Transactivation by the Thyroid Hormone Receptor Is Dependent on the Spacer Sequence in Hormone Response Elements Containing Directly Repeated Half-Sites

Abstract

The thyroid hormone receptor (TR) regulates the transcription of its target genes by interacting with specific hormone response elements consisting usually of directly repeated half-sites with the consensus sequence AGGTCA. To investigate the role of the spacer sequences separating the half-sites, heterodimers formed by TRalpha and the retinoid-X receptor (RXR) were used in a PCR based selection and amplification assay. The TRalpha/RXR heterodimer selected for elements with directly repeated half-sites having a spacer of 4 nucleotides (DR4). Preferences for nucleotides in the TR binding half-site motif as well as for the 4 nucleotides separating the two half-sites were found. DNA binding and transfection studies using DR4 elements with different spacer sequences showed the importance of these nucleotides for the activity of the response element: some spacer sequences allowed little or no transactivation from the element, whereas other sequences supported strong transactivation. A pyrimidine nucleotide in position three of the spacer enhanced TRalpha binding and transactivation. Additional experiments showed that heterodimers between RXR and other putative receptors exhibited a similar but distinct specificity for the spacer sequence. Our results thus suggest that the four nucleotides separating the two half-sites in hormone response elements have a major role in determining induction of hormone responsive genes.

Modulation of Mouse and Human Phenobarbital-Responsive Enhancer Module by Nuclear Receptors

“…Indeed, hepatocytes transfected with a PBREM construct showed only 2-fold activation after PCN treatment (Xie et al, 2000b;Smirlis et al, 2001); hepatic CYP2B10 was induced 37-fold by PB but only 7-fold by PCN (Pellinen et al, 1994); CYP2B10 mRNA induction was not affected either by thyroid hormone or by retinoic acid in mouse hepatocytes ); T3 does not seem to affect PBREM or its associated factors in rats (Ganem et al, 1999). The identity of 5Ј-flanking nucleotides in DR4 motifs is important for TR-mediated activation (Harbers et al, 1996;Zhang and Lazar, 2000) and this property may explain the discrepancy between the strong binding and inefficient function by TR␣ on PBREM. Although TR isoforms are expressed in liver (Zhang and Lazar, 2000), and TR can inhibit CAR LBD, the levels of TR relative to CAR may be too low for significant suppression via competition for NR coregulators.…”

“…The sources of expression vectors for mRAR␣ and hRAR␣ (Zelent et al, 1989), cTR␣ (Harbers et al, 1996), hLXR␤ (Teboul et al, 1995), mCOUP-TFI (NR2F1; Cooney et al, 1993), hPPAR␣ and mPPAR␣ (NR1C1; Sher et al, 1993), hFXR (Forman et al, 1995), hCAR and mCAR Sueyoshi et al, 1999), hVDR (Quack and Carlberg, 2000) and hPXR and mPXR (Lehmann et al, 1998) have been described previously. The expression plasmid for coactivator hTIF2 (Voegel et al, 1996) was donated by Dr. Hinrich Gronemeyer (IGBMC, Illkirch, France).…”

Regulation of regional expression in rat brain PC2 by thyroid hormone/characterization of novel negative thyroid hormone response elements in the PC2 promoter

“…Recent studies have demonstrated that RXR heterodimerization can augment T 3 -mediated gene regulation to increase TR-DNA interactions (11,19). However, the magnitude of the augmentation varies significantly, especially when different cell lines were used.…”

Thyroid hormone regulation of prohormone convertase 1 (PC1): regional expression in rat brain and in vitro characterization of negative thyroid hormone response elements

“…Recent studies have demonstrated that RXR heterodimerization can augment T 3 -mediated gene regulation to increase TR/DNA interactions (Force et al 1994, Harbers et al 1996. However, the magnitude of the augmentation varies significantly, especially when different cell lines were used.…”