Characterization of the Promoter of the Rat Sarcoplasmic Endoplasmic Reticulum Ca2+-ATPase 1 Gene and Analysis of Thyroid Hormone Responsiveness

Abstract

Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid hormone (T3) stimulates the expression of the SERCA1 isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned and studied the first 962 base pairs of the 5'-flanking region of the rat SERCA1 gene. This sequence was tested for T3-regulated expression in transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) alpha in mobility shift assays. A construct of the 5'-flanking region and a reporter gene was unresponsive to T3 in the absence of co-transfected thyroid hormone receptor. In the presence of TRalpha, a T3 induction ratio of almost 4.0 was found, and this induction ratio was doubled with co-transfection of an RXR expression plasmid. Analysis of progressive 5'-deletion fragments of the sequence indicated multiple regions involved in T3 responsiveness. Three regions, R1, R2, and R3, were identified that bound TR complexes in mobility shift assays and conferred T3 responsiveness to a heterologous promoter. The most potent of these thyroid hormone response elements, R3, increased the 2-fold background T3 stimulation of the thymidine kinase promoter to nearly 6-fold. Detailed analysis of this element showed that four TR-binding half-sites, comprising two independent thyroid hormone response elements, interact cooperatively to give the maximal T3 response. T3 regulation of SERCA1 expression is mediated by a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor partners or cell-specific transcription factors.