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The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or “tRFs,” are an emerging class of regulatory RNAs with poorly understood functions. We recently identified a role for one specific tRF—5′ tRF-Gly-GCC, or tRF-GG—as a repressor of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs—snoRNAs, scaRNAs, and snRNAs—that are dependent on Cajal bodies for stability and activity. Among these noncoding RNAs, regulation of the U7 snRNA by tRF-GG modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, on activity of a histone 3′ UTR reporter, and ultimately on MERVL regulation could all be suppressed by manipulating U7 RNA levels. We additionally show that the related RNA-binding proteins hnRNPF and hnRNPH bind directly to tRF-GG, and are required for Cajal body biogenesis, positioning these proteins as strong candidates for effectors of tRF-GG function in vivo. Together, our data reveal a conserved mechanism for 5′ tRNA fragment control of noncoding RNA biogenesis and, consequently, global chromatin organization.

The X-linked geneRlimplays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemicRlimknockout (KO) die around implantation, maleRlimKO mice appear healthy and are fertile. Here, we report an important role forRlimin testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of theRlimgene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditionalRlimcKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions ofRlimin male reproduction specifically in round spermatids during spermiogenesis.

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

SummaryThe X-linked geneRlimplays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemicRlimknockout (KO) die around implantation, maleRlimKO mice appear healthy and are fertile, raising questions as to the pressures drivingRlimgene selection during evolution. Here we report an important role forRlimin testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of theRlimgene leads to lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility andin vitrofertilization rates. Targeting the conditionalRlimcKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions ofRlimin male reproduction specifically in round spermatids during spermiogenesis with likely evolutionary implications.

After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.

Inheritance of genomic DNA underlies the vast majority of biological inheritance, yet it has been clear for decades that additional epigenetic information can be passed on to future generations. Here, we review major model systems for transgenerational epigenetic inheritance via the germline in multicellular organisms. In addition to surveying examples of epivariation that may arise stochastically or in response to unknown stimuli, we also discuss the induction of heritable epigenetic changes by genetic or environmental perturbations. Mechanistically, we discuss the increasingly well-understood molecular pathways responsible for epigenetic inheritance, with a focus on the unusual features of the germline epigenome.

SUMMARYMajor cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small cell populations within developing embryos. To understand how TFs regulate cell fate it is important to identify their genomic binding sites in these populations. However, current methods cannot profile TFs genome-wide at or near the single cell level. Here we adapt the CUT&RUN method to profile chromatin proteins in low cell numbers, mapping TF-DNA interactions in single cells and individual pre-implantation embryos for the first time. Using this method, we demonstrate that the pluripotency TF NANOG is significantly more dependent on the SWI/SNF family ATPase BRG1 for association with its genomic targets in vivo than in cultured cells—a finding that could not have been made using traditional approaches. Ultra-low input CUT&RUN (uliCUT&RUN) enables interrogation of TF binding from low cell numbers, with broad applicability to rare cell populations of importance in development or disease.

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI is dependent on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and -independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single-cell level. Here we adapt the cleavage under targets and release using nuclease (CUT&RUN) method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments suggest that only a fraction of TF binding sites are occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos. DOI: http://dx.doi.org/10.7554/eLife.19127.001

Offspring affected by sperm small RNAs Paternal dietary conditions in mammals influence the metabolic phenotypes of offspring. Although prior work suggests the involvement of epigenetic pathways, the mechanisms remains unclear. Two studies now show that altered paternal diet affects the level of small RNAs in mouse sperm. Chen et al. injected sperm transfer RNA (tRNA) fragments from males that had been kept on a high-fat diet into normal oocytes. The progeny displayed metabolic disorders and concomitant alteration of genes in metabolic pathways. Sharma et al. observed the biogenesis and function of small tRNA-derived fragments during sperm maturation. Further understanding of the mechanisms by which progeny are affected by parental exposure may affect human diseases such as diet-induced metabolic disorders. Science , this issue p. 397 , p. 391

Early embryonic development is characterized by dramatic changes in cell potency and chromatin organization. The role of histone variants in the context of chromatin remodeling during embryogenesis remains under investigated. In particular, the nuclear distribution of the histone variant H2A.Z and its modifications have not been examined. Here we investigated the dynamics of acetylation of H2A.Z and two other active chromatin marks, H3K9ac and H3K36me3, throughout murine and bovine pre-implantation development. We show that H2A.Z distribution is dynamic during the earliest stages of mouse development, with protein levels significantly varying across stages and lowest at the 2-cell stage. When present, H2A.Z localizes preferentially to euchromatin at all stages analyzed. H2A.Z is acetylated in pre-implantation blastomeres and is preferentially localized to euchromatin, in line with the known role of H2A.Zac in transcriptional activation. Interestingly, however, H2A.Zac is undetectable in mouse embryos at the 2-cell stage, the time of major embryonic genome activation (EGA). Similarly, H3K36me3 is present exclusively in the maternal chromatin immediately after fertilization but becomes undetectable in interphase nuclei at the 2-cell stage, suggesting uncoupling of these active marks with global embryonic transcription activation. In bovine embryos, which undergo EGA at the 8-cell stage, H2A.Zac can be detected in zygotes, 4-, 8- and 16-cell stage embryos as well as in blastocysts, indicating that the dynamics of H2A.Zac is not conserved in mammals. In contrast, H3K36me3 displays mostly undetectable and heterogeneous localization pattern throughout bovine pre-implantation development. Thus, our results suggest that 'canonical' active chromatin marks exhibit a dynamic behavior in embryonic nuclei, which is both stage- and species-specific. We hypothesize that chromatin of early embryonic nuclei is subject to fine-tuning through differential acquisition of histone marks, allowing for proper chromatin remodeling and developmental progression in a species-specific fashion.

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 addition to the well-studied epigenetic mechanisms associated with DNA methylation and histone modifications, histone variants have emerged as major regulators of chromatin activity. Apart from the major core histones, whose synthesis and incorporation into chromatin is linked to the S-phase of the cell cycle, histone ʻvariantsʼ are synthesised and incorporated into chromatin independently of DNA synthesis. These replacement histones confer distinct properties to nucleosomes and appear to be involved in important epigenetic processes. A significant role for histone variants in specialised chromatin signatures after fertilisation has emerged in the recent years. Here we review our knowledge on the involvement and the function of histone variants during the reprogramming phase occurring after mammalian fertilisation in vivo. We postulate that addressing the reprogramming mechanisms in its natural context, where this process occurs with a high efficiency to give rise to a new developmental programme, will help us to understand how we can modulate cell plasticity in induced and experimental models. Although there is still much to learn on how specific histone variants regulate reprogramming mechanistically, histone variants provide a remarkably versatile and exquisitely powerful way of regulating chromatin function in different biological contexts. Thus, the usage of histone variants provides an extra layer of regulation to the complexity of the reprogramming process.

The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.

Producing competent gametes is essential for transmitting genetic information throughout generations. Spermatogenesis is a unique example of rearrangements of genome packaging to ensure fertilization. After meiosis, spermatids undergo drastic morphological changes, perhaps the most dramatic ones occurring in their nuclei, including the transition into a protamine-packaged genome. In this issue of Genes & Development, Montellier and colleagues (pp. 1680–1692) shed new light on the molecular mechanisms regulating this transition by ascribing for the first time a function to a histone variant, TH2B, in the regulation of this process.