Abstract
Macrohistones (mH2As) are unusual histone variants found exclusively in vertebrate chromatin. In mice, the H2afy gene encodes two splice variants, mH2A1.1 and mH2A1.2 and a second gene, H2afy2, encodes an additional mH2A2 protein. Both mH2A isoforms have been found enriched on the inactive X chromosome (Xi) in differentiated mammalian female cells, and are incorporated into the chromatin of developmentally-regulated genes. To investigate the functional significance of mH2A isoforms for X chromosome inactivation (XCI), we produced male and female embryonic stem cell (ESC) lines with stably-integrated shRNA constructs that simultaneously target both mH2A1 and mH2A2. Surprisingly, we find that female ESCs deficient for both mH2A1 and mH2A2 readily execute and maintain XCI upon differentiation. Furthermore, male and female mH2A-deficient ESCs proliferate normally under pluripotency culture conditions, and respond to several standard differentiation procedures efficiently. Our results show that XCI can readily proceed with substantially reduced total mH2A content.
Highlights
The most extreme epigenetic modification that occurs on the nucleosome level is the substitution of core histones with noncanonical variants
Alternative splicing occurs for H2afy mRNAs in mice [4]
Redundancy has complicated the production of knock out mice that lack all macroH2As
Summary
The most extreme epigenetic modification that occurs on the nucleosome level is the substitution of core histones with noncanonical variants. Macrohistones (mH2As) are non-allelic variants of the conventional histone H2A and are defined by the presence of a large (,30 kDa) C-terminal non-histone domain connected to the H2A-like domain through a short linker [1]. The mouse genome contains two genes, H2afy and H2afy, that encode separate proteins called macroH2A1 and macroH2A2 (abbreviated mH2A1 and mH2A2) [2,3]. The mRNA product of H2afy is subject to alternative splicing to produce two distinct protein isoforms, mH2A1.1 and mH2A1.2 that differ in the nonhistone region [4]. The mouse genome databases indicate the existence of a third macrohistone gene (termed H2afy3), but this locus is most likely a processed pseudogene that does not encode protein [5]
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