cDNA Array Analysis of Pineal Gene Expression Reveals Circadian Rhythmicity of the Dominant Negative Helix-Loop-Helix Protein-Encoding Gene, Id-1

The pineal gland is a major output of the endogenous vertebrate circadian clock, with melatonin serving as the output signal. In many species, elevated nocturnal melatonin production is associated with changes in pineal gene expression. In the current study, cDNA array analysis was used in an attempt to identify additional genes that exhibit day/night differential expression in the rat pineal gland. This revealed 38 candidate genes, including Id‐1 (inhibitor of DNA binding and differentiation). Id‐1 encodes a helix‐loop‐helix (HLH) protein that lacks a basic DNA binding domain and could affect pineal physiology via a dominant negative trans‐acting regulatory activity. For this reason Id‐1 was selected for further analysis. Id‐1 was expressed in a major population of pineal cells and the Id‐1 protein was associated with a nuclear complex. The levels of Id‐1 mRNA and protein exhibit approximately six‐fold day/night rhythms. In contrast, the related genes Id‐2 and Id‐3 do not exhibit marked day/night differences in pineal expression. Rhythmic Id‐1 expression is primarily limited to a C‐terminally extended splice variant of Id‐1, which would restrict the functional output of the rhythm to protein binding partners of this isoform of Id‐1. Our findings add to the body of evidence indicating that transcriptional regulators play a role in neuroendocrine rhythms, and extend this by introducing the concept of a dominant negative HLH involvement. The rhythm in Id‐1 in the pineal gland provides an experimental opportunity to identify Id‐1‐binding partners which may also be involved in Id‐1 activity in other functional contexts.