2006 Fiscal Year Final Research Report Summary
Toward understanding higher order chromatin structure and epigenomic regulation in ES cell differentiation
| Project/Area Number |
16570111
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Functional biochemistry
|
|---|
| Research Institution | Tokyo Metropolitan Organization for Medical Research |
|---|
Principal Investigator |
SATO Noriko Tokyo Metropolitan Organization for Medical Research, The Tokyo Metropolitan Institute of Medical Science, Chief Investigator, 東京都臨床医学総合研究所, 主任研究員 (70280956)
|
|---|
| Project Period (FY) |
2004 – 2006
|
| Keywords | higher order chromatin structure / nuclear organization / epigenomic regulation / Oct-3 / 4 / GCNF / DNA methylation |
|---|
| Research Abstract |
In order to understand various functions and dynamics of mammalian genomes, it is necessary to analyze not only epigenomic information but also higher-order chromatin structure and nuclear organization. We examined whether spontaneous alteration of chromatin structure, especially of its large scale unfolding, correlates with variation in gene expression, using cell clones carrying tandem repeats of the GFP-tagged reporter genes driven by a tetracycline responsive promoter. Flow cytometry and live-recording fluorescence microscopy revealed that, although fully activated by a saturating amount of doxycycline, GFP signal fluctuated in individual cell clones, regardless of the cell cycle stage. 3D-FISH analyses revealed that the degree of chromosome unfolding of the integrated array is stochastically altered, which significantly influences gene expression activities. This may be the first demonstration of stochastic fluctuation of chromosome unfolding. Next we examined the dynamics of Oct-
… More
3/4 chromatin status during ES cell differentiation. Oct-3/4 plays a crucial role in maintaining pluripotency. Although Oct-3/4 expression declines as ES cells differentiate, 3D-FISH analyses revealed that there are little changes in higher order chromatin structure (i.e. intranuclear positioning relative to constitutive heterochromatin and folding levels) of the Oct-3/4 locus during the differentiation. In contrast, DNA methylation following to deacetylation of H3K9K14 and demethylation and H3K4 in the Oct-3/4 promoter region was prominent. We asked how site-specific DNA methylation was induced. In the Oct-3/4 promoter, there is a binding site for GCNF, a transcriptional repressor and an orphan nuclear receptor. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggested a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene. Less
|
