Transcript 슬라이드 1

Placenta-specific NOS3 expression is mediated by a LTR10A element
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.
http://www.primate.or.kr
2Department
ABSTRACT
RESULTS & DISCUSSION
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 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
SINE
13%
Other region
16%
Retroelement
LINE
20%
- LTR element
+ LTR element
Retroposon
- env
Gene-related Sequence
36%
+ RT
LTR
SINE
LTR
P
Te r min al
LTR
LTR
Retrovirus
Rep e at
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 bisulfitemodified 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.
.
LINE
LINE: Long Integrated nuclear element
SINE: Short
ORF2
Human THE1
Poly(A)
Human Alu
Long
ORF1
Yeast Ty1/copia/truncated HERVs
3%
LT R :
+ env
Retrotransposon
- RT
DNA element
3%
Pseudogene
Coding sequence
1%
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
N O S 3 g e n e w i t h the a n tis e n s e
orientation on human chromosome
7q36.1.
.
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
b a s i c v e c to r wa s i n d i c a t e d a s
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.
.
RNA intermediate
HERV element
8%
of Psychiatry, Hyung Ju Hospital.
LTR
gag
pol
env
LTR
Integrated nuclear element
H E RV: H u m a n E n d o g e n o u s R e t r o - Vi r u s
P
ORF1
ORF2
L1
Poly(A)
Full-length HERVs/exogenous retrovirus
MATERIALS & METHODS
Bioinformatics
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
.
Luciferase assay
PCR, RT-PCR
Bisulfite Sequencing PCR
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.
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 pGL2mNOS3-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.
.