BRG1によるクロマチン構造変化を介した細胞初期化促進メカニズムの解明
| Project/Area Number |
11F01388
|
|---|
| Research Category |
Grant-in-Aid for JSPS Fellows
|
| Allocation Type | Single-year Grants |
|---|
| Section | 外国 |
|---|
| Research Field |
Cell biology
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
山中 伸弥 京都大学, iPS細胞研究所, 教授
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KIM Shin-ii 京都大学, ips細胞研究所, 外国人特別研究員
KIM Shin-Il 京都大学, iPS細胞研究所, 外国人特別研究員
|
|---|
| Project Period (FY) |
2011 – 2013
|
| Project Status |
Completed (Fiscal Year 2013)
|
| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2013: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2012: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2011: ¥400,000 (Direct Cost: ¥400,000)
|
|---|
| Keywords | Inducible Pluripotent Stem (iPS) cell / Reprogramming / Chromatin Remodeling / Epigenetic Regulation / Embryonic Stem (ES) cell / Regenerative Medicine / Induced Pluripotent Stem Cell(iPSC) / Epigenetic Modification / Embryonic Stem Cell(ESC) |
|---|
| Research Abstract |
Induced pluripotent stem (iPS) cells can be generated from somatic cells by ectopic expression of four transcription factors : Oct4, Sox2, Klf4 and c-Myc. Examination of the start (fibroblasts) and end (iPSC) points, it is apparent that factor expression induces broad changes to the epigenome, yet how these changes are initiated and maintained during reprogramming remains largely unknown. Recent studies in ES cells showed that BRG1 is crucial for ES cell self-renewal and binding core ES cell genes. Further studies revealed that overexpression of BAF complex components BRGI and BAF155 enhanced cellular reprogramming, presumably by facilitating OCT4 binding to target gene promoters. I hypothesize that the chromatin remodeling activity of BRG1 is required in an early stage of iPS cell reprogramming, and may be equally critical at later stages of pluripotency maintenance.
The main purpose of this research proposal is to shed light on the dynamic chromatin changes elicited by factor expression, highlighting the role of nucleosome remodeling through BRG1 as somatic cells transition to pluripotency.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
A. The goal in the first year of the project was to define the molecular characteristics of the drug-inducible reprogramming system. I have carefully established several assays, and collected data over a time-course of drug treatment.
1) I observed and recorded morphological changes in cells treated with doxycycline. Fibroblasts that express Yamanaka factors (Oct4 Sox2, Klf4, and c-Myc) were tracked throughout reprogramming by fluorescence-activated cell sorting (FACS) analysis.
2) Kinetics and levels of Oct4, Sox2, Klf4, and c-Myc protein expression upon dox-induction were measured by Western Blotting. Differences in factor expression levels correlated with differences in cell proliferation, morphology, and colony formation.
3) Measuring the intermediate markers Thy1, SSEA-1, and EPCAM by FACS has led us to define sub-populations in our reprogramming system.
4) Global gene expression changes in the reprogramming process have been examined by microarray analysis and then confirmed by qRT-PCR. Both cell-cell interaction and proper control of proliferation may be the key determinants of the efficient iPSC generation.
B. In the second year of the project, I aimed to implement a system to test the role of BRG-I during different stages of iPS reprogramming.
1) RNAi was planned to knockdown endogenous BRGI to define molecular events of iPS derivation in the absence of BRGI activity. This was not measured during the project.
: RNAi technology has been established to knockdown endogenous Cdhl, which is critical for successful iPS reprogramming. From our results, we are now considering other targets/strategies to regulate reprogramming.
2) Time-course microarray analyses will be performed to measure gene expression changes at different days after BRGI knockdown.
: I established a time-course microarray analyses by FACS sorting of transgene-expressing cells (mCherry positive) and applying RNA from these cells to microarray analysis. Addition to this, I have obtained significant amount of knowledge about how to analyze the array results. The results of my experiments have been written as a manuscript, and will be submitted shortly.
|
| Strategy for Future Research Activity |
Since BRG-1 is important for transcription factor binding across the genome, whole-genome BRG-1, Oct4, Sox2, Klf4, and c-myc occupancy at the promoters of several target genes will be measured by ChIP (Chromatin Immunoprecipitation) -seq. Now, using our well-characterized drug-inducible system and next-generation sequencing analysis. we can address the question of how Oct4 and Sox2 occupancy across the genome controls ES cells.
|
Report
(3 results)
Research Products
(3 results)
