F to VCP and activity of VCP as well as mislocalization of the protein in the cytoplasm. We also provided evidence that the ATPase activity of VCP promotes cytoplasmic aggregation of transactivation responsive region (TAR) DNA-binding protein 43, which is commonly observed in degenerating neurons in ALS patients. Given that mutations of VCP identified in ALS patients also increase its ATPase activity, our results suggest that Cyclin F mutations may contribute to ALS pathogenesis by increasing the ATPase activity of VCP in the cytoplasm, which in turn increases TDP-43 aggregates.

Effectiveness of assertive case management on repeat self-harm in patients admitted for suicide attempt: Findings from ACTION-J study.

BACKGROUND: Self-harm is an important risk factor for subsequent suicide and repetition of self-harm, and a common cause of emergency department presentations. However, there still remains limited evidence on intervention in emergency department settings for individuals who self-harm. METHODS: This multicentre, randomised controlled trial was conducted at 17 general hospitals in Japan. In total, 914 adult patients admitted to emergency departments for a suicide attempt and had a DSM-IV-TR axis I disorder were randomly assigned to two groups, to receive either assertive case management (intervention) or enhanced usual care (control). Assertive case management was introduced by the case manager during emergency department admissions for suicide attempts, and continued after discharge. Interventions were provided until the end of the study period (for at least 18 months and up to 5 years). RESULTS: The number of overall self-harm episodes per person-year was significantly lower in the intervention group (adjusted incidence risk ratio (IRR) 0.88, 95%CI 0.80-0.96, p=0.0031). Subgroup analysis showed a greater reduction of overall self-harm episodes among patients with no previous suicide attempt at baseline (adjusted IRR 0.73, 95% CI 0.53-0.98, p=0.037). LIMITATIONS: Patients younger than 20 years and patients who self-harmed but were not admitted to an emergency department were excluded. CONCLUSIONS: The present study showed that assertive case management following emergency admission for a suicide attempt reduced the incident rate of repeat overall self-harm.

Transforming Growth Factor ß-Induced Proliferative Arrest Mediated by TRIM26-Dependent TAF7 Degradation and Its Antagonism by MYC.

Recognition of gene promoters by RNA polymerase II is mediated by general transcription factor IID (TFIID), which has been thought to be a static complex and to play a passive role in the regulation of gene expression under the instruction of gene-specific transcription factors. Here we show that transforming growth factor ß (TGF-ß) induced degradation of the TFIID subunit TAF7 in cultured mouse mammary epithelial cells and that this effect was required for proliferative arrest in response to TGF-ß stimulation. TGF-ß stimulated transcription of the gene for the ubiquitin ligase TRIM26, which was shown to ubiquitylate TAF7 and thereby to target it for proteasomal degradation. Sustained exposure of cells to TGF-ß resulted in recovery from proliferative arrest in association with amplification of the Myc proto-oncogene, with MYC inhibiting TRIM26 induction by TGF-ß. Our data thus show that TFIID is not simply a general mediator of transcription but contributes to the regulation of transcription in response to cell stimulation, playing a key role in the cytostatic function of TGF-ß.

Regulation of mitosis-meiosis transition by the ubiquitin ligase ß-TrCP in male germ cells.

The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that ß-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of ß-TrCP2 in male germ cells of ß-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The ß-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in ß-TrCP-deficient testes. DMRT1 contains a consensus ß-TrCP degron sequence that was found to bind ß-TrCP. Overexpression of ß-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in ß-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that ß-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation.

S6 Kinase- and ß-TrCP2-Dependent Degradation of p19Arf Is Required for Cell Proliferation.

The kinase mTOR (mammalian target of rapamycin) promotes translation as well as cell survival and proliferation under nutrient-rich conditions. Whereas mTOR activates translation through ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP), how it facilitates cell proliferation has remained unclear. We have now identified p19(Arf), an inhibitor of cell cycle progression, as a novel substrate of S6K that is targeted to promote cell proliferation. Serum stimulation induced activation of the mTOR-S6K axis and consequent phosphorylation of p19(Arf) at Ser(75). Phosphorylated p19(Arf) was then recognized by the F-box protein ß-TrCP2 and degraded by the proteasome. Ablation of ß-TrCP2 thus led to the arrest of cell proliferation as a result of the stabilization and accumulation of p19(Arf). The ß-TrCP2 paralog ß-TrCP1 had no effect on p19(Arf) stability, suggesting that phosphorylated p19(Arf) is a specific substrate of ß-TrCP2. Mice deficient in ß-TrCP2 manifested accumulation of p19(Arf) in the yolk sac and died in utero. Our results suggest that the mTOR pathway promotes cell proliferation via ß-TrCP2-dependent p19(Arf) degradation under nutrient-rich conditions.

