Ken Takamatsu

CLOCK-mediated acetylation of BMAL1 controls circadian function

Regulation of circadian physiology relies on the interplay of interconnected transcriptional–translational feedback loops. The CLOCK–BMAL1 complex activates clock-controlled genes, including cryptochromes (Crys), the products of which act as repressors by interacting directly with CLOCK–BMAL1. We have demonstrated that CLOCK possesses intrinsic histone acetyltransferase activity and that this enzymatic function contributes to chromatin-remodelling events implicated in circadian control of gene expression. Here we show that CLOCK also acetylates a non-histone substrate: its own partner, BMAL1, is specifically acetylated on a unique, highly conserved Lys 537 residue. BMAL1 undergoes rhythmic acetylation in mouse liver, with a timing that parallels the downregulation of circadian transcription of clock-controlled genes. BMAL1 acetylation facilitates recruitment of CRY1 to CLOCK–BMAL1, thereby promoting transcriptional repression. Importantly, ectopic expression of a K537R-mutated BMAL1 is not able to rescue circadian rhythmicity in a cellular model of peripheral clock. These findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.

Distribution of hippocalcin mRNA and immunoreactivity in rat brain

Distribution of hippocalcin in rat brain was analysed by in situ hybridization and immunohistochemical methods. Hippocalcin mRNA and immunoreactivity were expressed more intensely in the pyramidal cells of the hippocampus, intensely in the Purkinje cells of the cerebellum, moderately in the dentate granule cells and pyramidal cells of cerebral cortex layers II-VI and weakly in the large neuronal cells of the caudate-putamen. Some discrepancies in the localization of hippocalcin mRNA and immunoreactivity were noted in the mamillary nuclei, anterior part of the thalamus and the septal nuclei. In most cell types, hippocalcin immunoreactivity was localized in the cytoplasm and plasma membrane of cell bodies and dendrites.

Purification and characterization of S-modulin, a calcium-dependent regulator on cGMP phosphodiesterase in frog rod photoreceptors.

S-modulin is a 26 kDa protein that regulates light sensitivity of cGMP phosphodiesterase in a Ca(2+)-dependent manner in frog rod outer segments (ROSs). In the present study, we purified S-modulin by taking advantage of a hydrophobic interaction between Phenyl Sepharose and S-modulin at high Ca2+ concentrations. The yield was greater than 90%. 45Ca(2+)-binding experiment showed that S-modulin is a Ca(2+)-binding protein. At high Ca2+ concentrations, S-modulin binds to ROS membranes. The binding target of the Ca2+/S-modulin complex is possibly a ROS membrane lipid(s), but it was difficult to identify. The binding was observed mainly at greater than 1 microM Ca2+. The amino acid sequence deduced from proteolytic fragments of S-modulin was approximately 80% and 60% identical to those of recovering and visinin, respectively.

Molecular Genetic Analysis of Myelin-Deficient Mice: Shiverer Mutant Mice Show Deletion in Gene(s) Coding for Myelin Basic Protein

Abstract: The gene expression of myelin basic proteins (MBPs) in shiverer mutant mice was investigated by the Northern and Southern hybridization techniques. In the control mice RNA molecules from the brains which were about 2,300 nucleotides in length were hybridized to cDNA of 1.8 kb encoding for a mouse MBP, but RNA from the brains of 3‐week‐old shiverer mutant mice contained no detectable amount of MBP transcripts hybridizing to this probe. Moreover the shiverer mutant mice lost several restriction fragments that hybridized to the same probe in the control mice when each of the five restriction enzymes, i.e., HindIII, PstI, PvuII, AccI, and StuI, was used. These data suggest that the shiverer mutation may correspond to the deletion of a large portion of MBP exon(s) in the gene, and this deletion causes inefficient transcription leading to the depletion of MBPs in the myelin and the dysmyelination observed in these mice.

CK2|[alpha]| phosphorylates BMAL1 to regulate the mammalian clock

Clock proteins govern circadian physiology and their function is regulated by various mechanisms. Here we demonstrate that Casein kinase (CK)-2α phosphorylates the core circadian regulator BMAL1. Gene silencing of CK2α or mutation of the highly conserved CK2-phosphorylation site in BMAL1, Ser90, result in impaired nuclear BMAL1 accumulation and disruption of clock function. Notably, phosphorylation at Ser90 follows a rhythmic pattern. These findings reveal that CK2 is an essential regulator of the mammalian circadian system.

