Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Drug Treatments
2.3. RNA Isolation and Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR)
2.4. DNA Isolation and PCR
2.5. Chromatin Immunoprecipitation (ChIP)
2.6. Western Blot Analyses
2.7. Immunostaining
2.8. Statistical Analysis
3. Results
3.1. Small Molecules Targeting H3K9 Methylation, by Themselves, are not Very Effective at Activating FMR1 Expression in FXS-Patient-Derived Cells
3.2. Chaetocin Potentiates the Expression of AZA-Reactivated FM Alleles in FXS Patient Cells
3.3. HMT Inhibitors Delay Re-Silencing of AZA-Reactivated Alleles in FXS Lymphoblastoid Cells
3.4. Effect of HMT Inhibitors on H3K9 Methylation of the FMR1 Gene in FXS Patient Cells
4. Discussion and Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Cell Line Name | Cell Type | FXS/Control | CGG-Repeat Size/MethyLation |
---|---|---|---|
BJ | fibroblast | Control | 26 |
SC207 | fibroblast | Control | 20 |
SC126 | fibroblast | FXS | Mosaic for PM/ methylated FM |
SC128 | fibroblast | FXS | Mosaic for PM/ methylated FM |
C10259 | fibroblast | FXS | Methylated FM (1363 by SB) |
C10700 | fibroblast | FXS | Mosaic for PM/ methylated FM |
GM05848 | fibroblast | FXS | Methylated FM (~700) |
GM06865 | lymphoblastoid | Control | 30 |
GM0032B | lymphoblastoid | FXS | Methylated FM (570) |
GM04025 | lymphoblastoid | FXS | Methylated FM (645) |
GM07294 | lymphoblastoid | FXS | Methylated FM (745) |
13-1 | iPSCs | FXS | Methylated FM (463) |
15C | iPSCs | FXS | Methylated FM (330) |
SC128 | iPSCs | FXS | Methylated FM (300) |
BC1 NSC | NSC | Control | NA |
SC128 NSC | NSC | FXS | Methylated FM (300) |
SC128 neurons | neurons | FXS | Methylated FM (300) |
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Kumari, D.; Sciascia, N.; Usdin, K. Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells. Genes 2020, 11, 356. https://doi.org/10.3390/genes11040356
Kumari D, Sciascia N, Usdin K. Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells. Genes. 2020; 11(4):356. https://doi.org/10.3390/genes11040356
Chicago/Turabian StyleKumari, Daman, Nicholas Sciascia, and Karen Usdin. 2020. "Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells" Genes 11, no. 4: 356. https://doi.org/10.3390/genes11040356