Characterization of Human MacroH2A Through Gene Targeting

Macro histone H2A (macroH2A) is a variant of core histone H2A that differs primarily by an extensive carboxy-terminal tail of unknown function that makes up two-thirds of the protein's mass. The histone variant is distributed throughout the nucleus, but in female mammalian cells, it has been found to be associated with the inactive X chromosome (Xi) in a local accumulation referred to as a macrochromatin body. The association of macroH2A with facultative heterochromatin of the Xi is suggestive of a role for the variant in gene silencing. MacroH2A1 was the first form of macroH2A discovered and is encoded by the H2AFY gene. Two splice isoforms exist due to two alternate versions of exon-6, giving rise to macroH2A1.1 and macroH2A1.2. A second form of macroH2A encoded by H2AFY2 gene, known as macroH2A2, shares 80% amino acid identity with macroH2A1 and also accumulates at Xi, suggesting the possibility of functional redundancy between the two proteins. In order to further investigate macroH2A, we have generated knockouts of macroH2A1 and targeted a single allele of macroH2A2 in a human female telomerase immortalized retinal pigment epithelial cell line (RPE1). Targeted clones were generated by exchanging exon-2 of one or both alleles with a promoter-trap construct containing a promoterless neomycin selection cassette flanked by arms of homology designed to the intronic sequences immediately adjacent to exon-2. The selection cassette contains a splice-acceptor, internal ribosome entry site and polyadenylation signal, which when exchanged with exon-2 results in the inclusion of the neomycin coding sequence in the resulting truncated messenger RNA and its subsequent translation, allowing for correct targeting to be selected for through neomycin resistance. To enhance targeting, Zinc Finger Nucleases (ZFNs) were engineered to create a double strand break at or close to exon-2. In order to target the genes, cells were co-nucleofected with the targeting construct and ZFNs before seeding cells in media containing neomycin. Single cell clones were screened for correct targeting and loss of macroH2A assessed by Western blotting. In order to explore the impact of macroH2A1 loss, RNA was extracted from a macroH2A1 knockout as well as parental RPE1 and changes to the transcriptome assessed by massively paralleled sequencing of complementary DNA (RNAseq). Genes that showed a significant change in expression between the wildtype and knockout cells were selected for further study. Quantitative Chromatin Immunoprecipitation (qChIP) was performed on an affected gene to evaluate any local chromatin changes due to macroH2A1 loss. Additionally, to examine the possibility that macroH2A1 splice isoforms fulfill different roles, full-length MYC-tagged expression constructs for macroH2A1.1 and macroH2A1.2 were reintroduced into cells and rescue of wild-type expression assessed for genes that displayed altered expression in response to macroH2A1 loss.