| Project/Area Number |
15390458
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Orthopaedic surgery
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
TSUMAKI Noriyuki Osaka University, Graduate School of Medicine, Assistant, 医学系研究科, 助手 (50303938)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YOSHIKAWA Hideki Osaka University, Graduate School of Medicine, Professor, 医学系研究科, 教授 (60191558)
|
|---|
| Project Period (FY) |
2003 – 2005
|
| Project Status |
Completed (Fiscal Year 2005)
|
| Budget Amount *help |
¥12,400,000 (Direct Cost: ¥12,400,000)
Fiscal Year 2005: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2004: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2003: ¥5,700,000 (Direct Cost: ¥5,700,000)
|
|---|
| Keywords | collagen / Bone morphogenetic proteins / animal model / joint / gene / 軟骨 / トランスジェニックマウス / 骨 / 転写因子 |
|---|
| Research Abstract |
"Insulator" is the name given to a class of DNA sequence that can block an enhancer of one gene from activating a promoter on another nearby gene. The a2(XI) collagen chain gene (Col11a2) is specifically expressed in cartilage. We previously isolated the Col11a2 gene and identified its first intron sequence as a tissue-specific enhancer. In addition, we disclosed that the Col11a2 gene resided very closely to the 3'-end of retinoid-X-receptor gene (Rxrb) which is ubiquitously expressed. This structure of the Rxrb / Col11a2 locus may raise the possibility of the existence of insulators between these two genes. Because almost all of the characterized enhancer-blocking elements identified in vertebrates bind the transcription factor CTCF, we here first searched for sequences that bound CTCF by electrophoretic mobility-shift assays (EMSA). The rat 2.1 kb stretch from termination codon of the Rxrb gene to the translational start codon of the Col11a2 gene was cloned. Twelve overlapping DNA fr
… More
agments 300 bp) covering this stretch were prepared, incubated with in vitro translated CTCF protein. One fragment located upstream of the transcriptional start site of the Col11a2gene specifically interacted with CTCF. To confirm the binding of CTCF to this locus in vivo, chromatin immunoprecipitation assays were performed on rat chondrosarcoma cells (RCS) using anti-CTCF antibody. Co-precipitating sequences were detected quantitatively by real-time PCR using 9 sets of primers spaced across 14 kb of the Rxrb / Col11a2 locus. The CTCF-binding site detected by EMSA was greatly enriched in anti-CTCF immunoprecipitates. Because it is reported that insulator acts as a block to spreading of acetylation of histones from the enhancer to the gene, we next performed chromatin immunoprecipitation assays using anti-acetylated histone H3 antibody. In RCS cells, the level of acetylation was relatively higher around the first intron of the Col11a2 gene than those of the rest of the locus. In contrast to the high level acetylation of histon H3 around the first intron, that of the CTCF binding site was dramatically low. These results suggest that the CTCF binding site in the Col11a2 promoter is involved in the regulation of histone H3 acetylation around the Rxrb/ Col11a2 locus. Less
|
