Cloning of a human thyroid hormone responsive gene

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3,3', 5-Triodothyronine (T3), an active form of the thyroid hormone, plays an important role in fetal development and throughout life in humans. T3 exerts its effect through binding to its nuclear receptor and regulating expression of target genes. Thus, identification of T3-responsive genes in various tissue is important to elucidate T3 action at a molecular level in human. Utilizing a method called "differential display of mRNAs by means of polymerase chain reaction", the cDNA fragment of a T3-responsive gene named ZAKI-4 was cloned from cultured human skin fibroblasts. Northern blot analysis revealed that there were tow ZAKI-4mRNA species (3.4 and 1.4kb) and both mRNAs were increased by a physiological concentration of T3 within 12hr. This increase was due to an increased transcription of ZAKI-4 gene rather than increased stability of ZAKI-4 mRNA by T3. The T3 effect on ZAKI-4 expression was abolished by the treatment with cycloheximide, indicating the possibility that the regulatio … More n of ZAKI-4 mRNA by T3 is indirect, through an intermediate product induced by T3. ZAKI-4 mRNA was detected in heart, brain, liver, and skeletal muscle but not in placenta, lung, kidney and pancreas. In skin fibroblasts and skeletal muscle, 3.4-kb mRNA was the major species, whereas 1.4-kb mRNA was dominant in heart, brain, and liver. By using Rapid Amplification of cDNA Ends (RACE), the full-length cDNA was cloned. The sequence data confirmed the presence of two mRNA species which are possibly generated by an altermative polyadenylation and showed that these mRNAs code a single protein of 192 amino acids. Transcription coupled in vitro translation of the two mRNA species revealed both the two mRNA species code a protein with a molecular mass of 26 kDa, that roughly corresponds to the data of sequence analysis od ZAKI-4 cDNA.In fibroblasts from a patient with thyroid hormone resistance due to deletion of beta isoform of T3 receptor (TR) gene, T3 was able to induce ZAKI-4 mRNA like in normal fibroblasts. It is thus suggested that the other isoform, TR alpha can mediate the up-regulation of ZAKI-4 mRNA expression. Less