PIWI-interacting RNAs (piRNAs) are germline-specific small RNAs that form effector complexes with PIWI proteins to preserve genomic integrity by repressing transposable elements (TEs). Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets via heterochromatin formation characterized by H3K9me3 marks and the linker histone H1. Recently, we showed that Piwi forms a complex with Nxf2, a nuclear RNA export factor variant. Here, we performed proteomics analysis and Lamin DamID-seq and identified that Piwi and Nxf2 localize chromatin regions that code piRNA target TEs to the nuclear periphery. Furthermore, Hi-C analysis revealed that the depletion of Piwi or Nxf2 results in decreased intra-TAD interactions at those regions. Ectopic targeting of Nxf2 indicated that the regulation initiates by co‐transcriptional repression of the target reporter coupling with the removal of active histone marks and nuclear periphery localization. Continuous silencing involves the increase of H3K9me3 marks and H1 and the decrease of intra-TAD interactions. These results suggest that Piwi–piRNA complexes promote heterochromatin formation by causing step-wise changes in nuclear architecture.

Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed. They differ from mRNAs in their production, structure and turnover, and thereby have unique cellular functions and potential biomedical applications. However, individual circRNA functions still remain poorly understood due to inadequate methods, such as RNAi and genome engineering, in distinguishing circRNAs from linear cognate mRNAs with overlapped exons. In this talk, I will discuss our recent work that has established the programable RNA-guided, RNA-targeting CRISPR-Cas13, RfxCas13d, as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.

Single-cell analysis is a valuable tool to dissect cellular heterogeneity in complex systems. We used single-cell RNA sequencing to determine the cell-type composition of all major mouse and human organs to construct cell landscapes for the mammalian systems. We revealed single-cell hierarchies for many tissues that have not been well characterised. We established a cell mapping pipeline that helps to define mammalian cell identity. Finally, we performed a single-cell comparative analysis of landscapes from both human and mouse to reveal the conserved genetic networks. In the mammalian systems, stem and progenitor cells exhibt strong transcriptomic stochasticity, while the differentiated cells are more distinct.

Transcriptional bursting is emerging as a pervasive feature of gene expression conserved across species. It is thought to arise from consecutive recruitment and release of multiple RNA polymerase II complexes followed by a refractory period, resulting in successive bursts of de novo transcription. This phenomenon was directly visualized more than 40 years ago in electron micrographs of chromatin spreads in early Drosophila embryos, in which active transcription units are separated by internal fiber-free gaps (McKnight and Miller, 1979). Recent quantitative imaging studies suggested that enhancers regulate the frequency of transcriptional bursting. However, considerably less is known about the role of core promoters in the process. My lab uses quantitative live-imaging approaches to visualize transcription dynamics in early Drosophila embryos. In this talk, I will discuss mechanisms and functions of burst regulation during embryogenesis.

Phase separations involving biomolecular liquids are emerging as a fundamental mechanism of intracellular organization. Liquid-liquid phase separation (LLPS) of biomolecules compartmentalizes intracellular space into multiple coexisting condensates to facilitate spatio-temporal regulation of biological processes. The list of intracellular structures driven to form by LLPS is growing rapidly, and include canonical membrane-less organelles such as nucleoli, nuclear speckles and stress granules, as well as recently described structures such as transcriptional and synaptic clusters. In an effort to investigate biophysical mechanisms of intracellular condensation, I have been developing technologies to navigate intracellular phase space. In this talk, I will first discuss a strategy to control phase separation in living cells, in particular within nuclear space. We have developed an optogenetics-based technology where genome targeting capacity of the Cas9 system is combined with the optogenetic controllability. This approach enables targeted liquid-liquid phase separation at a specific genomic locus, and suggests the role of liquid condensates of transcriptional regulators in shaping genome structure. I will also briefly discuss an imaging technique enabling quantitative measurement of biomolecular concentrations in condensates.

Both microRNAs (miRNAs) and long noncoding RNAs have been shown to play important roles in regulating the self-renewal and differentiation of pluripotent stem cells. In this presentation, I will briefly summarize our work in understanding the function of miRNAs in mouse embryonic stem cells (ESCs). In addition, I will introduce our work on developing miRNA-inducible CRISPR-Cas9 platform (MICR). Using MICR, we successfully tracked the differentiation status of stem cells and identified a novel heterogeneous state defined by the activity of miR-21. Finally, I will present our recent work on elucidating the function of long noncoding RNAs in ESCs.

Imbalanced allelic gene expression has been widely observed and plays important roles in mammalian development and disease progress. How it is established, regulated and functioning, remains to be explored. Here by combining the haploid stem cell and genome editing technologies, we identified the imprinting-related factors necessary for crossing same-sex reproduction barriers in mammals, and then established a unisexual reproduction method for producing normally growing bimaternal mice and live bipaternal mice. We also identified the aberrantly expressed non-canonical imprinting genes in cloned mice, which can significantly increase the animal cloning efficiency up to 14% after being rescued.

The germ-cell lineage ensures the creation of new individuals, perpetuating/diversifying the genetic and epigenetic information across the generations. We have been investigating the mechanism for germ-cell development, and have shown that mouse embryonic stem cells (mESCs)/induced pluripotent stem cells (miPSCs) are induced into primordial germ cell-like cells (mPGCLCs) with a robust capacity both for spermatogenesis and oogenesis and for contributing to offspring. These works have served as a basis for exploring the mechanism of key events during germ-cell development such as epigenetic reprogramming/programming, sex determination, and meiotic entry.

By investigating the development of cynomolgus monkeys, we have defined a developmental coordinate of the spectrum of pluripotency among mice, monkeys, and humans, and have identified the origin of the germ-cell lineage in cynomolgus monkeys in the amnion. Accordingly, we have succeeded in inducing human iPSCs (hiPSCs) with a primed pluripotency into human PGCLCs (hPGCLCs) and then into oogonia and early oocytes with appropriate epigenetic reprogramming. More recently, we have shown that hPGCLCs can be propagated to ~1,000,000-fold over a period of 4 months under a defined condition. These studies serve as a key foundation for human in vitro gametogenesis.