Abstract
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.
Highlights
Nents: a self-sustainable pacemaker that generates 24-h rhythmicity; an input pathway that allows the clock to be reset and entrained by temporal cues in the environment; and mechanisms of output that regulate molecular and biochemical pathways and translate to rhythms in physiology and behavior
The CLOCK-BMAL1 heterodimer binds to the E-box element(s) within the period gene promoter region, and in addition to its function in driving rhythmic patterns of period gene expression (12), may contribute, in a permissive manner, to the gating of pCREB induction of transcription (19, 20)
We first sought to identify whether interactions occurred between ID2 and the positive elements of the circadian clock, CLOCK and BMAL1, and whether they would complex together
Summary
Nents: a self-sustainable pacemaker that generates 24-h rhythmicity; an input pathway that allows the clock to be reset and entrained by temporal cues in the environment; and mechanisms of output that regulate molecular and biochemical pathways and translate to rhythms in physiology and behavior. The positive loop contains the basic helix-loop-helix (bHLH)/PAS (Per-Arnt-Sim) proteins BMAL1 (brain and muscle ARNT-like 1) and CLOCK (Circadian locomotor output cycle kaput) and/or NPAS2 (neuronal PAS domain-containing protein 2). These transcription factors interact through their PAS and HLH regions and bind to the promoter region of their target genes of the negative loop at specific E-box regulatory elements (CACGTG), resulting in transcriptional activation. ID (Inhibitor of DNA binding) (ID1– 4) proteins are a family of HLH transcription factors previously implicated in the regulation of cell cycle, apoptosis, and development and whose misregulation is associated with tumorigenesis (22–25) Their mechanistic role within these processes is as a transcriptional repressor, by binding to partner bHLH proteins, but as they do not contain the basic domain necessary to bind to DNA, transcription is not elicited (22–25). Whereas Id genes continue to be expressed throughout the body in the adult animal, few roles in normal postmitotic adult tissues have been described (23, 25, 27)
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