Cellular plasticity is essential for early embryonic cells. Unlike pluripotent cells, which form embryonic tissues, totipotent cells can generate a complete organism including embryonic and extraembryonic tissues. Cells resembling 2-cell-stage embryos (2C-like cells) arise at very low frequency in embryonic stem (ES) cell cultures. Although induced reprogramming to pluripotency is well established, totipotent cells remain poorly characterized, and whether reprogramming to totipotency is possible is unknown. We show that mouse 2C-like cells can be induced in vitro through downregulation of the chromatin-assembly activity of CAF-1. Endogenous retroviruses and genes specific to 2-cell embryos are the highest-upregulated genes upon CAF-1 knockdown. Emerging 2C-like cells exhibit molecular characteristics of 2-cell embryos and higher reprogrammability than ES cells upon nuclear transfer. Our results suggest that early embryonic-like cells can be induced by modulating chromatin assembly and that atypical histone deposition may trigger the emergence of totipotent cells.

In mammals, oocyte fertilisation by sperm initiates development. This is followed by epigenetic reprogramming of both parental genomes, which involves de-novo establishment of chromatin domains. In the mouse embryo, methylation of histone H3 1 establishes an epigenetic asymmetry and is predominant in the maternal pronucleus [2][3][4][5] . However, the role of (i) differential incorporation of histone H3 variants in the parental chromatin and of (ii) modified residues within specific histone variants has not been addressed. Here we show that the histone variant H3.3, and in particular lysine 27, is required for the establishment of heterochromatin in the mouse embryo. H3.3 localises to paternal pericentromeric chromatin during S-phase at the time of transcription of pericentromeric repeats. Mutation of H3.3K27, but not H3.1K27, results in aberrant accumulation of pericentromeric transcripts, HP1 mislocalisation, dysfunctional chromosome segregation and developmental arrest. This phenotype is rescued by injection of dsRNA derived from pericentromeric transcripts, indicating a functional link between H3.3K27 and silencing of such regions via an RNAi pathway. Our work demonstrates a role for a modifiable residue within a histone variant-specific context during reprogramming and identifies a novel function for mammalian H3.3 in the initial formation of dsRNA-dependent heterochromatin.Fertilisation of the oocyte by the sperm constitutes the first event of embryogenesis and results in the formation of the zygote. The creation of such a totipotent cell from two differentiated ones involves epigenetic reprogramming of the parental genomes. Throughout the complete first cell cycle, the male and female pronuclei behave as two distinct sets of chromatin that coexist as separate nuclear entities. Both pronuclei evolve differently, showing different chromatin signatures, histone marks and replication and transcription timing 2-4, 6, 7 . How the chromatin is assembled, specified and reprogrammed after fertilisation remains a central question in biology. Before fertilisation, the sperm nucleus is condensed six-fold higher than a somatic cell nucleus, and most of its histones are replaced by protamines 8 . Immediately following the entry of the sperm nucleus into the oocyte cytoplasm, protamines are removed from the sperm and replaced by maternally provided Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts histones 9 . In mice, histones H3 and H4 are translated from maternal mRNAs stored in the oocyte, whereas histones H2A and H2B are already present as proteins in the oocyte 10 .Apart from the canonical histones, which are synthesised exclusively during S-phase, histone variants can be incorporated into chromatin throughout the cell cycle. The replication-independent H3 variant H3.3 is preferentially incorporated into the male pronucleus following fertilisation 5 , while the replication-dependent H3.1/2 variants are found predominantly in the female pronucleus 11 , suggesting a role of differe...

The spatiotemporal organization of genomes in the nucleus is an emerging key player to regulate genome function. Live imaging of nuclear organization dynamics would be a breakthrough toward uncovering the functional relevance and mechanisms regulating genome architecture. Here, we used transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences. We established TALE-mediated genome visualization (TGV) to label genomic sequences and follow nuclear positioning and chromatin dynamics in cultured mouse cells and in the living organism. TGV is highly specific, thus allowing differential labeling of parental chromosomes by distinguishing between single-nucleotide polymorphisms (SNPs). Our findings provide a framework to address the function of genome architecture through visualization of nuclear dynamics in vivo.

