Fig. 1.

IDH mutation are associated with dysregulation of glial differentiation and global histone methylation.

1. From sequencing results revealed a high frequency of IDH mutations in tumour of oligodendroma (33 samples were IDH1 mutation, 2 samples were IDH2 mutation and 6 were wild type for IDH1/2).

2. IDH-associated changes in histone methylation could be observed in 293 cells. The mutant IDH1 and IDH2 had markedly increase in histone methylation and it correlated with 2HG production.

3. Histone lysine methlation (H3K9me3 and H3K27me3) significantly increase in glioma with IDH mutation by Immunohistochemistry.

Fig. 2.

Differentiation arrest induced by mutant IDH or 2HG treatment is associated with increased global and promoter specific H3K9 and H3K27 methylation.

(3T3-L1 cells tranduced with R172K-IDH2 mutant)

1. The IDH2 mutant cells produced 2HG whereas wild type and vector did not.

2. The IDH2 mutant cells had reduced lipid accumulation ( less differentiated).

3. By quantifying the absorbance, treatment of 3T3-L1 cells with octyl-2HG led to a dose-dependent inhibition of lipid accumulation.

4. IDH mutant cells exhibited a defect of transcriptional factor essential for adipgenesis (PPARg, Cebpa and Adipoq).

5. Treatment of octyl-2HG was sufficient to show a defect of above transcriptional factor.

6. By ChIP analysis using Ab against H3K9me3 and H3K27me3 and QPCR with primer targeting promoters of Cebpa and Adipoq showed significantly increase in IDH mutant cell at day 4 differentiation.

7. A global increase in H3K9 methylation and a reciprocal decrease in H3 acetylation of IDH mutant.

Fig . 3

IDH mutation induces histone methylation increase in CNS-derived cells and can alter cell lineage gene expression.

Fig a-e, NHA (Normal Human Astrocytes) cells tranduced with mutant IDH1.

1. Compared to parental cells, late-passage cells expressing mutant IDH showed elevated levels of a variety of histone methylation marks.

2. And it correlated with an enhanced expression of the neural marker nestin.

c-e. Relationship of histone and DNA methylation in IDH-expressing astrocytes. H3K9me3 levels were significantly increase by passage 12 after cells were infected with mutant IDH. Increase in DNA methylation never observed before passage 17 and significantly increased after P22.

f. Under conditions that promote astrocyte differentiation, neurosphere cultures infected with mutant IDH failed to induced expression of the astrocytic marker GFAP and showed expression of the neural marker tubulin.

Fig. 4.

2HG-inhibitable H3K9-demethylase KDM4C is required for cell differentiation.

(fig.b-c, 3T3-L1 cells with recombinant human GST-tagged KDM4C; fig d, 3T3-L1 transfected with siRNA against KDM4C)

1. KDM4C (a.k.a JMJD2C), an H3K9-spesific JHDM, was induced in 3T3-L1 cells during differentiation.

2. KDM4C effectively removed H3K9me2 and H3K9me3 in the presence of αKG, conversely 2HG inhibited the demethylation reaction in a dose dependent manner.

3. Increasing the conc.of αKG reversed the inhibition of H3K9 demethylation by 2HG.

4. H3K9 demethylation is a required component of adipocyte differentiation.

This study demonstrate 2HG can inhibit histone demethylation through IDH mutation and that inhibition of histone demethylation can be sufficient to block the differentiation of non-transformed cells.

（担当：Royhan）