Structure and promoter activity of the human glia maturation factor-gamma gene: a TATA-less, GC-rich and bidirectional promoter Yoko Kawai a,b, * , Kiyofumi Asai b , Yutaka Miura b , Yuichiro Inoue c , Manami Yamamoto b , Akihiko Moriyama d , Naoki Yamamoto b , Taiji Kato b a Nagoya City University School of Nursing, Mizuho-ku, Nagoya 467-8601, Japan b Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan c Department of Psychiatry, Nara Medical University, Kashihara, Nara 634-8521, Japan d Division of Biomolecular Science, Graduate School of Natural Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8501, Japan Received 3 May 2002; received in revised form 28 November 2002; accepted 17 December 2002 Abstract Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5V -flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5V RACE (rapid amplification of cDNA 5Vends) identified multiple transcription initiation sites within the region À 84 to À 70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Human glia maturation factor-gamma; Primer extension; 5V RACE; Promoter regulation; Bidirectional promoter 1. Introduction The predicted amino acid sequence of human glia matu- ration factor-gamma (hGMFG) is 82% identical to that of glia maturation factor-beta (GMFB) and it has the three cysteine residues thought to be crucial for GMFB biological activity [1]. Independently, a cDNA corresponding to GMFG was identified as a GMFB homolog, from a collec- tion of cDNAs synthesized from hematopoietic stem/pro- genitor cells [2]. The gene has been mapped to 19q13.2, which is frequently deleted in human malignant gliomas [3]. Although GMFB is expressed in the nervous system [4,5], hGMFG is predominantly expressed in human lung, heart and placenta [1]; in rat, GMFG is expressed in thymus, testis and spleen [6]. Both GMFB and GMFG mRNAs were detected in retina as early as embryonic day 18 and they persisted until adulthood. GMFB is synthesized and is mainly localized in Mu ¨ller cells in rat retina, suggesting that it contributes to glial cell development [7]. hGMFB can be phosphorylated by a variety of protein kinases and it plays a role in intracellular signal transduction. Recombi- nant GMFB, a 17-kDa brain protein, inhibits the activity of mitogen-activated protein (MAP) kinase in a test tube assay [8]. It is also a strong enhancer of p38 MAP kinase activity in vitro [9]. In spite of their similar structures and speculated func- tions, the patterns of GMFG and GMFB gene expression are different. The expression of GMFG is not limited to the nervous system and it is found in various organs. In this study, we characterized the promoter region of the hGMFG gene and found the putative cis-acting regulatory elements responsible for promoter activity. 0167-4781/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-4781(02)00627-9 Abbreviations: GMFG, glia maturation factor-gamma; GMFB, glia maturation factor-beta; 5V RACE, rapid amplification of cDNA 5Vends * Corresponding author. Nagoya City University School of Nursing, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81-52-853-8049; fax: +81-52- 853-8049. E-mail address: yokawai@med.nagoya-cu.ac.jp (Y. Kawai). www.bba-direct.com Biochimica et Biophysica Acta 1625 (2003) 246 – 252