Epigenetic regulation limits competence of pluripotent stem cell-derived oocytes.

Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro-generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development in vitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro-derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5-hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements.

Nucleosomes in mammalian sperm: conveying paternal epigenetic inheritance or subject to reprogramming between generations?

The genome of mammalian sperm is largely packaged by sperm-specific proteins termed protamines. The presence of some residual nucleosomes has, however, emerged as a potential source of paternal epigenetic inheritance between generations. Sperm nucleosomes bear important regulatory histone marks and locate at gene-regulatory regions, functional elements, and intergenic regions. It is unclear whether sperm nucleosomes are retained at specific genomic locations in a deterministic manner or are randomly preserved due to inefficient exchange of histones by protamines. Recent studies indicate heterogeneity in chromatin packaging within sperm populations and an extensive reprogramming of paternal histone marks post fertilization. Obtaining single-sperm nucleosome distributions is fundamental to estimating the potential of sperm-borne nucleosomes in instructing mammalian embryonic development and in the transmission of acquired phenotypes.

HP1 proteins regulate nucleolar structure and function by secluding pericentromeric constitutive heterochromatin.

Nucleoli are nuclear compartments regulating ribosome biogenesis and cell growth. In embryonic stem cells (ESCs), nucleoli containing transcriptionally active ribosomal genes are spatially separated from pericentromeric satellite repeat sequences packaged in largely repressed constitutive heterochromatin (PCH). To date, mechanisms underlying such nuclear partitioning and the physiological relevance thereof are unknown. Here we show that repressive chromatin at PCH ensures structural integrity and function of nucleoli during cell cycle progression. Loss of heterochromatin proteins HP1α and HP1ß causes deformation of PCH, with reduced H3K9 trimethylation (H3K9me3) and HP1γ levels, absence of H4K20me3 and upregulated major satellites expression. Spatially, derepressed PCH aberrantly associates with nucleoli accumulating severe morphological defects during S/G2 cell cycle progression. Hp1α/ß deficiency reduces cell proliferation, ribosomal RNA biosynthesis and mobility of Nucleophosmin, a major nucleolar component. Nucleolar integrity and function require HP1α/ß proteins to be recruited to H3K9me3-marked PCH and their ability to dimerize. Correspondingly, ESCs deficient for both Suv39h1/2 H3K9 HMTs display similar nucleolar defects. In contrast, Suv4-20h1/2 mutant ESCs lacking H4K20me3 at PCH do not. Suv39h1/2 and Hp1α/ß deficiency-induced nucleolar defects are reminiscent of those defining human ribosomopathy disorders. Our results reveal a novel role for SUV39H/HP1-marked repressive constitutive heterochromatin in regulating integrity, function and physiology of nucleoli.

Mechanisms of maternal intergenerational epigenetic inheritance.

Mammalian embryos are formed by fusion of eggs and sperm. Here we provide an integrated overview of the major dynamic changes in transcriptional processes, chromatin composition and 3D organization which the maternal and paternal genomes undergo during oocyte and early embryonic development in mice. We derive mechanistic insights into molecular hierarchies and crosstalk between the various chromatin-associated processes that define three distinct types of maternal epigenetic memory states in oocytes to support pre-implantation and post-implantation development. Firstly, H3 lysine 4 trimethylation (H3K4me3) licenses early embryonic transcription by counteracting repressive H3K9 methylation. Secondly, an extensive interplay between transcription, H3K36me3 and H3K4 demethylation drives de novo DNA methylation required for embryogenesis. Thirdly, maternally inherited Polycomb-mediated H3K27me3 is a potent regulator of post-implantation development. Additionally, Polycomb Group proteins and H3K4me3 regulate 3D organization in oocytes and embryos.

SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin.

Chromatin integrity is essential for cellular homeostasis. Polycomb group proteins modulate chromatin states and transcriptionally repress developmental genes to maintain cell identity. They also repress repetitive sequences such as major satellites and constitute an alternative state of pericentromeric constitutive heterochromatin at paternal chromosomes (pat-PCH) in mouse pre-implantation embryos. Remarkably, pat-PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2-modified CBX2-containing Polycomb Repressive Complex 1 (PRC1) recruits the H3.3-specific chaperone DAXX to pat-PCH, enabling H3.3 incorporation at these loci. Deficiency of Daxx or PRC1 components Ring1 and Rnf2 abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis-segregation. Complementation assays show that DAXX-mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical role for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development.

Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins.

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased imaging time intervals - addressing major concerns in the field of volumetric embryo imaging.