Transcript 슬라이드 1

Transcriptional regulation of mammalian LTR-retrotransposon element on the human Dorfin gene
Ja-Rang Lee1, Jae-Won Huh1, Dae-Soo Kim2, Kung Ahn1, Hong-Seok Ha1, Yun-Ji Kim1, Won-Ho Lee1, and Heui-Soo Kim1,2,*
2
1Division
of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Korea
PBBRC, Interdisciplinary Research Program of Bioinformatics, Pusan National University, Busan 609-735, Korea
Abstract
Results & Discussion
Dorfin containing RING-finger and IBR motifs is an E3 ubiquitin ligase that is localized in Lewy bodies, a
characteristic neuronal inclusion in Parkinson’s disease brains. The Dorfin gene located on human
chromosome 8q22.2 has showed 4.4 kb transcript and expressed ubiquitously in various tissues
including brain. Here we found its alternatively spliced transcript variants which derived from MaLR
(mammalian LTR-retrotransposon) element using bioinformatic tools. The MaLR-derived promoter
transcripts are detected as two different types in all tissues examined, while breast tissue only showed
three variant types. Reporter gene assay of the promoter activity of MaLR element on Dorfin gene
indicated good activity in human colon carcinoma cells (HCT-116). These findings suggest that the
MaLR element acquired the role of transcriptional regulation of Dorfin gene during primate evolution..
8
Human
Human
100
100
Dog
Dog
77
Pig
Pig
42
Cattle
Cattle
77
100
100
42
Mouse
Mouse
100
RING-finger/IBR
Rat
Rat
100
100
1a
1
Chicken
Chicken
Dorfin
(RNF19)
Zebrafish
Zebrafish
8q22.2
1b
MaLR
2
4
5
S1
AS1
S3
6
7
8
9
10
AluJ
S2
Introduction
3
ORF
AS2
AS3
0.05
category
research
- cloned from the anterior horn tissues
Parkinson’s disease
Amyotrophic lateral sclerosis
genome
Not searched in genome level
transcriptome
cDNA sequence decision
Expression pattern identification by nothern blot
Transcripts expression research in ALS
proteome
Identification of E3 activity
Location decision of Dorfin in cell
Phylogeny research in RBRfamily
Homology research related with parkin
Research about activity mechanism (VCP releated)
Dorfin
494 bp
1
Other region
SINE
13%
2
3
4
5
6
7
8
9 10
11
12 13
14
15
16
17
18 19
16%
NM_0154
35.3
LINE
Gapdh
195 bp
20%
HERV element
Gene-related
Sequence
DNA element Pseudogene
8%
36%
Marker
Coding sequence
Primer S2
(B)
3%
A G A A G C T G G A A G AG T C A A A G G A CA C A T T C T C C C C T C A A G C C C C A G T G G G A
A G A A G C T G G A A G AG T C A A A G G A CA C A T T C T C C C C T C A A G C C C C A G T G G G A
Breast (C)
1%
(A)
Breast (N)
3%
G C A C G G C C C A G C T G G A T T T T G G A C T T C T G G C C T C C A G A A C T A G A C A G G G C
G C A C G G C C C A G C T G G A T T T T G G A C T T C T G G C C T C C A G A A C T A G A C A G G G C
C T C A C T G T G T C A C C C A G G G T G G A A T A C A G T G G T G T G A T C A T A G C T C A C T G
C T C A C T G T G T C A C C C A G G G T G G A A T A C A G T G G T G T G A T C A T A G C T C A C T G
0.04
AluJ element
C A G C C T G G A A T T C C T G G G C T C A A G C A A C C C T G C C A C C T C A G C C T T C C A A G
C A G C C T G G A A T T C C T G G G C T C A A G C A A C C C T G C C A C C T C A G C C T T C C A A G
Materials & Methods
T T T C A A G A A T T C T C T G A A G G A A C C A A G T A G G A T A T T C T T A C A T C A T G A C T
T T T C A A G A A T T C T C T G A A G G A A C C A A G T A G G A T A T T C T T A C A T C A T G A C T
M
RT-PCR
Luciferase assay
Luciferase
assay
1
MaLR
2
MaLR
11
AluJ
2
10
I
F
K
S
Y
N
E
L
MaLR
V
C
T
N
P
D
V
I
S
L
S
T
I
D
L
M
S
M
S
L
H
R
H
R
Q
M
G
M
G
S
D
R
D
R
D
L
D
L
Q
S
S
C A T G A G T T T A C A T C G G C A A A T G G G T T C A G A T C G A G A T C T T C A G T C C T C T G
C A T G A G T T T A C A T C G G C A A A T G G G T T C A G A T C G A G A T C T T C A G T C C T C T G
