Epigenetic Modulation of Circadian Rhythms: <em>Bmal1</em> Gene Regulation

Circadian rhythms that function in behaviour and physiology have adaptive significance for living organisms from bacteria to humans and reflect the presence of a biological clock. The engine of circadian rhythms is a transcription-translation feedback loop that is fine-tuned by epigenetic regulation in higher eukaryotes. We elucidated the chromatin structure of the Bmal1 gene, a critical component of the mammalian clock system, and have continued to investigate transcriptional regulation including DNA methylation. Various ailments including metabolic diseases can disrupt circadian rhythms, and many human diseases are associated with altered DNA methylation. Therefore, regulated circadian rhythms are important for human health. Here, we summarise the importance of epigenetic clock gene regulation, including DNA methylation of the Bmal1 gene, from the viewpoint of relationships to diseases

A novel E4BP4 element drives circadian expression of mPeriod2

Period2 (Per2) is an essential component of the mammalian clock mechanism and robust circadian expression of Per2 is essential for the maintenance of circadian rhythms. Although recent studies have shown that the circadian E2 enhancer (a non-canonical E-box) accounts for most of the circadian transcriptional drive of mPer2, little is known about the other cis-elements of mPer2 oscillatory transcription. Here, we examined the contribution of E4BP4 to Per2 mRNA oscillation in the cell-autonomous clock. Knockdown experiments of E4BP4 in both Northern blots and real-time luciferase assays suggested that endogenous E4BP4 negatively regulates Per2 mRNA oscillation. Sequence analysis revealed two putative E4BP4-binding sites (termed A-site and B-site) on mammalian Per2 promoter regions. Luciferase assays with mutant constructs showed that a novel E4BP4-binding site (B-site) is responsible for E4BP4-mediated transcriptional repression of Per2. Furthermore, chromatin immunoprecipitation assays in vivo showed that the peak of E4BP4 binding to the B-site on the Per2 promoter almost matched the trough of Per2 mRNA expression. Importantly, real-time luciferase assays showed that the B-site in addition to the E2 enhancer is required for robust circadian expression of Per2 in the cell-autonomous clock. These findings indicated that E4BP4 is required for the negative regulation of mammalian circadian clocks

Molecular characterization of Mybbp1a as a co-repressor on the Period2 promoter

The circadian clock comprises transcriptional feedback loops of clock genes. Cryptochromes are essential components of the negative feedback loop in mammals as they inhibit CLOCK-BMAL1-mediated transcription. We purified mouse CRY1 (mCRY1) protein complexes from Sarcoma 180 cells to determine their roles in circadian gene expression and discovered that Myb-binding protein 1a (Mybbp1a) interacts with mCRY1. Mybbp1a regulates various transcription factors, but its role in circadian gene expression is unknown. We found that Mybbp1a functions as a co-repressor of Per2 expression and repressed Per2 promoter activity in reporter assays. Chromatin immunoprecipitation (ChIP) assays revealed endogenous Mybbp1a binding to the Per2 promoter that temporally matched that of mCRY1. Furthermore, Mybbp1a binding to the Per2 promoter correlated with the start of the down-regulation of Per2 expression and with the dimethylation of histone H3 Lys9, to which it could also bind. These findings suggest that Mybbp1a and mCRY1 can form complexes on the Per2 promoter that function as negative regulators of Per2 expression

ALMA 0.02"-resolution observations reveal HCN-abundance-enhanced counter-rotating and outflowing dense molecular gas at the NGC 1068 nucleus

We present ALMA ~0.02"-resolution observations of the nucleus of the nearby (~14 Mpc) type-2 AGN NGC 1068 at HCN/HCO+/HNC J=3-2 lines, as well as at their 13C isotopologue and vibrationally excited lines, to scrutinize the morphological/dynamical/chemical/physical properties of dense molecular gas in the putative dusty molecular torus around a mass-accreting supermassive black hole. We confirm almost east-west-oriented dense molecular gas emission both morphologically and dynamically, which we regard as coming from the torus. Bright emission is compact (<3 pc), and low-surface-brightness emission extends out to 5-7 pc. These dense molecular gas properties are not symmetric between the eastern and western torus. The HCN J=3-2 emission is stronger than the HCO+ J=3-2 emission within the ~7 pc torus region, with an estimated dense molecular mass of (0.4-1.0)x10^6Msun. We interpret that HCN abundance is enhanced in the torus. We detect signatures of outflowing dense molecular gas and a vibrationally excited HCN J=3-2 line. Finally, we find that in the innermost (<1 pc) part of the torus, the dense molecular line rotation velocity, relative to the systemic velocity, is the opposite of that in the outer (>2 pc) part, in both the eastern and western torus. We prefer a scenario of counter-rotating dense molecular gas with innermost almost-Keplerian-rotation and outer slowly rotating (far below Keplerian) components. Our high-spatial-resolution dense molecular line data reveal that torus properties of NGC 1068 are much more complicated than the simple axi-symmetrically rotating torus picture in the classical AGN unification paradigm.Comment: 45 pages, 20 Figures. Accepted for publication in Ap

