HA Hong-seok, HUH Jae-Won, KIM Dae-Soo1, JOO Myung-Jin2 and KIM Heui-Soo*

Division of Biological Sciences, College of Natural Sciences, Pusan National University. 

1PBBRC, Interdisciplinary Research Program of Bioinformatics, College of Natural Sciences, Pusan National University.  

2Department of Psychiatry, Hyung Ju Hospital.

ABSTRACTEndothelial 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 that derived from LTR10A belonging to HERV-I family on human NOS3 gene.  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 in placenta tissue only.  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 indicated good promoter activity of in human colon carcinoma cells (HCT-116).  These findings suggest that the LTR10A element acquired the role of placenta-specific regulation of NOS3 gene during primate evolution. .

INTRODUCTION

MATERIALS & METHODS

RESULTS & DISCUSSION

The genomic structure of NOS3 gene including LTR10A element. Exons were represented by solid box with the exon numbers. Arrows indicate the primer location. The LTR10A element was integrated into the NOS3 gene with the antisense orientation on human chromosome 7q36.1. .

REFERENCES

J. Boeke, J. Stoye, Retrotransposons, endogenous retroviruses, and the evolution of retroelements, In Retroviruses (1997) 343–435.

J. Jurka, Repbase update: a database and an electronic journal of repetitive elements, Trends Genet. 16 (2000) 418-420.

Luciferase reporter gene assay for LTR10A-derived promoter of NOS3 gene in transient transfected HCT116 and COS7 cells (A) and sequence analysis of LTR10A-derived promoter of NOS3 gene for the prediction of transcription factor binding sites (B). Relative activity of luciferase assay for pGL2-hNOS3-LTR10A in forward and reverse orientation or the pGL2 basic vector was indicated as schematic diagram. Results are expressed as ratios of the luciferase activity to that of the promoterless pGL2 reporter plasmid. The average and standard error of the mean are presented as error bars. Putative transcription binding sites, AREB6, MEF-2, NF-Y, AP-1, FOXO4, are indicated. The transcription start sites (GAAAC) are represented by an arrow. .

PCR analysis for the presence of LTR10A-derived promoter region of NOS3 gene using the various primate genomic DNAs. Hominoids and Old World monkeys showed PCR products that were cloned and sequenced .

Nucleotide sequence alignment of LTR10A-derived promoter region of NOS3 gene from human (NOS3-HU), chimpanzee (NOS3-CH), gorilla (NOS3-GO), orangutan (NOS3-OR), gibbon (NOS3-GI), Japanese monkey (NOS3-JM), crab-eating monkey (NOS3-CR), and rhesus monkey (NOS3-RM). The dot represents the same nucleotides as the human NOS3-HU sequences. TSS indicates transcription start sites of NOS3 gene. TSD (AATTT) represents target sites duplication of the boundary of LTR10A element of NOS3 gene. Specific mutations of Old world monkeys are highlighted by the solid box. Binding sites for the transcription factors are also indicated .

Luciferase reporter gene assay for LTR10A-derived promoter of NOS3 gene derived from the crab-eating monkey in transient transfected HCT116 and COS7 cells. Relative activity of luciferase assay for pGL2-mNOS3-LTR10A in forward and reverse orientation or the pGL2 basic vector was indicated as schematic diagram. Results are expressed as ratios of the luciferase activity to that of the promoterless pGL2 reporter plasmid. The average and standard error of the mean are presented as error bars. .

Other region

16%

Gene-related Sequence

36%

LINE20%

SINE13%

Coding sequence

3%

Pseudogene1%

HERV element

8%DNA element

3%

PPoly(A)

Human Alu

Retroelement

Retroposon

SINE

Retrotransposon

Retrovirus

LINE

RNA intermediate

- LTR element + LTR element

- env + env

- RT + RT

Yeast Ty1/copia/truncated HERVs

LTR ORF1 ORF2 LTR

LTR LTR

Human THE1

ORF1 ORF2PPoly(A)L1

gag pol envLTR LTR

Full-length HERVs/exogenous retrovirus

LTR: Long Terminal Repeat

LINE: Long Integrated nuclear element

SINE: Short Integrated nuclear element

HERV: Human Endogenous Retro-Virus

PCR, RT-PCR

Bisulfite Sequencing PCR

Luciferase assayBioinformatics

Placenta-specific NOS3 expression is mediated by a LTR10A element Placenta-specific NOS3 expression is mediated by a LTR10A element

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RT-PCR analysis of LTR10A derived transcript (A) and methylation analysis (B) from different human tissues. Methylation state of all cytosines in the CpG sequences was analyzed by the bisulfite-modified DNA sequencing method. Each nucleotide position is symbolized by a circle representing the results of seven clones analyzed. Black sectors indicate the percentage of methylated cytosine. .