Constitutive heterochromatin is typically defined by high levels of DNA methylation and H3 lysine 9 trimethylation (H3K9Me3), whereas facultative heterochromatin displays DNA hypomethylation and high H3 lysine 27 trimethylation (H3K27Me3). The two chromatin types generally do not coexist at the same loci, suggesting mutual exclusivity. During development or in cancer, pericentromeric regions can adopt either epigenetic state, but the switching mechanism is unknown. We used a quantitative locus purification method to characterize changes in pericentromeric chromatin-associated proteins in mouse embryonic stem cells deficient for either the methyltransferases required for DNA methylation or H3K9Me3. DNA methylation controls heterochromatin architecture and inhibits Polycomb recruitment. BEND3, a protein enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, allows Polycomb recruitment and H3K27Me3, resulting in a redundant pathway to generate repressive chromatin. This suggests that BEND3 is a key factor in mediating a switch from constitutive to facultative heterochromatin.

How a more plastic chromatin state is maintained and reversed during development is unknown. Heterochromatin-mediated silencing of repetitive elements occurs in differentiated cells. Here, we used repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. RNA sequencing throughout early mouse embryogenesis revealed that repetitive-element expression is dynamic and stage specific, with most repetitive elements becoming repressed before implantation. We show that LINE-1 and IAP retrotransposons become reactivated from both parental genomes after fertilization. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicates that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. Furthermore, short LINE-1 RNAs regulate LINE-1 transcription in vivo. Our data indicate that reprogramming after mammalian fertilization comprises a robust transcriptional activation of retrotransposons and that repetitive elements are initially regulated through RNA.

PR-Set7 is the sole monomethyltransferase responsible for H4K20 monomethylation (H4K20me1) that is the substrate for further methylation by Suv4-20h1/h2. PR-Set7 is required for proper cell cycle progression and is subject to degradation by the CRL4 Cdt2 ubiquitin ligase complex as a function of the cell cycle and DNA damage. This report demonstrates that PR-Set7 is an important downstream effector of CRL4Cdt2 function during origin of DNA replication licensing, dependent on Suv4-20h1/2 activity. Aberrant rereplication correlates with decreased levels of H4K20me1 and increased levels of H4K20 trimethylation (H4K20me3). Expression of a degradationresistant PR-Set7 mutant in the mouse embryo that is normally devoid of Suv4-20 does not compromise development or cell cycle progression unless Suv4-20h is coexpressed. PR-Set7 targeting to an artificial locus results in recruitment of the origin recognition complex (ORC) in a manner dependent on Suv4-20h and H4K20me3. Consistent with this, H4K20 methylation status plays a direct role in recruiting ORC through the binding properties of ORC1 and ORCA/LRWD1. Thus, coordinating the status of H4K20 methylation is pivotal for the proper selection of DNA replication origins in higher eukaryotes.

Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a modification that is enriched at pericentric heterochromatin and associated with repeat sequences and transcriptionally inactive genomic regions. We show that this new mark is dynamic during the two main epigenetic reprogramming events in mammals. In primordial germ cells, H3K64me3 is present at the time of specification, but it disappears transiently during reprogramming. In early mouse embryos, it is inherited exclusively maternally; subsequently, the modification is rapidly removed, suggesting an important role for H3K64me3 turnover in development. Taken together, our findings establish H3K64me3 as a previously uncharacterized histone modification that is preferentially localized to repressive chromatin. We hypothesize that H3K64me3 helps to 'secure' nucleosomes, and perhaps the surrounding chromatin, in an appropriately repressed state during development.

Development of the germline requires consecutive differentiation events. Regulation of these has been associated with germ cell-specific and pluripotency-associated transcription factors, but the role of general transcription factors (GTFs) remains elusive. TATA-binding protein (TBP) is a GTF involved in transcription by all RNA polymerases. During ovarian folliculogenesis in mice the vertebrate-specific member of the TBP family, TBP2/ TRF3, is expressed exclusively in oocytes. To determine TBP2 function in vivo, we generated TBP2-deficient mice. We found that Tbp2 À/À mice are viable with no apparent phenotype. However, females lacking TBP2 are sterile due to defective folliculogenesis, altered chromatin organization, and transcriptional misregulation of key oocyte-specific genes. TBP2 binds to promoters of misregulated genes, suggesting that TBP2 directly regulates their expression. In contrast, TBP ablation in the female germline results in normal ovulation and fertilization, indicating that in these cells TBP is dispensable. We demonstrate that TBP2 is essential for the differentiation of female germ cells, and show the mutually exclusive functions of these key core promoter-binding factors, TBP and TBP2, in the mouse.[Keywords: TBPL2; TRF3; TBP; Zp3; RNA Polymerase II; ovary] Supplemental material is available at http://www.genesdev.org.