Histone macroH2A1.2 relocates to the inactive X chromosome after initiation and propagation of X-inactivation

The histone macroH2A1.2 has been implicated in X chromosome inactivation on the basis of its accumulation on the inactive X chromosome (Xi) of adult f emale mammals. We have established the timing of macroH2A1.2 association wi th the Xi relative to the onset of X-inactivation in differentiating murine embryonic stem (ES) cells using immuno-RNA fluorescence in situ hybridizat ion (FISH). Before X-inactivation we observe a single macroH2A1.2-dense reg ion in both undifferentiated XX and XY ES cells that does not colocalize wi th X inactive specific transcript (Xist) RNA, and thus appears not to assoc iate with the X chromosome(s). This pattern persists through early stages o f differentiation, up to day 7. Then the frequency of XY cells containing a macroH2A1.2-rich domain declines. In contrast, in XX cells there is a stri king relocalization of macroH2A1.2 to the Xi, Relocalization occurs in a hi ghly synchronized wave over a 2-d period, indicating a precisely regulated association. The timing of macroH2A1.2 accumulation on the Xi suggests it i s not necessary for the initiation or propagation of random X-inactivation.