The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-daydependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.

Bood POZ containing gene type 2 (BPOZ‐2), which contains ankyrin repeats, NLS, BTB/POZ domains and LXXLL motifs, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. We isolated a cDNA encoding eukaryotic elongation factor 1A1 (eEF1A1) as a BPOZ‐2 binding protein by screening a human thymus cDNA library using a yeast two‐hybrid system. eEF1A1 is essential for translation and is also involved in the 26S proteasome‐dependent degradation of misfolded or unfolded proteins. The binding between BPOZ‐2 and eEF1A1 was confirmed by pull‐down and immunoprecipitation assays in vitro and in vivo, respectively. BPOZ‐2 binds to eEF1A1 through the ankyrin repeats and both BTB/POZ domains in BPOZ‐2 and Domains I and III in eEF1A1. BPOZ‐2 and eEF1A1 over‐expressed in HEK 293T cells co‐localized as speckles within the cytoplasm. BPOZ‐2 promoted eEF1A1 ubiquitylation and degradation, suggesting that eEF1A1 is a substrate of BPOZ‐2. BPOZ‐2 inhibited GTP binding to eEF1A1 and prevented translation in in vitro translation assay using rabbit reticulocytes.

No abstract