Regulation of the JMJD3 (KDM6B) histone demethylase in glioblastoma stem cells by STAT3

Abstract

The growth factor and cytokine regulated transcription factor STAT3 is required for the self-renewal of several stem cell types including tumor stem cells from glioblastoma. Here we show that STAT3 inhibition leads to the upregulation of the histone H3K27me2/3 demethylase Jmjd3 (KDM6B), which can reverse polycomb complex-mediated repression of tissue specific genes. STAT3 binds to the Jmjd3 promoter, suggesting that Jmjd3 is a direct target of STAT3. Overexpression of Jmjd3 slows glioblastoma stem cell growth and neurosphere formation, whereas knockdown of Jmjd3 rescues the STAT3 inhibitor-induced neurosphere formation defect. Consistent with this observation, STAT3 inhibition leads to histone H3K27 demethylation of neural differentiation genes, such as Myt1, FGF21, and GDF15. These results demonstrate that the regulation of Jmjd3 by STAT3 maintains repression of differentiation specific genes and is therefore important for the maintenance of self-renewal of normal neural and glioblastoma stem cells.

Fig 1. STAT3 represses Jmjd3 expression in glioblastoma stem cells.

A. RT-qPCR of GS6-22 cells treated with S3I-201 or STA-21 demonstrates that Jmjd3 mRNA is upregulated at both 4 hours and 24 hours after inhibitor treatment. Values represent the fold change relative to DMSO treated cells for three experiments; bars SD (**p<0.01). B. Knockdown of STAT3 using an shRNA-containing lentivirus leads to the upregulation of Jmjd3 mRNA in GS6-22 cells, two days after selection. Values represent the fold change relative to DMSO treated cells for three experiments; bars SD (**p<0.01). C. Chromatin immunoprecipitation using anti-STAT3 antibody, followed by PCR using primers to the Jmjd3 promoter demonstrates STAT3 binding in GS7-2 and GS6-22 cells. Enrichment of DNA in STAT3 immunoprecipitation (relative to input) was quantified by ImageJ. D. Treatment of GS6-22 cells with retinoic acid increases Jmjd3 mRNA levels. GS6-22 cells were treated for 1 and 4 hours with 1 μM RA. Values represent the fold change relative to DMSO treated cells for three experiments; bars SD (*p<0.05, **p<0.01). E. S3I-201 treatment decreases NCor binding at the Jmjd3 promoter. GS6-22 cells were treated with S3I-201 (50 μM) or DMSO for 4 hours, fixed, lysed, and sonicated. Sonicated lysates were incubated overnight with anti-NCor or IgG control and subjected to chromatin immunoprecipitation. The resulting DNA was subjected to PCR using primers to the Jmjd3 promoter. F. Densitometry of E was performed using ImageJ.

Fig 2. STAT3 controls GBM-SC neurosphere formation and proliferation through repression of Jmjd3.

A. Knockdown of Jmjd3 rescues neurosphere formation in the presence of S3I-201. Representative images of GS6-22 and GS7-2 cells infected with either control or shJmjd3 containing lentivirus treated with DMSO or S3I-201 (50 μM). Images for GS6-22 cells were taken after 6 days and GS7-2 cells were taken after 4 days, based on differences in sphere formation rate in these lines. B. Quantification of neurosphere formation in GS6-22 cells. After 6 days of inhibitor treatment, the number of spheres per 100 cells was counted. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01). C. After 4 days of inhibitor treatment, GS7-2 cell sphere formation was quantified as described for GS6-22 cells. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01 relative to DMSO control, upon shJMJD3 infection). D. Representative images of GS6-22 and GS7-2 cells infected with overexpression retrovirus containing either a control (empty) plasmid, Jmjd3, or a catalytic domain mutant of Jmjd3. After three days of selection, cells were dissociated and replated at 100 cells per ml. Pictures were taken at 50X seven days after replating. E. Quantification of neurosphere formation capacity in GS6-22 and GS7-2 cells infected with the Jmjd3 overexpression retrovirus. Neurosphere formation assay was performed in triplicate (**p<0.01). F. GS6-22 and G. GS7-2 cells were infected with either control, Jmjd3, or Jmjd3 mutant retroviruses as previously described. Cells were pulsed with 30 μM BrdU for 16 hours. After 24 hours, cells were fixed and stained with an anti-BrdU antibody. Cells were also stained with 7-AAD at this time. The percentage of BrdU positive cells was analyzed using flow cytometry. Values represent the mean of 3 experiments; bars SD of the mean (*p<0.05).

Fig 3. STAT3 regulates H3K27 trimethylation and neural gene induction in GBM-SC.

A. RT-qPCR analysis of FGF21, GDF15 and JMJD3 expression in GS6-22 cells treated with BP-1-102 (15uM) for 6 hours. Values represent the fold change relative to control cells for three experiments; bars SD (**p<0.01, ***p<0.005). B. GS6-22 cells were treated with BP-1-102 (15 μM) or DMSO for 6 hours, fixed, lysed, and subjected to chromatin immunoprecipitation with an antibody to H3K27 trimethylation or total histone H3. Quantitative PCR using primers to FGF21 or GDF15 or Myt1 genes was performed after immunoprecipitation, amplification and DNA purification. Data is displayed as percentage of input DNA of H3K27me3/H3 ChIP, normalized relative to control cells for three experiments (*p<0.05, **p<0.01, ***p<0.005). C. Demethylation of H3K27 at the Myt1 promoter confers BMP4 inducibility of the gene. RT-qPCR analysis of Myt1 and JMJD3 expression in GS6-22 cells treated with BP-1-102 (15uM), recombinant BMP4 (10 ng/ml) or a combination of both for 6 hours. Values represent the fold change relative to DMSO treated control cells for three experiments; bars SD (*p<0.05, **p<0.01).

Fig 4. STAT3 inhibition in human neural stem cells leads to a decrease in neurosphere formation and proliferation, and an upregulation of Jmjd3 expression.

A. STAT3 activation in human neural stem cells was assessed by immunoblotting with phospho-specific antibodies. B. Human neural stem cells were plated in serum free media supplemented with EGF and FGF2. 24 hours after plating, cells were treated with S3I-201 (50 μM) or DMSO control. Pictures were taken 3 days after treatment. C. STAT3 regulation of Jmjd3 was assessed by qRT-PCR. Human neural stem cells were treated with S3I-201 (50 μM) for 24 hours. Values represent the fold change relative to control cells for three experiments; bars SD (**p<0.01). D. Cells were treated with DMSO or S3I-201 for 24 hours, and then subjected to a 15 μM BrdU pulse for 20 hours. Values represent the average of three experiments; bars SE (*p<0.05, **p<0.01).