Xiao-Ou Zhang

Transposable elements (TEs) are temporally activated during mammalian early embryogenesis and are closely linked to zygotic genome activation (ZGA), yet their nascent transcriptional dynamics remain incompletely resolved owing to limited sensitivity and maternal RNA background. Here, we develop single-cell TimeLapse sequencing (scTL-seq), enabling allele-resolved detection of nascent RNA in individual embryos. Applying scTL-seq to mouse ZGA, we generate a refined transcriptional atlas of genes and TEs and identify the pronuclear (PN) stage activated genes and TEs. We found that transcription at PN stage is largely contributed by the maternal genome and enriched for genes highly transcribed in germinal vesicle oocytes. Similarly, Mouse Transcripts type-A (MTA) elements are transcribed in oocytes and highly active in PN stage. MTA knockdown disrupts zygotic gene activation and dysregulates hundreds of TE subfamilies, resulting in 1-cell embryo arrest. Using nascent transcripts, we further identify 662 TE-driven chimeric transcripts and show that TEs are essential for regulating key developmental genes, including Nfya, Setd2 and Tead4. Finally, we identify maternal transcription factors, including NOBOX, as potential regulators of MTA activation. Together, our study provides a high-resolution nascent transcriptome landscape of mouse ZGA and reveals vital regulatory roles of MTA retrotransposon in orchestrating early embryonic development.

Small nuclear RNAs (snRNAs) are essential components of the spliceosome and are encoded by large, multicopy gene families. However, their genome-wide identification and quantification have remained challenging due to high sequence similarity among family members. To address this, we utilized RAMPAGE (Rapid Amplification of cDNA Ends) data from the ENCODE project to comprehensively profile nascent transcription of spliceosomal snRNAs across 115 human biosamples. We identified 74 expressed snRNA variants, characterized by canonical promoter features including bidirectional transcription flanking a positioned nucleosome, active histone modifications, and evolutionary conservation- features largely absent from unexpressed variants. These transcriptional events were corroborated by total RNA-seq and Bru-seq data, yet the majority of these variants showed extremely low levels in small RNA-seq, indicating post-transcriptional bottlenecks for snRNA processing and maturation. Our findings reveal new layers of regulation in snRNA variant expression and suggest that selective post-transcriptional processing plays a critical role in shaping the functional snRNA repertoire and its contribution to splicing regulation.

One of the leading causes of human subfertility is the continuous decline in semen quality, contributing to a global fertility crisis. Over half of subfertile men suffer from asthenozoospermia and teratozoospermia, with mechanisms still largely unknown. Traditional small noncoding RNA sequencing (sncRNA-seq) primarily targets miRNAs, failing to capture the broader spectrum of small noncoding RNAs (sncRNAs), including abundant transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs). These sncRNAs possess complex RNA modifications and non-canonical terminal structures, impeding their accurate profiling. In this prospective cohort study, we addressed these limitations by combining our state-of-the-art PANDORA-seq with traditional sncRNA-seq, which generated the most comprehensive sncRNA landscape of human sperm from 25 participants with asthenozoospermia, teratozoospermia, or normozoospermia. PANDORA-seq significantly improved the annotation efficiency of sncRNAs and delivered a more detailed characterization for tsRNAs and rsRNAs, which were strongly correlated with key clinical indicators of sperm quality, thereby enhancing our understanding of the landscape of human sperm sncRNAome and its association with male subfertility. Importantly, machine learning with Lasso regression identified specific tsRNA/rsRNA signatures as highly effective clinical biomarkers (AUC ≥ 0.83) for predicting sperm abnormalities, offering significant improvements over World Health Organization-based semen quality assessments and novel insights for clinical diagnosis.

Translational control is vital during the maternal-to-zygotic transition (MZT), yet the landscape of embryonic transfer RNA (tRNA) pools has been difficult to explore. Here, we develop Optimized Reaction for Accurate Capture of Low-input Entities tRNA Sequencing (ORACLE-tRNAseq), enabling robust tRNA profiling from as few as five mouse oocytes. We map tRNA landscapes from oocyte to blastocyst, identifying a distinct transition to embryonic tRNA repertoires and upregulation of tRNA pseudogenes at the 4-cell stage. Integrated multi-omics analyses reveal that zygotic tRNA gene activation coincides with zygotic genome activation (ZGA) and correlates with H3K4me3 establishment and chromatin remodeling. By coupling ORACLE-tRNAseq with Ribo-seq, we demonstrate that embryonic tRNA anticodon pools coordinate with high translation-efficiency gene pools to preferentially establish the zygotic translation machinery, particularly from major ZGA onwards. Collectively, these findings provide a resource for understanding translational regulatory networks during early embryogenesis.

