Regulation of mineralocorticoid receptor activation by circadian protein TIMELESS

The mineralocorticoid receptor (MR) is a ligand-activated transcription factor that regulates cardiorenal physiology and disease. Ligand-dependent MR transactivation involves a conformational change in the MR and recruitment of coregulatory proteins to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate the circadian protein TIMELESS as a selective regulator of MR transactivation. TIMELESS has an established role in cell cycle regulation and DNA repair. TIMELESS may not be central to mammalian clock function and does not bind DNA; however, RNA and protein levels oscillate over 24 h. Co-expression of TIMELESS down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. TIMELESS markedly inhibited MR transactivation of synthetic and native gene promoters and expression of MR target genes in H9c2 cardiac myoblasts. Immunofluorescence showed aldosterone induces colocalisation of TIMELESS and MR, although a direct interaction was not confirmed by coimmunoprecipitation. Potential regulation of circadian clock targets cryptochrome 1 and 2 by TIMELESS was not detected. However, our data suggest that these effects may involve TIMELESS coactivation of oestrogen receptor alpha (ERα). Taken together, these data suggest that TIMELESS may contribute to MR transcriptional outputs via enhancing ERα inhibitory actions on MR transactivation. Given the variable expression of TIMELESS in different cell types, these data offer new opportunities for the development of MR modulators with selective actions.