A high throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation
Significance In mammalian female cells, nearly all genes are silenced on one of two X chromosomes. Heterozygous females with "dominant" X linked diseases, such as Rett syndrome, may benefit from pharmacological reactivation of the silent, healthy allele in affected organs. Toward establishing proof of concept, here we carry out a primed screen of a large library of small molecules for compounds that can reactivate expression from the inactive X (Xi) We identify a combination of compounds that inhibits the DNA methylation and Aurora kinase pathways and demonstrate that the two pathways act synergistically to repress genes on the Xi, including genes involved in X linked disease. 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.
Proceedings of the National Academy of Sciences