| Project/Area Number |
16K14675
|
|---|
| Research Category |
Grant-in-Aid for Challenging Exploratory Research
|
| Allocation Type | Multi-year Fund |
|---|
| Research Field |
Molecular biology
|
|---|
| Research Institution | Tokyo Metropolitan Institute of Medical Science |
|---|
Principal Investigator |
MASAI Hisao 公益財団法人東京都医学総合研究所, ゲノム医科学研究分野, 所長 (40229349)
|
|---|
| Project Period (FY) |
2016-04-01 – 2019-03-31
|
| Project Status |
Completed (Fiscal Year 2018)
|
| Budget Amount *help |
¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
|
|---|
| Keywords | Rif1タンパク質 / クロマチンループ / 核内染色体構造 / 複製タイミング / グアニン４重鎖構造 / 核膜 / 染色体ループ構造 / ゲノム改変 / グアニン4重鎖 / Rif1 / 染色体核内配置 / DNA結合 / クロマチン動態 / 核機能 / グアニン４重鎖 |
|---|
| Outline of Final Research Achievements |
In order to manipulate chromatin loop formations, we used G-quadruplex binding protein, Rif1, which is known to regulate chromatin loops. We first identified L848S mutant, which cannot bind to chromatin. Overexpression of Rif1 resulted in inhibition of S phase progression and aberrant chromosome structures, as well as mitotic arrest with short spindles. The effects can be observed with Rif1 incapable of binding to phosphatase, but not with L848S deficient in chromatin binding. Thus, chromatin binding of Rif1 is responsible for these effects. By using the L848S mutant, we tried to tether Rif1 at a specific location on the genome. We used Gal4DBD-Gal4BS, and replaced a cellular Rif1BS with Gal4BS, and expressed Rif1L848S-Gal4BD at the endogenous locus. However, significant effects on the replication timing near the binding site were not observed, presumably due to low expression of the Gal4DBD fusion protein
|
| Academic Significance and Societal Importance of the Research Achievements |
Rif1はTAD(Topologically Associated Domain)等に関連したクロマチン高次構造を、G4結合を介して形成する。このクロマチンドメインは、複製タイミングや転写等の制御ユニットとなる。ゲノム操作が容易な分裂酵母を用いて、今回、それ自身ではクロマチンに結合しない変異Rif1を同定した。これを用いて、別のDNA結合ドメインを介して、任意の場所にRif1を局在化し、近傍のクロマチン高次構造を変化させうる可能性が生まれた。この方法は、特定の遺伝子のみを標的とするのではなく、クロマチンドメイン全体を制御可能であり、従来のゲノム操作技術とは異なる、新しい細胞改変技術をもたらす。
|
