Diverse factors are involved in maintaining X chromosome inactivation

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 (HP1 alpha)-in Xi silencing. Immunofluorescence studies indicate that Orc2 and HP1 alpha localize on Xi in mouse cells. Depletion of Orc2 by shRNA leads to the loss of both Orc2 and HP1 alpha localization on Xi. Furthermore, the silencing of genes on Xi is disrupted in both Orc2- and HP1 alpha-depleted cells. Finally, we show, using ChIP assay, that the localization of HP1 alpha 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 HP1 alpha impact Xi silencing, and provide a basis for future studies on the maintenance of Xi silencing.