Department of Biological Sciences, Galvin Life Science Center, University of Notre Dame, Notre Dame, Indiana 46556, USA.
J Biol Chem. 2010 Dec 10;285(50):38987-9000. doi: 10.1074/jbc.M110.175182. Epub 2010 Sep 22.
ID2 is a rhythmically expressed HLH transcriptional repressor. Deletion of Id2 in mice results in circadian phenotypes, highlighted by disrupted locomotor activity rhythms and an enhanced photoentrainment response. ID2 can suppress the transactivation potential of the positive elements of the clock, CLOCK-BMAL1, on mPer1 and clock-controlled gene (CCG) activity. Misregulation of CCGs is observed in Id2(-/-) liver, and mutant mice exhibit associated alterations in lipid homeostasis. These data suggest that ID2 contributes to both input and output components of the clock and that this may be via interaction with the bHLH clock proteins CLOCK and BMAL1. The aim of the present study was to explore this potential interaction. Coimmunoprecipitation analysis revealed the capability of ID2 to complex with both CLOCK and BMAL1, and mammalian two-hybrid analysis revealed direct interactions of ID2, ID1 and ID3 with CLOCK and BMAL1. Deletion of the ID2 HLH domain rendered ID2 ineffective at inhibiting CLOCK-BMAL1 transactivation, suggesting that interaction between the proteins is via the HLH region. Immunofluorescence analysis revealed overlapping localization of ID2 with CLOCK and BMAL1 in the cytoplasm. Overexpression of CLOCK and BMAL1 in the presence of ID2 resulted in a significant reduction in their nuclear localization, revealing that ID2 can sequester CLOCK and BMAL1 to the cytoplasm. Serum stimulation of Id2(-/-) mouse embryonic fibroblasts resulted in an enhanced induction of mPer1 expression. These data provide the basis for a molecular mechanism through which ID2 could regulate aspects of both clock input and output through a time-of-day specific interaction with CLOCK and BMAL1.
ID2 是一种节律表达的 HLH 转录抑制因子。在小鼠中缺失 Id2 会导致昼夜节律表型，表现为运动活动节律紊乱和光适应反应增强。ID2 可以抑制时钟的正性元件 CLOCK-BMAL1 对 mPer1 和时钟控制基因 (CCG) 活性的转录激活潜力。在 Id2(-/-) 肝脏中观察到 CCG 的失调，突变小鼠表现出相关的脂质稳态改变。这些数据表明 ID2 有助于时钟的输入和输出组件，并且这可能是通过与 bHLH 时钟蛋白 CLOCK 和 BMAL1 相互作用实现的。本研究的目的是探索这种潜在的相互作用。共免疫沉淀分析显示 ID2 能够与 CLOCK 和 BMAL1 形成复合物，哺乳动物双杂交分析显示 ID2、ID1 和 ID3 与 CLOCK 和 BMAL1 之间存在直接相互作用。ID2 的 HLH 结构域缺失使 ID2 无法有效抑制 CLOCK-BMAL1 的转录激活，表明蛋白质之间的相互作用是通过 HLH 区域进行的。免疫荧光分析显示 ID2 与 CLOCK 和 BMAL1 在细胞质中存在重叠定位。在存在 ID2 的情况下过表达 CLOCK 和 BMAL1 会导致它们的核定位显著减少，表明 ID2 可以将 CLOCK 和 BMAL1 隔离到细胞质中。Id2(-/-) 小鼠胚胎成纤维细胞的血清刺激导致 mPer1 表达的显著诱导增强。这些数据为 ID2 通过与 CLOCK 和 BMAL1 的特定时间依赖性相互作用调节时钟输入和输出的各个方面提供了分子机制的基础。