S
L
S
V
S
Q
L
P
S
S
V
K
K
A
P
Q
K
S
K
2
11
3
AluJ
S
S
V
S
L
P
S
V
K
K
A
P
K
K
0.03
Testis
Cerebellum
0.02
Kidney
Bone marrow
0.01
S
R
R
C T T C A T C T G T G A G C T T G C C T T C A G T C A A A A A G G C A C C C A A A A A A A G A A G A
C T T C A T C T G T G A G C T T G C C T T C A G T C A A A A A G G C A C C C A A A A A A A G A A G A
R
R
R
I
S
I
G
S
L
F
R
K
K
D
N
K
R
K
S
R
I
S
I
G
S
L
F
R
K
K
D
N
K
R
K
S
0
1
A T T T C A A T A G G C T C C C T G T T T C G G A G G A A A A A A G A T A A C A A A C G T A A A T C
A T T T C A A T A G G C T C C C T G T T T C G G A G G A A A A A A G A T A A C A A A C G T A A A T C
R
E
L
N
G
G
V
D
G
I
A
S
I
E
S
I
R
E
L
N
G
G
V
D
G
I
A
S
I
E
S
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
A A G G G A G C T A A A T G G C G G G G T G G A T G G A A T T G C A A G T A T T G A A A G T A T A C
A A G G G A G C T A A A T G G C G G G G T G G A T G G A A T T G C A A G T A T T G A A A G T A T A C
Qiaquick Gel-extraction, Promega T-easy Vector,
EcoR-I(DH5α)
G
A
3)
G
I
E
Q
G G G C T G T G T G T A A A C A C T G A C C C T G T C T C A A T T C T A A C A A G C A T T T T A G A
G G G C T G T G T G T A A A C A C T G A C C C T G T C T C A A T T C T A A C A A G C A T T T T A G A
A
AluJ
1
Gene
cloning
3
1
2)
E
Q
T A A T G T G A A T G C A A G A A C A A G A A A T A G G T T T T A T C T C T A A A T A T A A T G A A
T A A T G T G A A T G C A A G A A C A A G A A A T A G G T T T T A T C T C T A A A T A T A A T G A A
(C)
1)
Modified PGL2-vector, HCT116, Cos7,
Lipofectamine, Dual luciferase Assay
T A G C T A G G A C T A C A G A A C A T C C A T G A T A G C A G T C T T C T G T A A A T C G A A C T
T A G C T A G G A C T A C A G A A C A T C C A T G A T A G C A G T C T T C T G T A A A T C G A A C T
1
2
3
556 bp
430 bp
315 bp
Relative Expression
Lung
126 bp
115 bp
MaLR-derived promoter
transcript (430 bp)
MaLR-derived promoter
transcript (556 bp)
Primer AS2
Real-time PCR
Gene cloning
Primer
Design
RT-PCR
In silico analysis of transcription binding sites of MaLR element
35cycle
94 ℃ : 40sec 56 ℃ : 30sec 72 ℃ : 40sec.
GATA-1
CCCCACAAAA
GATATGTTCA
TGTCCTAATC
GCAAATGTTA
TTTGGAAAAA
AGATCATCCT
GGATTATCCA
ACAAGTGTCT TTGTAAGGGA CAAGTAGACC
CATTACAGAG
GATA-2
Reverse
Twenty
Different
Human tissue
cDNAs
Quantitative
Analysis
Real-time
PCR
40cycle (Sybr green)
CCCAGAATCT
GGGGTTTTGC
AGATGTAATT
AAGTTAAGAA
TCTTGAGATA
Cos7
94 ℃ : 10sec 56 ℃ : 15sec 72 ℃ : 15sec
Nkx2-5
Forward
GGTAGCCTCA AAATCAAGTG
Whn
Nkx2-5
ELF-1
AAGACGACGC GCAGAAAAGG AGGAAGCAGT GTGCTCATGG AGGCGGAGAT TGGAGTGATG
Reverse
HCT116
+1 TRANSCRIPTION START SITE
References
1. Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS. 2006. Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells.
Arch. Virol. [Epub ahead of print]
2. Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G. 2003. Dorfin localizes to the ubiquitylated inclusions in Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,
and amyotrophic lateral sclerosis. Am J Pathol. 163(2):609-619.
TAACCGCAAG
Forward
CCGAGGAATG
CTCCCCTCAA GCCCCAGTGG
Control
CTTATAGTCA CCAGAAGCTG
GAGCACGGCC
CAGCTGGATT
GAAGAGTCAA AGGACACATT
TTGGACTTCT
GGCCTCCAGA
Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS. 2006. Transcriptional control of the HERV-H LTR element of the GSDML gene in
ACTGTAAGAG AAATGTCCAT TGTCTTAAGC CAACCAGTTT GTGGTAGTTT GTTACAGCAG
0
2
4
6
8
10 12 14 16 18
20 22
human tissues and cancer cells. Arch. Virol. [Epub ahead of print]
Relative Luciferase Activity
(Fold of pGL-2 control)
CCCCAGGAAA
CTACTA
.
Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G. 2003. Dorfin localizes to the ubiquitylated inclusions in Parkinson's disease, dementia with Lewy bodies,
multiple system atrophy, and amyotrophic lateral sclerosis. Am J Pathol. 163(2):609-619.