Double gene knockdown leads to X‐chromosome reactivation (939.7)

• DOI: 10.1096/fasebj.28.1_supplement.939.7
• Publication Date: 2021-06-16T09:41:54Z
• Authors: Stacy Thomas; Smitha Sripathy; Vid Leko; Eric Foss; Uyen Lao; Antonio Bedalov
• Keywords: 0301 basic medicine; 03 medical and health sciences

Rett Syndrome is a X‐linked brain disorder caused by a mutation in one copy of the MeCP2 gene. This gene is only expressed in some cells due to X‐chromosome silencing, a phenomenon that occurs in all cells with two X chromosomes. After four cell divisions, each cell of the embryo randomly inactivates one X‐chromosome (Xi) and the other (Xa) provides the X‐linked genetic code for the cell. This choice is propagated through subsequent cell divisions. The exact mechanism of the initiation and maintenance of X‐chromosome silencing is unknown, but some factors are known. Xist RNA coats Xi, acting as a signal to initiate and maintain X‐silencing. DNA methylation at CpG islands makes the chromatin less available and hypoacteylated histones alter the chromatin packaging to maintain X‐silencing. We hypothesize that a future treatment option for Rett Syndrome will be to use Small hairpin RNAs (shRNAs) to knockdown genes fundamental to X‐chromosome silencing to reactivate Xi and cause more cells to produce functional. shRNAs can reduce the expression of a gene by imposing post‐transcriptional control on mRNAs, with which they complementary base‐pair. A shRNA library screen using (Xa)MeCP2/(Xi)MeCP2‐Luciferase‐Hygromycin Resistance mouse tail fibroblasts generated 82 possible genes involved with X‐silencing. In order to validate these screens, the knockdown efficiency of genes has been tested using qPCR and was used to measure the amount of X‐reactivation. Reducing the expression of Acvr1 and dnmt1 in cells results in some X‐reactivation. In combination with a dnmt1 knockdown, a Xist knockdown increases the frequency of reactivation.Grant Funding Source: This research is supported in part by the Cancer Center Support Grant (CCSG) CURE Supplement: NCI 5