Diverse factors are involved in maintaining X chromosome inactivation

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 40; pp. 16699 - 16704
• Main Authors: Chan, Kui Ming, Zhang, Hui, Malureanu, Liviu, van Deursen, Jan, Zhang, Zhiguo
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 04.10.2011; National Acad Sciences
• Subjects: Animals; Biological Sciences; Cell division; Cell lines; Cells; Chromatin Immunoprecipitation; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Chromosomes; DNA; DNA Primers - genetics; Epigenetics; eukaryotic cells; Fluorescence; Fluorescent Antibody Technique; Gene silencing; Genes; Genetic screening; Genomics; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Heterochromatin; Heterochromatin protein 1; Histones; Immunofluorescence; In Situ Hybridization, Fluorescence; Mice; Oligonucleotide Array Sequence Analysis; Origin Recognition Complex - genetics; Origin Recognition Complex - metabolism; promoter regions; Promoters; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA Interference; RNA, Small Interfering - genetics; RNA-mediated interference; Transgenes; X chromosome; X Chromosome inactivation; X Chromosome Inactivation - physiology; X linked genes
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1107616108

X chromosome inactivation (XCI) is the most dramatic example of epigenetic silencing in eukaryotes. Once established, the inactivated X chromosome (Xi) remains silenced throughout subsequent cell divisions. Though the initiation of XCI has been studied extensively, the protein factors involved in Xi silencing and maintenance are largely unknown. Here we report the discovery of a diverse set of 32 proteins involved in maintenance of Xi silencing through a genome-wide RNAi screen. In addition, we describe the mechanistic roles of two proteins—origin recognition complex 2 (Orc2) and heterochromatin protein 1 (HP1α)—in Xi silencing. Immunofluorescence studies indicate that Orc2 and HP1α localize on Xi in mouse cells. Depletion of Orc2 by shRNA leads to the loss of both Orc2 and HP1α localization on Xi. Furthermore, the silencing of genes on Xi is disrupted in both Orc2- and HP1α-depleted cells. Finally, we show, using ChIP assay, that the localization of HP1α and Orc2 to the promoter regions of Xi-silenced genes is interdependent. These findings reveal a diverse set of proteins involved in Xi silencing, show how Orc2 and HP1α impact Xi silencing, and provide a basis for future studies on the maintenance of Xi silencing.