Transactivation by the thyroid hormone receptor is dependent on the spacer sequence in hormone response elements containing directly repeated half-sites.
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.
Harbers
M
M
Karolinska Institute, Medical Nobel Institute, Department of Cell and Molecular Biology, Laboratory of Developmental Biology, Stockholm, Sweden.
Wahlström
G M
GM
Vennström
B
B
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D047488:Retinoid X Receptors; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D017931:DNA Primers; D004275:DNA, Ribosomal; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D047488:Retinoid X Receptors; D014157:Transcription Factors