Histone macroH2A1.2 (macroH2A) is an unusual histone H2A variant with a large non-histone macrodomain at its carboxyl terminal. MacroH2A1.2 is enriched in facultative heterochromatin, including inactivated X chromosomes in mammalian females and senescence-associated heterochromatin foci. We show here that a small population of macroH2A1.2 is mono-ubiquitinated in human HeLa cells. Mass spectrometry analysis revealed that the specific targeting sites for the mono-ubiquitination are Lysl 15 and Lysl 16 of the historic domain. A corresponding Lysl 19 conserved in histone H2A is also mono-ubiquitinated by Ring protein in the polycomb group complex. We suggest that the mono-ubiquitination of macroH2A1.2 and histone H2A has similar or synergistic implications, but that the multiple ubiquitination sites in macroH2A1.2 might confer a variety of functions upon macroH2A1.2 to modulate chromatin states. (c) 2005 Elsevier Inc. All rights reserved.