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Tissue-specific NOS3 expression is mediated by a LTR10A element belonging to HERV-I family Hong-Seok Ha1, Jae-Won Huh1, Dae-Soo Kim2, Kung Ahn1, Yun-Ji Kim1Ja-Rang Lee1, Dong-Woo Kang1, Do-Sik Min1, Myung-Jin Joo3, and Heui-Soo Kim1,2 1 Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Korea 2 PBBRC, Interdisciplinary Research Program of Bioinformatics, Pusan National University, Busan 609-735, Korea 3 Department of Psychiatry, Hyung Ju Hospital, Yangsan 626-851, Korea -2704 -2761 -2825 -3143 -2859 -3158 -3148 -2909 -3104 -3170 Brain 3´ 5´ Placenta 5´ 3´ LTR region MATERIALS & METHODS ABSTRACT Bioinformatics Genomic DNA PCR & Gene cloning Luciferase assay Transfac 6.0 RT-PCR Bisulfite Sequencing PCR Endothelial nitric oxide synthase (NOS3) is a key enzyme in the regulation of vascular wall homeostasis and regulation of vasomotor tone, which has been identified to consist of 26 exons spanning 21 kb of genomic DNA and encoding an mRNA of 4052 nucleotides which is translated into a 1203 amino acids. Here we found new transcript variant derived from LTR10A belonging to HERV-I family on human NOS3 gene, which has promoter activity. The LTR10A element located on the upstream of the original promoter elements (Sp1 and GATA motifs) of NOS3 gene seems to be inserted into primate genome approximately 33 Myr ago. The LTR10A-derived promoter transcripts by RT-PCR amplification are detected only in human placenta tissue. Methylation study using the sodium bisulfied DNA sequencing indicated that LTR10A element of placenta tissue showed hypomethylated pattern. Reporter gene assay of LTR10A element on NOS3 gene from humans (pGL2-hNOS3-LTR10A) and crab-eating monkeys (pGL2-mNOS3-LTR10A) indicated good promoter activity in HCT116 cells and HCT116/COS7 cells, respectively. Transversion or transition mutations on C/EBP, AP-2, 1st-NF-Y, Sox-5, and E12 binding sites in comparison with LTR10A sequences between humans and monkeys have been occurred, indicating that they allowed the activity differences of luciferase reporter gene assay in HCT116 and COS7 cells. The reverse-oriented promoter in humans and monkeys showed higher activity than the forward one. Taken together, our findings suggest that the LTR10A element acquired the role of tissue-specific regulation of NOS3 gene during primate evolution. . PNU PNU PNU PNU PNU RESULTS & DISCUSSION Skeletal muscle (A) (B) (C) Brain Prostate Testis Heart Kidney Liver Lung Spleen Thymus Uterus Placenta Marker S2 (A) (B) S1 2 27 1 MS MAS 442bp LTR10A Current study 195bp -3216 -2522 AS2 AS1 New promoter region: 868bp -3413/-2545 INTRODUCTION 495bp +23: same ATG sites for amino acid 195bp -2723 -104/-95 -4843/-4684 1 27 -144/-115 LTR10A Previous study -2606 -3107 Promoter region Enhancer region Karantzoulis-Fegaras et al., 1999 Laumonnier et al., 2000 Retroelements have been subjected to many amplification and transposition events resulting in a widespread distribution of complete or partial retroviral sequences throughout the human genome. The human genome comprises approximately 8% of the human endogenous retroviruses (HERVs) and other long terminal repeat (LTR)–like elements. Most HERVs seem to have entered the genome between 10 and 50 million years ago, and they comprise over 200 distinct groups and subgroups. Expression of retroelements can influence the outcome of infections in different ways that can be either beneficial or detrimental to the host. A function of the multiple copy families, scattered throughout the genome, has been reported regulatory functions on the gene expression of nearby located genes. A small minority of such sequences has acquired a role in regulating gene expression, and some of these may be related to differences between individuals, and to expression of disease. Nitric oxide accounts for the biologic activity of endothelium-derived relaxing factor, which is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). In the human central nervous system, three NOS isoforms have been identified. NOS1 has been detected in different types of neurons of the cerebellum, hypothalamus, striatum, cerebral cortex and hippocampus, while NOS2 has been identified in activated macrophages, astroglia, and microglia. In case of NOS3, it has been originally detected in microvessels, but is also found in neurons and glial cells. The NOS3 gene contains 26 exons distributed over 21 kb of human genomic DNA and encodes an mRNA of 4052 nucleotides which is translated into a 1203 amino acids. Characterization of 5´-flanking genomic region indicated that NOS3 gene showed TATA-less promoter elements (Sp1 and GATA motifs) and was constitutively expressed in endothelial cells, especially the endotherial layer of medium to large sized arterial blood vessels (Marsden et al., 1993). Here we found a LTR10A element of the HERV-I family on human NOS3 gene, which showed promoter activity and placenta-specific expression. . (A) (B) 10 TSD 8 6 Relative Luciferase Activity (Fold of pGL-2 control) C/EBP AREB6 AP-2 NF-Y 4 Crab-eating monkey Common marmoset Squirrrel monkey Japanese monkey Ring-tailed lemur Rhesus monkey Night monkey Chimpanzee Orangutan 2 Human Marker Gibbon Gorilla Sox-5 MEF-2 NF-Y AREB6 AP-1 E12 AP-1 SINE Other region 13% 0 16% 869 bp Vector only Forward Reverse Vector only Forward Reverse TSS AREB6 FOXO4 LINE 20% HCT116 COS7 TSD Gene-related Sequence HERV element 36% 8% DNA element Pseudogene Coding sequence 1% 3% 3% (B) (A) pGL2-mNOS3-LTR10A COS7 160 Genome Information Lab HTTP://WWW.PRIMATE.OR.KR TTTTT 16 120 REFERENCES 12 1 2 3 4 5 6 7 8 Relative Luciferase Activity (Fold of pGL-2 control) 1. HU - 80 2. CH 98.3 - 8 3. GO 97.5 97.9 - 4. OR 95.2 95.6 95.3 - 40 4 5. GI 95.2 95.7 95.6 96.1 - 6. CR 89.4 89.9 90.3 90.9 90.7 - 7. JM 89.5 90.1 90.2 91.1 90.8 99.6 - 0 0 8. RM 89.4 89.9 90.1 90.7 90.7 99.2 99.3 - 1. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar, K, Doyle M, Fitz Hugh W et al: Initial sequencing and analysis of the human genome. Nature 2001, 409:860–921. 2. Jurka J: Repbase update: a database and an electronic journal of repetitive elements. Trends Genet 2000, 16:418–420. 3. Akopov SB, Nikolaev LG, Khil PP, Lebedev YB, Sverdlov ED: Long terminal repeats of human endogenous retrovirus K family (HERV-K) specifically bind host cell nuclear proteins. FEBS Lett 1998, 421:229-233. 4. Sverdlov ED: Perpetually mobile footprints of ancient infections in human genome. FEBS Lett 1998, 428:1-6. 5. Forstermann U, Kleinert H: Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn Schmiedebergs Arch Pharmacol 1995, 352:351-364. Vector only Vector only Forward Reverse Vector only Forward Reverse Vector only Forward Forward Reverse Reverse HCT116 COS7 pGL2-hNOS3-LTR10A pGL2-mNOS3-LTR10A