CRL4(VprBP) E3 ligase promotes monoubiquitylation and chromatin binding of TET dioxygenases.

DNA methylation at the C-5 position of cytosine (5mC) regulates gene expression and plays pivotal roles in various biological processes. The TET dioxygenases catalyze iterative oxidation of 5mC, leading to eventual demethylation. Inactivation of TET enzymes causes multistage developmental defects, impaired cell reprogramming, and hematopoietic malignancies. However, little is known about how TET activity is regulated. Here we show that all three TET proteins bind to VprBP and are monoubiquitylated by the VprBP-DDB1-CUL4-ROC1 E3 ubiquitin ligase (CRL4(VprBP)) on a highly conserved lysine residue. Deletion of VprBP in oocytes abrogated paternal DNA hydroxymethylation in zygotes. VprBP-mediated monoubiquitylation promotes TET binding to chromatin. Multiple recurrent TET2-inactivating mutations derived from leukemia target either the monoubiquitylation site (K1299) or residues essential for VprBP binding. Cumulatively, our data demonstrate that CRL4(VprBP) is a critical regulator of TET dioxygenases during development and in tumor suppression.

Trait impulsivity in suicide attempters: preliminary study.

Suicide attempt is a risk factor for suicide. To investigate trait impulsivity among suicide attempters, 93 attempters admitted to an emergency department and 113 healthy controls were evaluated using the Japanese version of the Barratt Impulsiveness Scale (BIS-11J). Impulsivity was analyzed in relation to clinical data in the attempters. Total BIS-11J, attention impulsiveness, and motor impulsiveness scores were significantly higher in the attempters than in the controls. Both total BIS-11J and non-planning impulsiveness scores were significantly higher in attempters with schizophrenia and other psychotic disorders among the diagnostic groups. Control of impulsivity should be considered as one of the targets for suicide prevention.

Reliability, validity and clinical utility of a Japanese version of the Social Adaptation Self-evaluation Scale as calibrated using the Beck Depression Inventory.

AIM: The Social Adaptation Self-evaluation Scale (SASS) was developed to assess the social impairment caused by depression. The purposes of this study were to develop a Japanese version of the SASS (SASS-J) and to evaluate its reliability and validity. METHODS: The SASS-J and the 21-item Beck Depression Inventory (BDI) were administered to 322 participants (95 working patients who were working while under treatment for depression, 99 non-working patients who were absent from their work due to depression, and 128 healthy controls). The healthy controls underwent both questionnaires twice, at baseline and 2 weeks later, in order to assess test-retest reliability. RESULTS: Cronbach's alpha was 0.81. Significance correlations were found between SASS-J scores at baseline and 2 weeks later in healthy controls (R = 0.845, P < 0.001). There were negative correlations between the SASS-J and BDI scores (ρ = -0.683, P < 0.001). Mean SASS-J scores differed significantly among the three groups (working patients: 33.7 ± 7.9; non-working patients: 25.2 ± 7.8; healthy controls: 36.1 ± 6.0 [mean ± SD]). The best compromise between the true positive and the false negative rate in this study was at a cut-off point of 25/26. CONCLUSION: SASS-J showed sufficient reliability and validity, and could be considered a suitable instrument to evaluate social functioning in depressive patients.

Comparison of characteristics of suicide attempters with schizophrenia spectrum disorders and those with mood disorders in Japan.

Suicidality in patients with schizophrenia is high. To clarify the characteristics of suicidal behavior in patients with schizophrenia, we investigated suicide attempters with schizophrenia spectrum disorders in comparison with patients with mood disorders. One hundred patients with schizophrenia spectrum disorders and 155 patients with mood disorders admitted to an emergency department after a suicide attempt were interviewed in detail on items concerning 1) demographic characteristics, 2) previous suicidal behavior, and 3) index suicidal behavior. Differences between the two groups were subsequently analyzed. Patients with schizophrenia spectrum disorders showed a lower incidence of previous deliberate self-harm, and a higher incidence of a subsequent suicide attempt more than 1 year after the previous suicide attempt as well as a higher lethality of index suicide attempt compared to patients with mood disorders. Furthermore, the most common motive for making a suicide attempt in patients with schizophrenia spectrum disorders was having a mental problem. This study revealed the factors associated with suicide attempts among Japanese patients with schizophrenia spectrum disorders, and the nature of these factors makes it difficult to predict future attempts. This makes clear the importance of continuous long-term follow-up with careful attention to the mental symptoms and psychological burden for such patients.

