Expression of Xist RNA is sufficient to initiate macrochromatin body formation

Abstract

MacroH2A1 is a histone variant that is found as a component of the inactive X chromosome where it is detected as a dense accumulation called a macrochromatin body (MCB). Macrochromatin bodies co-localize with Xist RNA, which is an untranslated RNA that is expressed exclusively from the inactive X chromosome of placental mammals. However, no studies to date have investigated whether Xist RNA expression is necessary or sufficient to cause the formation of MCBs. Here we show that expression of Xist RNA is sufficient to cause the formation of MCBs even when Xist is expressed from an inducible transgene at ectopic autosomal sites. Macrochromatin bodies form at sites of transgenic Xist expression in differentiating mouse ES cell lines and transgenic fibroblasts, but MCBs cannot form in undifferentiated ES cells even after prolonged Xist expression. The kinetics of MCB formation revealed that Xist expression precedes MCB formation and that differentiating ES cells undergo a rapid and synchronous transition that renders them competent to form MCBs. Once MCBs have formed, continued expression of Xist is required for their maintenance. These results show that Xist RNA and macroH2A1 function in a common pathway. Expression of Xist in a permissive nuclear environment is sufficient to initiate a chromatin-remodeling event culminating in the incorporation of macroH2A1. The results also strongly suggest the existence of additional regulatory factors for X inactivation that are regulated developmentally. In addition, we present evidence that macroH2A1 density is not simply a measure of the general degree of DNA compaction.