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Tissue-specific NOS3 expression is mediated by a LTR10A element belonging to HERV-I family Hong-Seok 1 Ha , Jae-Won 1 Huh , Dae-Soo 2 Kim , 1 Kung Ahn , Yun-Ji 1 Kim Ja-Rang 1 Lee , Dong-Woo 1 Kang , Do-Sik 1 Min , Myung-Jin 3 Joo , and Heui-Soo 1,2 Kim 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 MATERIALS & METHODS Bioinformatics 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-mNOS3LTR10A) 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. . Luciferase assay Transfac 6.0 RT-PCR Genomic DNA PCR & Gene cloning Bisulfite Sequencing PCR TTTTT RESULTS & DISCUSSION 2 1 Uterus Thymus Spleen Placenta Lung Liver Kidney Heart Testis Skeletal muscle S1 Brain S2 Prostate (A) Marker ABSTRACT (A) 27 442bp Current study MS MAS -3216 -2522 LTR10A 195bp (B) AS1 New promoter region: 868bp INTRODUCTION AS2 495bp -3413/-2545 +23: same ATG sites for amino acid -2723 -104/-95 -4843/-4684 1 -144/-115 195bp (C) (B) 27 0.0160 Previous study LTR10A 0.0120 0.0100 -3107 -2606 Enhancer region Laumonnier et al., 2000 -3158 -3170 0.0140 -3148 -3143 5´ 3´ 0.0060 0.0040 Placenta 5´ 3´ 0.0020 LINE 20% Gene-related Sequence 36% HERV element 8% DNA element Pseudogene Coding sequence 1% 3% 3% C VE HU sp le en ut er us liv er sk -m us cle th ym us ne y kid he ar t te st is pr os ta te br ai n lu ng pl ac en ta LTR region (B) TSD 0 Ring-tailed lemur Common marmoset Night monkey Squirrrel monkey Rhesus monkey Japanese monkey Gibbon 2 Gorilla 4 Orangutan 6 Crab-eating monkey 8 Chimpanzee (Fold of pGL-2 control) Relative Luciferase Activity 10 Human Other region 16% (A) C/EBP Sox-5 MEF-2 AREB6 AP-2 NF-Y NF-Y AREB6 AP-1 E12 AP-1 869 bp AREB6 HCT116 FOXO4 TSS COS7 TSD (B) (A) pGL2-mNOS3-LTR10A COS7 16 (Fold of pGL-2 control) Relative Luciferase Activity 160 REFERENCES 120 1 12 2 3 4 5 6 7 8 1. HU - 2. CH 98.3 - 3. GO 97.5 97.9 - 4. OR 95.2 95.6 95.3 - 5. GI 95.2 95.7 95.6 96.1 - 6. CR 89.4 89.9 90.3 90.9 90.7 - 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. 7. JM 89.5 90.1 90.2 91.1 90.8 99.6 - 2. Jurka J: Repbase update: a database and an electronic journal of repetitive elements. Trends Genet 2000, 16:418–420. 8. RM 89.4 89.9 90.1 90.7 90.7 99.2 99.3 80 8 40 4 0 0 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. HCT116 COS7 pGL2-hNOS3-LTR10A -2704 0.0080 Promoter region Karantzoulis-Fegaras et al., 1999 Marker SINE 13% -2761 Brain 0.0000 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. . -2825 -2859 -2909 -3104 pGL2-mNOS3-LTR10A Genome Information Lab HTTP://WWW.PRIMATE.OR.KR -