Characteristics of suicide attempters with family history of suicide attempt: a retrospective chart review.

BACKGROUND: Family history of suicide attempt is one of the risks of suicide. We aimed at exploring the characteristics of Japanese suicide attempters with and without a family history of suicide attempt. METHODS: Suicide attempters admitted to an urban emergency department from 2003 to 2008 were interviewed by two attending psychiatrists on items concerning family history of suicide attempt and other sociodemographic and clinical information. Subjects were divided into two groups based on the presence or absence of a family history of suicide attempt, and differences between the two groups were subsequently analyzed. RESULTS: Out of the 469 suicide attempters, 70 (14.9%) had a family history of suicide attempt. A significantly higher rate of suicide motive connected with family relations (odds ratio 2.21, confidence interval 1.18-4.17, p < .05) as well as a significantly higher rate of deliberate self-harm (odds ratio 2.51, confidence interval 1.38-4.57, p < .05) were observed in patients with a family history of suicide compared to those without such history. No significant differences were observed in other items investigated. CONCLUSION: The present study has revealed the characteristics of suicide attempters with a family history of suicide attempt. Further understanding of the situation of such individuals is expected to lead to better treatment provision and outcomes, and family function might be a suitable focus in their treatment.

Dopamine D2 receptor gene polymorphisms are associated with suicide attempt in the Japanese population.

BACKGROUND: Some reports have suggested the involvement of the D2 dopaminergic function in the expression of suicidal behavior. Here, we examined associations between suicide attempts and two kinds of functional polymorphisms in the dopamine D2 receptor (DRD2) gene, namely, TaqIA and -141C Ins/Del. METHODS: Subjects included 120 suicide attempters and 123 unrelated volunteers. Those who attempted suicide were severely injured and were transferred to the emergency unit in our university hospital. To determine each genotype, we performed polymerase chain reaction and restriction fragment length polymorphism analyses. RESULTS: We found significant differences in genotypic and allelic frequencies of -141C Ins/Del and TaqIA polymorphisms between suicide attempters and healthy controls (-141C Ins/Del, p = 0.01; TaqIA,p = 0.036). The Ins allele of -141C Ins/Del was significantly more frequent in suicide attempters (p = 0.011), as well as the A2 allele of TaqIA (p = 0.017). Haplotype analysis revealed no significant linkage disequilibrium between -141C Ins/Del and TaqIA polymorphisms (D' = 0.226, r(2) = 0.016, p = 0.10). CONCLUSIONS: These findings suggest that DRD2 gene polymorphisms may be involved in the biological susceptibility to suicide.

Trait aggression in suicide attempters: a pilot study.

Suicide attempt is a potent risk factor of subsequent suicide. Understanding the characteristics of suicide attempters is important for preventing suicide. The authors investigated aggression in medically serious suicide attempters at an emergency department. Trait aggression was evaluated in 55 suicide attempters and 71 healthy individuals as a control group using the Japanese version of the Buss-Perry Aggression Questionnaire (BAQ). Total BAQ scores (t = 2.782, P = 0.006) and the hostility scores (t = 3.735, P < 0.001) were significantly higher in the suicide attempters than the controls. It suggested that to focus on aggression and its management is one of the key components for preventing suicide.

Atmospheric nitrogen dioxide gas is a plant vitalization signal to increase plant size and the contents of cell constituents.

We report the unexpected novel finding that exogenously supplied atmospheric NO2 at an ambient concentration is a plant vitalization signal to double shoot size and the contents of cell constituents. When seedlings of Nicotiana plumbaginifolia were grown for 10 wk under natural light and irrigation with 10 mm KNO3 in air containing (+NO2 plants) or not containing (-NO2 plants) 15NO2 (150 +/- 50 ppb), shoot biomass, total leaf area, and contents per shoot of carbon (C), nitrogen (N), sulphur (S), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), free amino acids and crude proteins were all approximately 2 times greater in +NO2 plants than in -NO2 plants. In mass spectrometric analysis of the 15N/14N ratio, it was found that NO2-derived N (NO2-N) comprised < 3% of total plant N, indicating that the contribution of NO2-N to total N was very minor. It thus seems very likely that the primary role of NO2 is as a multifunctional signal to stimulate plant growth, nutrient uptake and metabolism.