Fetal growth restriction (FGR), a major complication of pregnancy caused by placental insufficiency, substantially increases neonatal morbidity and mortality. The mechanisms by which disrupted cellular signaling at the maternal–fetal interface contributes to FGR remain poorly understood. We performed single-cell RNA sequencing to generate a high-resolution atlas of the maternal–fetal interface in monochorionic diamniotic twins with type III selective FGR, minimizing genetic and environmental confounders to reveal FGR-specific cellular alterations. Compared with controls, extravillous trophoblasts (EVTs) in FGR placentas exhibit differentiation arrest and enhanced senescence, with a distinct EVT subset co-expressing vascular-remodeling and hypoxia-adaptation genes undergoing abnormal expansion, indicative of a hypoxia–angiogenesis imbalance. Maternal vascular smooth muscle cells are enriched, whereas fetal vascular smooth muscle cells are depleted and show increased senescence. Notably, FGR placentas harbor abundant SPP1-high Hofbauer (HB) cells, with enhanced SPP1 signaling at the macrophage–trophoblast interface. Integrated single-cell analyses and in vitro co-culture assays indicate that HB-derived SPP1 is associated with suppressed trophoblast migratory and invasive programs, potentially contributing to trophoblast dysfunction and vascular abnormalities in FGR. Circulating SPP1 is elevated in mothers carrying FGR fetuses, highlighting its potential as a non-invasive biomarker. These observations are further supported by bulk transcriptomic analyses across 17 independent cohorts comprising 679 placental samples. Together, our study highlights a coordinated triad of trophoblast arrest, vascular maladaptation, and HB-derived SPP1 signaling that collectively contributes to placental dysfunction in FGR.

Transposable elements are abundant in the human genome and have been increasingly recognized as sources of alternative promoters. Yet, the extent of their transcriptional activity in human tissues and the features that govern their regulatory potential remain unclear. Here, we integrated high-resolution RAMPAGE data from 115 human biosamples to construct a comprehensive atlas of 26,056 transcription start sites derived from transposable elements. These sites contribute to tissue-specific gene expression, with a notable fraction originating from primate- and hominid-specific elements. Transposable element–derived transcription start sites exhibit focused, narrow-peak architectures enriched for TATA boxes and depleted of CpG islands. Phylogenetic analyses reveal a continuous gradient in promoter strength and transcriptional precision across transposable element subfamilies, with evolutionarily younger elements retaining intrinsic promoter motifs that drive focused and robust transcription, whereas older, more divergent elements exhibit broader initiation patterns and lower intrinsic activity. Together, these findings advance our understanding of how the evolution and preservation of promoter features shape the capacity of transposable elements to be exapted as functional promoters, potentially contributing to lineage-specific regulatory innovation in primates.

Alternative cleavage and polyadenylation (APA) in the 3′‐untranslated region (3′‐UTR) of mRNA produces transcripts with varied 3′‐UTR and plays a key role in development and organogenesis. This work characterizes 3′‐UTR APA using 85 high‐quality RNA‐seq datasets encompassing 12 tissue types and eight developmental stages of mouse fetuses. Results show that 46.4% of expressed genes undergo APA in a tissue‐specific manner. Changes in polyadenylation site (pAS) usage often operate beyond transcriptional control, revealing APA as an additional layer of gene regulation. Sequence analysis demonstrates that pAS selection, governed by polyadenylation signal strength and adenine preferences, is evolutionarily conserved between mice and humans. Intriguingly, brain tissues display complex 3′‐UTR APA dynamics during development, potentially regulated by RNA‐binding proteins such as Rbm38, potentially impacting 3′ UTR extension by restricting distal pAS usage. These APA events are associated with a depletion of conserved miRNA binding sites and an enrichment of transposable elements within alternative 3′ UTRs. To facilitate further research, this work develops APApedia (http://xozhanglab.com/apapedia/), a comprehensive database cataloging identified 3′‐UTR APA events, which serves as a valuable resource for the community to study APA in development and tissue‐specific regulation. This comprehensive resource aids in deciphering the functional implications of APA in mouse fetal development.

Transposons constitute ~45% of the human genome, driving gene evolution and contributing to disease, but their repetitive nature complicates the identification of new insertions. We present LOCATE (Long-read to Characterize All Transposable Elements), an algorithm using long-read sequencing to detect and assemble transposon insertions. LOCATE outperforms existing tools on simulated datasets and achieves the best performance in two previous benchmarks, as well as in a new benchmark we constructed using real biological datasets. Applying LOCATE to public datasets revealed that pre-existing Alu copies create two hotspots for Alu and LINE1 insertions: the A-rich linker and the poly(A) tail. We further observed a preference for self-insertions over non-self-insertions in Alu and LINE1, suggesting a "feedforward" transposition mechanism in which Alu and LINE1 RNA transcripts target the hotspots of their source copies to generate new insertions. LOCATE enhances our ability to study transposons and their role in genome dynamics.

Totipotent cells can differentiate into three lineages: the epiblast, primitive endoderm, and trophectoderm. Naturally, only early fertilized embryos possess totipotency, and they lose this ability as they develop. The expansion of stem cell differentiation potential has been a hot topic in developmental biology for years, particularly with respect to the generation totipotent‐like stem cells. Here, the study describes the establishment of totipotency in embryonic stem cells (ESCs) via the deletion of a single gene, Pdzk1 . Pdzk1 ‐knockout (KO) ESCs substantially contribute to the fetus, placenta, and yolk sac in chimera assays but can also self‐organize to form standard blastocyst‐like structures containing the three lineages efficiently; thus, they exhibit full developmental potential as early blastomeres. Single‐cell transcriptome and bulk RNA‐seq comprehensive analyses revealed that Pdzk1 ‐KO activates several lineage inducers ( C1qa , C1qb , Fgf5 , and Cdx2 ) to break down barriers between embryonic and extraembryonic tissues, making these lineages switch smoothly and resulting in a totipotent‐like state. This versatile and scalable system provides a robust experimental model for differentiation potency and cell fate studies.