Repression of Cardiac Phospholamban Gene Expression Is Mediated by Thyroid Hormone Receptor-α1 and Involves Targeted Covalent Histone Modifications

Phospholamban (PLB) is a critical regulator of Ca2+ cycling in heart muscle cells, and its gene expression is markedly down-regulated by T-3. Nonetheless, little is known about the molecular mechanisms of T-3-dependent gene silencing in cardiac muscle, and it remains unclear whether thyroid hormone receptors (TRs) directly bind at the PLB gene in vivo and facilitate transcriptional repression. To investigate the regulatory role of TRs in PLB transcription, we used a physiological murine heart muscle cell line (HL-1) that retains cardiac electrophysiological properties, expresses both TR alpha 1 and TR beta 1 subtypes, and exhibits T-3-dependent silencing of PLB expression. By performing RNA interference assays with HL-1 cells, we found that TR alpha 1, but not TR beta 1, is essential for T3-dependent PLB gene repression. Interestingly, a PLB reporter gene containing only the core promoter sequences -156 to +64 displayed robust T-3-dependent silencing in HL-1 cells, thus suggesting that transcriptional repression is facilitated by TR alpha 1 via the PLB core promoter, a regulatory region highly conserved in mammals. Consistent with this notion, chromatin immunoprecipitationandin vitro binding assaysshowthat TR alpha 1 directly binds at the PLB core promoter region. Furthermore, addition of T3 triggered alterations in covalent histone modifications at the PLB promoter that are associated with gene silencing, namely a pronounced decrease in both histone H3 acetylation and histone H3 lysine 4 methylation. Taken together, our data reveal that T-3-dependent repression of PLB in cardiac myocytes is directly facilitated by TR alpha 1 and involves the hormone-dependent recruitment of histone-modifying enzymes associated with transcriptional silencing. (Endocrinology 151: 2946-2956, 2010)