Transcript 슬라이드 1

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
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