1. Abstract Results & Discussion Introduction Materials & Methods
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,*
1Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan , Korea
2 PBBRC, Interdisciplinary Research Program of Bioinformatics, Pusan National University, Busan , Korea
Abstract
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..
Results & Discussion
PNU
Dorfin gene structure
Evolutionary Conservation of the Dorfin Gene
100 77 42
Human
Dog
Pig
Cattle
Mouse
Rat
Zebrafish
Chicken 8
Dorfin
(RNF19) S1
8q22.2
MaLR 1 2 3 4 5 6 7 9 10 1a 1b
AS2 S2
AluJ
AS1
RING-finger/IBR
ORF S3
AS3
Introduction
- cloned from the anterior horn tissues
category
research
genome
transcriptome
proteome
Not searched in genome level
cDNA sequence decision
Expression pattern identification by nothern blot
Transcripts expression research in ALS
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)
Parkinson’s disease
Amyotrophic lateral sclerosis
NM_  
Other region
16%
Gene-related
Sequence
36%
LINE
20%
SINE
13%
Coding sequence 3%
Pseudogene 1%
HERV element 8%
DNA element
Expression of Dorfin gene by cellular promoter
Marker
Adrenal gland
Adult brain
Fetal brain
Fetal liver
Heart
Kidney
Liver
Lung
Bone marrow
Cerebellum
Skeletal muscle
Placenta
Spinal cord
Testis
Thymus
Thyroid
Trachea
Uterus
Prostate
Dorfin
Gapdh
494 bp
195 bp
Breast (N)
Breast (C)
Marker
AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC R L E G N D V I A S
GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC
CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG
CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG
TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT
TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT
TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA
GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA
CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG
CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA
ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC
AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA
Primer S2
Primer AS2 M Q F K Y C T P H
AluJ element
556 bp
430 bp
315 bp
(A)
(B)
(C) 3 11 1 2
MaLR
AluJ 10
126 bp
115 bp 1) 2) 3)
MaLR-derived promoter
transcript (556 bp)
transcript (430 bp)
Relative Expression
Bone marrow
Cerebellum
Kidney
Lung
Testis
Expression of Dorfin gene by MaLR-derived promoter
Materials & Methods
Luciferase assay
Gene cloning
RT-PCR
Primer Design
Twenty Different
Human tissue cDNAs
Quantitative
Analysis
Real-time PCR
Luciferase
assay
Modified PGL2-vector, HCT116, Cos7,
Lipofectamine, Dual luciferase Assay
Gene
cloning
Qiaquick Gel-extraction, Promega T-easy Vector,
EcoR-I(DH5α)
35cycle
94 ℃ : 40sec 56 ℃ : 30sec 72 ℃ : 40sec.
Real-time
PCR
40cycle (Sybr green)
94 ℃ : 10sec 56 ℃ : 15sec 72 ℃ : 15sec
In silico analysis of transcription binding sites of MaLR element
Promoter Activity of the MaLR Element
CCCCACAAAA GATATGTTCA TGTCCTAATC CCCAGAATCT GCAAATGTTA TTTGGAAAAA GGGGTTTTGC AGATGTAATT AAGTTAAGAA TCTTGAGATA AGATCATCCT GGATTATCCA GGTAGCCTCA AAATCAAGTG ACAAGTGTCT TTGTAAGGGA CAAGTAGACC CATTACAGAG AAGACGACGC GCAGAAAAGG AGGAAGCAGT GTGCTCATGG AGGCGGAGAT TGGAGTGATG TAACCGCAAG CCGAGGAATG CTTATAGTCA CCAGAAGCTG GAAGAGTCAA AGGACACATT CTCCCCTCAA GCCCCAGTGG GAGCACGGCC CAGCTGGATT TTGGACTTCT GGCCTCCAGA ACTGTAAGAG AAATGTCCAT TGTCTTAAGC CAACCAGTTT GTGGTAGTTT GTTACAGCAG CCCCAGGAAA CTACTA
GATA-1
Reverse
Forward
Control
Relative Luciferase Activity
(Fold of pGL-2 control)
Cos7
HCT116
GATA-2
Nkx2-5
Nkx2-5
Whn
ELF-1
References
+1 TRANSCRIPTION START SITE
Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells. Arch. Virol. [Epub ahead of print]
1. Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells. Arch. Virol. [Epub ahead of print]
Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G 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):
2. Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G 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):