2004 Fiscal Year Final Research Report Summary
Development of Retrovirus Vectors Permissive in Tissue Stem Cells
| Project/Area Number |
15609001
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
幹細胞生物学
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
MINOGUCHI Shigeru The University of Tokyo, Institute of Medical Science, Research associate, 医科学研究所, 助手 (60322757)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
IBA Hideo The University of Tokyo, Institute of Medical Science, Professor, 医科学研究所, 教授 (60111449)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Keywords | stem cells / ES cells / gene therapy / retrovirus vector / DNA methylation / MSCV / Brm / YY-1 |
|---|
| Research Abstract |
In order to develop a new retrovirus vector, which is able to express in various tissue stem cells for a long period of time, we attempted to reveal molecular mechanisms of murie leukemia virus (MLV) vector repression in mouse embryonic stem (ES) cells. Recently we found that MLV vector expression is significantly repressed in various human cell lines, which do not express the Brm protein, an ATPase catalytic subunit of the SWI/SNF chromatin-remodeling complex. Since usage of a modified vector without the YY-1 binding sequence of LTR or forced expression of exogenous Brm has been shown to suppress MLV silencing in the Brm-deficient cell lines, we first examined whether Brm would be also required for MLV and MSCV vector expression in ES cells. Although several tested ES lines showed undetectable Brm expression, disruption of the YY-1 binding element on LTR did not affect expression of MLV and MSCV vectors in ES cells. This result suggests that the Polycomb-G or trithorax-G complexes does not participate in the retrovirus silencing in ES cells. MSCV expression is permissive in ES cells but repressed gradually for a long period of culture. We next attempted to clarify the mechanism of this long-term silencing of MSCV in ES cells. In single cell-resolution expression analyses with a cell sorter, we found that the long-term silencing takes place in an all-or-none manner. Furthermore, we found that disappearance of MSCV expression is closely related to its provirus DNA methylation. Chromatin immunoprecipitation analyses showed that the covalent modifications such as acethylation and methylation of histone H3 and H4 lysine and arginine residues on provirus DNA appear not to be involved in extinction. A super-infection study suggested that promoter-specific repression mechanistically underlie the long-term silencing. Taken together, these findings indicate that provirus DNA methylation plays a pivotal role in the long-term silencing of MSCV in ES cells.
|
Research Products
(7 results)
