Females are genetically characterized by two copies of the X chromosome while male cells carry one X and one Y chromosome. As such, female cells carry twice the genetic information from the X chromosome compared to males. Dosage compensation aims to control this genetic imbalance by inactivating one copy of the X chromosome for the lifetime of a cell and its progeny. In some cases, X chromosome inactivation (Xi) can lead to diseases. For instance, individuals affected by Rett syndrome, a severe neurological disorder, possess two copies of the MeCP2 gene (methyl CpG binding protein 2) of which one is mutated and the wild type copy is on the inactivated X chromosome. As such, rescue of the mutated gene by the wild type copy is impossible, resulting in loss of MECP2 protein expression and disease development. Understanding the molecular biology of X chromosome inactivation may help identify solutions for reactivating expression of the functional MeCP2 gene.
The X chromosome inactivation relies on epigenetic mechanisms. A non-coding RNA, called Xist (X-inactive specific transcript), is expressed in cells carrying two X chromosomes, physically covers the X chromosome to be inactivated and recruits silencing proteins. After silencing has been established, it is maintained and clonally propagated for the lifetime of the cells. While the role of Xist RNA in the initiation of the X chromosome inactivation is known, its role in maintenance remains unclear. The laboratory of Dr. Antonio Bedalov (Clinical Research Division), who has long been interested in Rett syndrome and X chromosome inactivation, recently published a study in the journal Epigenetics and Chromatin, characterizing the role of Xist RNA in maintenance of the X chromosome inactivation.
Transgenic mice lacking expression of the Xist RNA in a brain-specific manner and carrying a MeCP2-GFP fusion gene specifically on the inactive chromosome were developed to measure the reactivation of MeCP2-GFP upon deletion of Xist RNA. At baseline, these mice do not express any MeCP2-GFP because inactivation is skewed toward the X-chromosome that carries the MeCP2-GFP reporter. After silencing has been established, Xist on the inactive X-chromosome is deleted, and reactivation is measured by counting MeCP2-GFP positive cells.
Figure provided by Dr. Bedalov.
Xist RNA recruits PRC1 and PRC2 (polycomb repressive complexes 1 and 2) proteins that are involved in modification of histone proteins to restrict transcription factor DNA accessibility. PRC1 and PRC2 recruitment can be identified by specific histone marks such as monoubiquitylation of lysine 119 on histone H2A and trimethylation of lysine 27 on histone H3. In mice lacking Xist RNA, these repressive histone marks could no longer be detected by immunofluorescence. Despite the complete loss of histone repressive marks in almost all brain cells, reactivation of MeCP2-GFP was found in only 2.3 to 4.8% of neurons, demonstrating the role of Xist RNA for maintaining tight transcriptional repression on the inactive X-chromosome.
Despite Xist RNA deletion, a majority of brain cells maintained MeCP2-GFP silencing and chromosomal inactivation. Global gene expression was slightly increased from the X chromosome but not from autosomes in Xist-deleted mice, suggesting that Xist RNA deletion influenced chromosomal inactivation only in a small subset of cells. Even more interesting was the finding that genes with higher levels of expression were more sensitive to Xist RNA deletion and reactivation relative to those with low expression. DNA methylation levels near transcription start sites was decreased on the X chromosome relative to that on autosomes and in wild-type mice. DNA methylation was also positively associated with the level of X-linked gene reactivation. These results support a role for Xist RNA in gene expression and DNA methylation status of the X chromosome to repress gene expression.
This study unravels a previously unknown function of Xist RNA in the maintenance of the chromosome inactivation and provides valuable information about potential consequences of expressing two active copies of genes that reside on the X chromosome. The results are encouraging and could be pursued from a therapeutic perspective. More investigations will be required to decipher the intricate mechanisms of X chromosome inactivation. Such studies are critical to provide clues for progress toward a treatment for diseases such as the Rett syndrome though reactivation of the functional copy of the gene on the inactive X-chromosome.
Funding for this study was provided by the Rett Syndrome Research Trust and the Shared Resource of the Fred Hutchinson/University of Washington Cancer Consortium.
Adrianse RL, Smith K, Gatbonton-Schwager T, Sripathy SP, Lao U, Foss EJ, Boers RG, Boers JB, Gribnau J, Bedalov A. 2018. Perturbed maintenance of transcriptional repression on the inactive X-chromosome in the mouse brain after Xist deletion. Epigenetics and Chromatin, 11(1), 50.