The chromatin template imposes an epigenetic barrier during the process of 
somatic cell reprogramming. Using fibroblasts derived from macroH2A double 
knockout (dKO) mice, here we show that these histone variants act cooperatively 
as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, 
we further demonstrate that macroH2A2 is the predominant barrier to 
reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together 
with H3K27me3, co-occupy pluripotency genes in wild-type (wt) fibroblasts. In 
particular, we find macroH2A isoforms to be highly enriched at target genes of 
the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. 
Finally, while macroH2A dKO-induced pluripotent cells are able to differentiate 
properly in vitro and in vivo, such differentiated cells retain the ability to 
return to a stem-like state. Therefore, we propose that macroH2A isoforms 
provide a redundant silencing layer or terminal differentiation 'lock' at 
critical pluripotency genes that presents as an epigenetic barrier when 
differentiated cells are challenged to reprogram.