TRα knockout reduces NCoR recruitment and increases target gene expression. (a) ChIP assay reveals reduced TR binding and corepressor recruitment at endogenous TREs in premetamorphic tadpoles. Seven-day-old tadpoles were genotyped, and two days after genotyping, at least five tadpoles of the same genotype were homogenized together for ChIP assays with antibodies against TR, corepressor NCoR, and ID14, an extracellular protein, as a nonspecific control. Note that TRα homozygous knockout dramatically reduced receptor binding or recruitment of the corepressor NCoR at the well-characterized TRE regions of TRβ and TH/bzip genes. Exon 5 of the TRβ gene was also analyzed as a control genomic region lacking TRE. Two asterisks (**) indicate a significant difference between homozygous knockout and the other groups (P < 0.01). (b) TRα knockout increases basal expression of T3 direct target genes in premetamorphic tadpoles. Total RNA from 7-day-old tadpoles was used for real-time PCR analysis of the expression of several well-known T3 direct target genes: TH/bzip (58), Klf9 (60), ST3 (61), and TRβ (57). The expression levels were normalized against that of EF1α. Two asterisks (**) indicate a significant difference between the homozygous knockout and other groups (P < 0.01).