Project description:CWO binding sites were genome-widely searched with Drosophila genome tiling array. Abstract: The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNAi system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix-ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tilling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation. Keywords: ChIP-chip
Project description:To investigate the temperature response in Thermosynechococcus elongatus (BP-1) and look for coordinated control in the cell, transcriptomes of BP-1 were measured using RNAseq following exposure to low and high temperature stress. The amount of temperature increase (53 to 61 °C) and decrease (53 to 45 °C) was based on the allowable range of continuous growth. The cells were growth in three separate culture tubes under LED light in a temperature-controlled submersible chamber under batch growth conditions. The temperature shift was conducted once the cells reached their target concentration of 1.5E7 cells/ml. A control experiment was conducted, in which the temperature remained at 53 °C, ensuring that the cellular physiology and light attenuation were comparable and that the only difference between the hot and cold treatments relative to the control treatment was temperature.
Project description:In the present study we show that the master myogenic regulatory factor, MYOD1, is a positive modulator of molecular clock amplitude and functions with the core clock factors for expression of clock-controlled genes in skeletal muscle. We demonstrate that MYOD1 directly regulates the expression and circadian amplitude of the positive core clock factor Bmal1. We identify a non-canonical E-box element in Bmal1 and demonstrate that is required for full MYOD1-responsiveness. Bimolecular fluorescence complementation assays demonstrate that MYOD1 colocalizes with both BMAL1 and CLOCK throughout myonuclei. We demonstrate that MYOD1 and BMAL1:CLOCK work in a synergistic fashion through a tandem Ebox to regulate the expression and amplitude of the muscle specific clock-controlled gene, Titin-cap (Tcap). In conclusion, these findings reveal mechanistic roles for the muscle specific transcription factor MYOD1 in the regulation of amplitude of the molecular clock as well as synergistic regulation of clock-controlled genes in skeletal muscle.
