DNA binding and transcriptional regulation by the orphan receptor Rev-Erb
Rev-Erb is an orphan member of the nuclear receptor superfamily conserved between mammalian and Drosophila suggesting that it has important functions is a sequence specific DNA binding protein and transcriptional regulator. In order to investigate Rev-Erb's potential transcriptional regulatory functions a non-biased selection method was used to identify a unique binding site for Rev-Erb which contained a 5 base pair A/T rich sequence followed by a single copy of the core binding site for estrogen and thyroid hormone receptors (AGGTCA). In spite of in vitro binding studies which indicated that Rev-Erb monomers were highly specific for the RevRE, no activity was seen on chloramphenicol acetyl transferase (CAT) reporters containing the RevRE. However, a potent repression domain was identified within the C-Terminus of Rev-Erb using chimeric proteins containing the DNA binding domain of the yeast GAL4 protein and various parts of Rev-Erb. Since the repression domain was not detected on the momomeric binding site which had been selected, a revised DNA binding site selection strategy was devised to test the hypothesis that Rev-Erb might function on a different site as a dimer. This approach identified a binding site containing a Rev-Erb monomer site followed by two base pair and a second AGGTCA motif (Rev-DR2). Remarkably, Rev-Erb bound as a homodimer to Rev-DR2 but not to other direct repeats or to a standard DR2 sequence. More importantly, Rev-Erb markedly repressed the basal activity of reporters with a strong Rev-DR2 specificity. Rev-Erb also repressed basal as well as retinoic acid-induced transcription from a naturally occurring Rev-DR2 in the CRBPI gene, which is a functional retinoic acid response element. Thus, although Rev-Erb is distinguished from other thyroid/steroid receptor superfamily by its ability to bind DNA as a monomer, it functions as a homodimer to repress transcription of genes containing a novel DR2 element.
Harding, Heather Parish, "DNA binding and transcriptional regulation by the orphan receptor Rev-Erb" (1995). Dissertations available from ProQuest. AAI9532193.