258. Differential expression of H2A and H3 variant histones in mouse preimplantation embryos and R1 ES cells

Abstract

Histone variants replace canonical histones in nucleosomes to serve numerous biological processes. This integration alters DNA properties to ultimately regulate gene expression. Previous mouse studies have indicated that some variants (H2AZ and H3.3) are essential for survival, but here we document and correlate histone expression patterns with key developmental events. Using quantitative reverse-transcribed PCR (qRT–PCR) we investigated the expression of 7 genes coding for H2A variants and 4 genes coding for H3 variants in mouse preimplantation embryos and in pluripotent R1 ES cells. Messenger RNA was extracted from pools of 3 embryos flushed from superovulated mice. Embryos were collected at five stages, zygotes, 2-cell embryos, morulae, blastocysts and hatching blastocysts (20 h, 44 h, 68 h, 92 h and 116 h post hCG respectively). The expression of H2A variant genes typically peaked within blastocysts. H2AZ and H2AX expression was 80 – 95% higher in blastocysts than other stages. Conversely, genes coding for H3 variants showed elevated expression in zygotes, where H3.3 expression was 85 – 95% higher and CENPA was ~75% higher than in later preimplantation stages. The expression profiles of histone remodellers SWI/SNF and CAF-1 correlated with the variants they are known to remodel (H2A and H3 variants respectively), suggesting an increased integration of those variants into nucleosomes. We also compared blastocyst and embryonic stem cell (ES cell) expression patterns. R1 ES cells express all histone variants, including H2A.Bbd, H3.1 and H3.2 which were not expressed in preimplantation embryos. Further, expression levels of every histone variant investigated differed significantly between R1 ES cells and hatching blastocysts (ANOVA, P < 0.05, n = 3 experiments). We conclude that histone variant expression reflects preimplantation developmental demands. Further, histone code expression profiles show significant change upon extended cell culture and maintenance of pluripotency as indicated by comparing in vivo hatching blastocysts to the R1 ES cell line.