Nucleocytoplasmic shuttling and phosphorylation of BMAL1 are regulated by circadian clock in cultured fibroblasts

Background:  Recent discoveries of clock proteins have unveiled an important part of the mammalian circadian clock mechanism. However, the molecular clockwork that cause these fundamental feedback loops to stably oscillate with a ∼24 h‐periodicity remain unclear.

Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro

Abstract The influence of the reeler mutation on the development of the cerebellum was examined morphologically and biochemically both in vivo and in vitro. SDS-polyacrylamide gel electrophoresis revealed that all cerebellar proteins which increase during development are found in the same amounts in reeler and control. The time schedule for migration of granule cells and formation of the granular layer in the reeler shows no significant difference from the control. Immunohistochemical methods using antisera against S-100 and glial fibrillary acidic (GFA) proteins reveal that most of the Bergmann cells are scattered around the molecular and granular layers. But in some part, the cells were aligned like those of the control though independently of the position of Purkinje cells. Proliferated astrocytes with finely arborized processes were observed in the central mass of the large neuron groups in the cerebellum from the reeler. High CNPase activity in the reeler cerebellum was suggested to be due to a decrease in granule cells. Autoradiography of the sections from the control cerebellum after intraperitoneal injection of 2-deoxy[14C]glucose revealed that the incorporation of 2-deoxyglucose was maximum in the granular layer. Little was incorporated in the white matter. In the reeler, incorporation of 2-deoxyglucose was found not only in the granular layer but also in the white matter. The primary cultures from the reeler cerebellum were generally comparable to those of the normal control in terms of neuritic outgrowth, schedule of general development, and quantity of myelin formation, except for the lack of laminar structure.

Immunohistochemical localization of neural visinin-like Ca2+-binding protein 2 in adult rat brain

The distribution of neural visinin-like Ca(2+)-binding protein 2 (NVP2) in adult rat brain was analyzed by immunoblot and immunohistochemical methods. NVP2 immunoreactivity was expressed intensely in the pyramidal cells of the CA1 and 2 regions of Ammons horn, the granule cells of the dentate gyrus and the pyramidal cells of the cerebral cortex layers II to VI, moderately in the pyramidal-shaped cells of the anterior olfactory nucleus and large spindle-shaped cells of the globus pallidus, and weakly in neurons in the nucleus accumbens and the anterior and dorsomedial thalamus. In most cell types, NVP2 immunoreactivity was located in the cytoplasm and plasma membrane of the cell bodies and dendrites.

Identification of recoverin-like immunoreactivity in mouse brain

Mouse brain was found by immunoblot analysis to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+ and had a Ca(2+)-dependent membrane-binding property. Immunohistochemical studies provided the specific distribution of P23k in cerebral cortex and cerebellum.

Synchronization of Circadian Per2 Rhythms and HSF1- BMAL1:CLOCK Interaction in Mouse Fibroblasts after Short-Term Heat Shock Pulse

Circadian rhythms are the general physiological processes of adaptation to daily environmental changes, such as the temperature cycle. A change in temperature is a resetting cue for mammalian circadian oscillators, which are possibly regulated by the heat shock (HS) pathway. The HS response (HSR) is a universal process that provides protection against stressful conditions, which promote protein-denaturation. Heat shock factor 1 (HSF1) is essential for HSR. In the study presented here, we investigated whether a short-term HS pulse can reset circadian rhythms. Circadian Per2 rhythm and HSF1-mediated gene expression were monitored by a real-time bioluminescence assay for mPer2 promoter-driven luciferase and HS element (HSE; HSF1-binding site)-driven luciferase activity, respectively. By an optimal duration HS pulse (43°C for approximately 30 minutes), circadian Per2 rhythm was observed in the whole mouse fibroblast culture, probably indicating the synchronization of the phases of each cell. This rhythm was preceded by an acute elevation in mPer2 and HSF1-mediated gene expression. Mutations in the two predicted HSE sites adjacent (one of them proximally) to the E-box in the mPer2 promoter dramatically abolished circadian mPer2 rhythm. Circadian Per2 gene/protein expression was not observed in HSF1-deficient cells. These findings demonstrate that HSF1 is essential to the synchronization of circadian rhythms by the HS pulse. Importantly, the interaction between HSF1 and BMAL1:CLOCK heterodimer, a central circadian transcription factor, was observed after the HS pulse. These findings reveal that even a short-term HS pulse can reset circadian rhythms and cause the HSF1-BMAL1:CLOCK interaction, suggesting the pivotal role of crosstalk between the mammalian circadian and HSR systems.