Timeless noncoding DNA contains cell- type preferential enhancers important for proper Drosophila circadian regulation

• 成果类型: Article
• 署名作者: Ma, Dingbang; Ojha, Pranav; Yu, Albert D.; Araujo, Maisa S.; Luo, Weifei; Keefer, Evelyn; Diaz, Madelen M.; Wu, Meilin; Joiner, William J.; Abruzzi, Katharine C.; Rosbash, Michael
• 署名单位: Chinese Academy of Sciences; Shanghai Institute of Organic Chemistry, CAS; Brandeis University; Howard Hughes Medical Institute; Brandeis University; Fundacao Oswaldo Cruz; Guangxi Academy of Sciences; University of Miami; University of California System; University of California San Diego; University of California System; University of California San Diego
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8639
• DOI: 10.1073/pnas.2321338121
• 发表日期: 2024-04-09
• 关键词: clock gene; transcriptional architecture; expression; rhythms; contributes; oscillator; promoter; platform; period; MODEL

摘要

To address the contribution oftranscriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR- mediated deletions within two regions of the circadian gene timeless (tim), an intronic E - box region and an upstream E - box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E - box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E - box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase- accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E - box region. Significance Drosophila circadian rhythms are governed by a feedback loop, in which the positive transcription factor CLK (CLOCK)/CYC (CYCLE) associates with two E - box- containing enhancers, one upstream and one intronic, within its negative regulatory gene timeless. Reporter gene experiments from more than 20 y ago showed that only the upstream enhancer affects timeless expression in tissue culture and fly heads. Consistent with these observations are current experiments indicating that the upstream enhancer is essential for timeless expression in glia. However, these deletions only modestly affect clock neuron timeless expression and circadian behavior. ATAC- seq experiments show that the intronic enhancer is much more active in neurons than glia, which indicate a discrete regulatory program for core clock genes in circadian neurons.