Genetic mosaics and time-lapse imaging identify functions of histone H3.3 residues in mouse oocytes and embryos

Abstract

During development from oocyte to embryo, genetic programs in mouse germ cells are reshaped by chromatin remodeling to orchestrate the onset of development. Epigenetic modifications of specific amino acid residues of core histones and their isoforms can dramatically alter activation and suppression of gene expression. H3.3 is a histone H3 variant that plays essential roles in mouse oocytes and early embryos, but the functional role of individual amino acid residues has been unclear because of technical hurdles. Here, we describe two strategies that successfully investigated the functions of three individual H3.3 residues in oogenesis, cleavagestage embryogenesis and early development. We first generated genetic mosaic ovaries and blastocysts with stochastic expression of wild-type or mutant H3.3 alleles and showed dominant negative effects of H3.3R26 and H3.3K27 in modulating oogenesis and partitioning cells to the inner cell mass of the early embryo. Timelapse imaging assays also revealed the essential roles of H3.3K56 in efficient H2B incorporation and paternal pronuclei formation. Application of these strategies can be extended to investigate roles of additional H3.3 residues and has implications for use in other developmental systems.

Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig

“…The core components of the PRC2 complex EZH2 and SUZ12 repress gene expression by establishing H3K27me3. Like in mouse, 34 the result indicates the expression of developmental genes is pre-patterned by the histone modification before ZGA. SETDB1 and SUV39H2 are responsible for establishment of H3K9me3 to repress gene expression.…”

“…The notion was validated using IF analysis of H3K9me3 from MII oocyte to blastocyst, and a reduction was observed from the 2-cell to 4-cell embryos ( Figure 5E). Like in mouse, 34 the result indicates the expression of developmental genes is pre-patterned by the histone modification before ZGA. The dynamic expression of epigenetic regulators suggests the transition of epigenetic profiles during the establishment of pluripotency in porcine embryos.…”

Enigma of Retrotransposon Biology in Mammalian Early Embryos and Embryonic Stem Cells

“…Concomitantly, ERV-enriched subpopulation of mouse/human ESCs activates a subset of early embryonic genes, differently from conventional ESCs [ 26 , 36 ]. In mouse ESCs, the absence of replacement histone variant H3.3, which is important for cell fate decision in early embryos [ 51 ], leads to reduced repressing histone mark and upregulated ERV expression, causing derepression of endogenous genes in vicinity [ 52 ]. It is also reported that depletion of miR-34a in mouse ESCs leads to significantly enhanced ERV expression and acquisition of totipotency, implicating that ERV is involved in a complicated molecular network with both transcriptional and posttranscriptional regulations [ 53 ].…”

Asymmetrical deposition and modification of histone H3 variants are essential for zygote development

“…Therefore, we hypothesized that the delay in DNA replication could be caused by the ectopic methylation of H3.1 and H3.2 at the K27me3 residue in the paternal perinucleolar region of H3.1/2-OEs. The physiological significance of histone modifications has successfully been probed by microinjection of embryos that contain cRNA encoding H3 variants with amino acid substitutions ( Santenard et al, 2010 ; Hatanaka et al, 2015 ; Zhou et al, 2017 ). To investigate the function of H3K27me3 in paternal pronuclei, we performed microinjection of cRNA encoding H3.1 and H3.2 with an arginine (R) substitution at residue 27 to replace K27 (H3.1K27R and H3.2K27R).…”