title: Diverse factors are involved in maintaining X chromosome inactivation.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Kui Ming
authors.initials: KM
authors.lastname: Chan
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Liviu
authors.initials: L
authors.lastname: Malureanu
authors.forename: Jan
authors.initials: J
authors.lastname: van Deursen
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D049951:X Chromosome Inactivation / Q000502:physiology*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D051738:Origin Recognition Complex; D034741:RNA, Small Interfering; D000090266:Chromobox Protein Homolog 5; D049452:Green Fluorescent Proteins