EXPERIMENTAL STUDY Genomic organization of mouse ZAKI-4 gene that encodes ZAKI-4 a and b isoforms, endogenous calcineurin inhibitors, and changes in the expression of these isoforms by thyroid hormone in adult mouse brain and heart Yutaka Mizuno 1 , Yasuhiko Kanou, Margarita Rogatcheva, Tsuneo Imai 1 , Samuel Refetoff 2 , Hisao Seo and Yoshiharu Murata Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, 1 Department of Endocrinology and Transplantation, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan and 2 Department of Medicine and Pediatrics, J P Kennedy Jr Mental Retardation Center and the Committee on Genetics, University of Chicago, Chicago, Illinois 60637, USA (Correspondence should be addressed to Y Murata, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; Email: ymurata@riem.nagoya-u.ac.jp) Abstract Objective: ZAKI-4 was identified as a thyroid hormone-responsive gene in cultured human fibroblasts. A single ZAKI-4 gene encodes two isoforms, ZAKI-4 a and b, both inhibiting calcineurin activity. ZAKI-4 a and b differ at their N termini, and show distinct distribution profiles in human tissues. The aim of this study was to elucidate the organization of the mouse ZAKI-4 gene and to determine the effect of thyroid hormone on the expression of ZAKI-4 isoforms in vivo. Design: We cloned mouse homologues of human ZAKI-4 a and b cDNA. Fluorescence in situ hybrid- ization and bioinformatics analysis were employed to determine the gene organization. The effect of thyroid hormone on the expression of ZAKI-4 isoforms in mouse brain and heart was also studied. Methods: Total RNA extracted from mouse cerebellum was used to clone ZAKI-4 a and b cDNAs by RT-PCR followed by rapid amplification of cDNA ends. Mice were rendered hypothyroid by feeding a low iodine diet supplemented with propylthiouracil for 2 weeks. In one group (hyperthyroid) L-T 3 was injected i.p. for the last 4 days whereas another group (hypothyroid) received vehicle only. Non- treated mice were controls. Results and conclusion: Mouse ZAKI-4 a and b cDNAs were highly homologous to the human isoforms. The gene was mapped on chromosome 17qC, syntenic to human chromosome 6 where the human ZAKI-4 gene is located. As observed in human, ZAKI-4 a mRNA was expressed only in brain whereas b mRNA was distributed in other tissues as well, such as heart and skeletal muscle. ZAKI-4 a mRNA was lower in the cerebral cortex of hypothyroid mice. Injection of L-T 3 caused an increase in ZAKI-4 b mRNA in heart; however, expression of neither ZAKI-4 a nor b mRNA was influenced by thyroid status in other tissues. These results indicate that expression of ZAKI-4 a and b isoforms is regulated by thyroid hormone in vivo, and the regulation is isoform- and tissue-specific. European Journal of Endocrinology 150 371–380 Introduction ZAKI-4 (DSCR1L1, according to the Human Gene Nomenclature Committee) was identified as a thyroid hormone-responsive gene in cultured human skin fibroblasts (1). In fibroblasts the expression of ZAKI-4 mRNA was enhanced transcriptionally by physiological concentrations of 3,3 0 ,5-triiodothyronine (T 3 ), the active form of thyroid hormone. This effect was indirect, requiring de novo protein synthesis. A recent study (2) demonstrated that a product of ZAKI-4 belongs to a family of proteins that inhibit calcineurin activity. This family includes DSCR1 (Down syndrome candidate region 1) (3) (also termed as MCIP1 (4) or Adapt 78 (5)) and DSCR1L2 (DSCR1 like protein 2) (6) in mam- mals, Rcnp1 in Saccharomyces cerevisiae (2) and Caenorhabditis elegans (7), and CBP1 (8) in Cryptococcus neoformans. Calcineurin is a calcium/calmodulin-dependent seri- ne/threonine protein phosphatase that exerts a variety of functions by dephosphorylating a family of transcrip- tion factors such as nuclear factor of activated T cells (NF-AT) (9) and myocyte-specific enhancer factor 2 (10). In brain, for example, calcineurin is involved in European Journal of Endocrinology (2004) 150 371–380 ISSN 0804-4643 q 2004 Society of the European Journal of Endocrinology Online version via http://www.eje.org Downloaded from Bioscientifica.com at 01/11/2022 03:14:22PM via free access