Aberrant Expression of Keratin 7 in Hepatocytes as a Predictive Marker of Rapid Progression to Hepatic Failure in Asymptomatic Primary Biliary Cirrhosis

A predictive marker of the rapid progression to hepatic failure is desired for patients with asymptomatic primary biliary cirrhosis (aPBC). We performed a systematic cohort analysis of 101 patients diagnosed as having aPBC and the rapid progression to liver failure in some, by focusing on cholestasis. Cholestasis was assessed by aberrant keratin7 (K-7) expressions in the patientsʼ hepatocytes. Intralobular expressions of K-7 were found in 9 of the 101 patients. The grades of K-7 expression were significantly associated with the levels of alanine aminotransferase, alkaline phosphatase, and total bilirubin at the time of diagnosis, but not with bile duct loss or cholestasis. Stepwise logistic regression analysis revealed that high grades of K-7 expression correlated positively with high levels of total bilirubin. During the follow-up period, 8 patients developed jaundice, and the mean period until the development of jaundice was 5.2 years. The proportional hazards models for the risk of developing jaundice identified a high grade of aberrant K-7 expression in hepatocytes as the only significant risk factor. Aberrant K-7 expression in hepatocytes can be used as an additional marker to predict rapid progression to liver failure in patients with aPBC at the time of diagnosis

Prognostic Model for Hepatocellular Carcinoma with Time-Dependent Factors

The purpose of this study was to build a prognostic model of hepatocellular carcinoma (HCC) using time-dependent covariates to re-evaluate the prognosis at any stage of the disease. The subjects were consecutive HCC patients who were treated at our institute between 1995 and 2007. We constructed time-fixed and time-dependent prognostic models with a training group (n＝336) and compared the prognostic abilities between conventional Cancer of the Liver Italian Program (CLIP) scores, Japan Integrated Staging (JIS) scores, an Okuda classification, and our prognostic models in the testing group (n＝227) with the c-index. The time-dependent prognostic model consisted of main tumor size, tumor number, portal vein invasion, distant metastasis, alpha-fetoprotein, des-gamma-carboxy prothrombin (DCP), bilirubin, and albumin and the weighted scores were set for each factor depending on the hazard ratio for the prognosis. The prognostic index was determined by summing the scores. The c-index values for the CLIP scores, JIS scores, Okuda classification, and our time-dependent model were 0.741, 0.727, 0.609, and 0.870, respectively. These results indicate that our time-dependent model can estimate the prognosis of HCC more precisely than traditional time-fixed models and can be used to re-predict the prognosis of HCC

Sub-millimeter Observations of Giant Molecular Clouds in the Large Magellanic Cloud: Temperature and Density as Determined from J=3-2 and J=1-0 transitions of CO

We have carried out sub-mm 12CO(J=3-2) observations of 6 giant molecular clouds (GMCs) in the Large Magellanic Cloud (LMC) with the ASTE 10m sub-mm telescope at a spatial resolution of 5 pc and very high sensitivity. We have identified 32 molecular clumps in the GMCs and revealed significant details of the warm and dense molecular gas with n(H2) $\sim$ 10$^{3-5}$ cm$^{-3}$ and Tkin $\sim$ 60 K. These data are combined with 12CO(J=1-0) and 13CO(J=1-0) results and compared with LVG calculations. We found that the ratio of 12CO(J=3-2) to 12CO(J=1-0) emission is sensitive to and is well correlated with the local Halpha flux. We interpret that differences of clump propeties represent an evolutionary sequence of GMCs in terms of density increase leading to star formation.Type I and II GMCs (starless GMCs and GMCs with HII regions only, respectively) are at the young phase of star formation where density does not yet become high enough to show active star formation and Type III GMCs (GMCs with HII regions and young star clusters) represents the later phase where the average density is increased and the GMCs are forming massive stars. The high kinetic temperature correlated with \Halpha flux suggests that FUV heating is dominant in the molecular gas of the LMC.Comment: 74 pages, including 41 figures, accepted for publication in ApJ