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Updated: Mar 7, 2026
Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome
Published on: March 2, 2018
A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X
Derek Lessing1,2,3, Thomas O Dial1,2,3, Chunyao Wei1,2,3
1Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.
Abstract:
X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.
Insights
Researchers screened over 367,000 compounds to find ways to reactivate the inactive X chromosome (Xi). They discovered that combining DNA methylation inhibitors with Aurora kinase inhibitors synergistically up-regulates genes on the Xi.
Area of Science:
- Genetics
- Epigenetics
- Pharmacology
Background:
- X-chromosome inactivation (Xi) silences one X chromosome in female mammals for dosage compensation.
- The inactive X chromosome retains over 1,000 functional genes, representing a potential therapeutic target for X-linked diseases.
- Reactivating genes on the Xi is challenging due to the robust nature of X-chromosome silencing.
Purpose of the Study:
- To identify small molecules capable of reactivating gene expression from the inactive X chromosome.
- To explore synergistic drug combinations for enhanced Xi reactivation.
Main Methods:
- A high-content screen of approximately 367,000 small molecules was performed using an Xi-linked GFP reporter in mouse fibroblasts.
- Cells were primed with 5-aza-2 acronym{prime}deoxycytidine (5azadC), a DNA methyltransferase inhibitor, to sensitize the screen.
- Compounds targeting Aurora kinase (VX680, MLN8237) and DNA methylation pathways were investigated.
Main Results:
- The screen identified compounds including VX680, MLN8237, and 5azadC as eliciting GFP activity, indicating potential Xi reactivation.
- Combinations of VX680 with 5azadC, and MLN8237 with 5azadC, demonstrated synergistic up-regulation of genes on the inactive X chromosome.
- A synergistic interaction between DNA methylation and Aurora kinase pathways was identified for Xi reactivation.
Conclusions:
- The study highlights the potential of targeting the interplay between DNA methylation and Aurora kinase pathways for pharmacological reactivation of the inactive X chromosome.
- This research opens avenues for developing novel therapeutic strategies for X-linked genetic disorders by tapping into the gene reservoir of the Xi.
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