Xiaoyuan Song

Age-related decline in male fertility, including diminished sperm quality, poses significant risks to offspring health and is exacerbated by modern trends toward delayed parenthood. Focusing on the hypothalamus–pituitary–testis axis, which governs hormonal control of male reproduction, we identified Synage , a long noncoding RNA expressed in the hypothalamic medial preoptic area (mPOA). We found that Synage expression in gonadotropin-releasing hormone (GnRH) neurons decreases with age, correlating with reduced sperm quality and male fertility. mPOA-targeted Synage _B (brain-specific isoform) supplementation increases serum testosterone and improves sperm count and progressive motility in 12-mo-old mice. Conversely, conditional deletion of Synage in GnRH neurons in young mice reduces GnRH neuron firing, decreases serum LH and testosterone levels, impaires sperm quality, and reduces male fertility. Mechanistically, Synage interacts with the heat shock protein HSPA8 and stabilizes the HSPA8–KCHIP4 interaction. Synage loss increases KCHIP4 abundance and its association with the Kv4.2 channel, promoting Kv4.2 membrane recruitment and enhancing A-type potassium currents ( I _A ), thereby reducing GnRH neuronal excitability. Consistent with this model, GnRH neurons from 12-mo-old mice also exhibit reduced excitability. Together, these findings support a role for age-dependent Synage loss in reproductive decline, at least in part through dysregulation of the HSPA8–KCHIP4–Kv4.2 pathway and reduced GnRH neuron excitability. These findings identify Synage as a candidate regulator of reproductive aging and motivate further investigation of its translational potential.

Chromatin interactions between cis-regulatory elements, especially enhancer–promoter interactions, are known to regulate the spatiotemporal dynamics of gene expression. Chromosome conformation capture (3C)-based methods can detect these interactions. Nicking enzyme-assisted open chromatin interaction capture (NicE-C) is a method we developed that leverages nicking enzyme-mediated open chromatin profiling with chromosome conformation capture to identify chromatin interactions specifically within open chromatin regions. This approach offers high efficiency, high resolution, low sequencing depth, and low requirements for starting material. Here, we describe a detailed step-by-step protocol for NicE-C.

Spermatogenesis is a complex differentiation process requiring precise spatiotemporal gene regulation, yet the functions of most testis-enriched long noncoding RNAs (lncRNAs) remain elusive. Here, we identify 1700008K24Rik as a pachytene-specific lncRNA that is essential for mouse spermatogenesis. Reanalysis of single-cell RNA-seq reveals that 1700008K24Rik expression is minimal in spermatogonia and preleptotene cells, sharply increases during the pachytene stage, and declines after meiosis, precisely coincides with the wave of pachytene piRNA production. In vivo knockdown of 1700008K24Rik in mouse testes via AAV9-shRNA severely disrupts spermatogenesis, causing a reduction in testis size, seminiferous epithelium thickness, and epididymal sperm count, suggesting its physiological necessity. Mechanistically, we demonstrate that 1700008K24Rik functions as a bona fide piRNA precursor: it harbors numerous piRNA sequences, and its overexpression in GC-2 spd(ts) cells specifically upregulates corresponding overlapping piRNAs. This processing relies on the core piRNA machinery, and co-overexpression of A-MYB, together with MOV10L1 or MIWI, promotes piRNA production. Notably, this piRNA precursor function is evolutionarily conserved, as the human homolog TDRG1, which is linked to asthenozoospermia, also generates piRNAs when expressed in GC-2 spd(ts) cells. Transcriptomic analysis following 1700008K24Rik overexpression in GC-2 spd(ts) cells reveals dysregulation of genes involved in metabolism and signaling, with several spermatogenesis-related genes (e.g., Msh5, Stard6) predicted as direct targets of 1700008K24Rik-derived piRNAs. Our study identifies 1700008K24Rik as a pachytene-specific lncRNA essential for male fertility and demonstrates that it functions as a conserved piRNA precursor. These findings provide mechanistic insight into how an individual lncRNA contributes to the piRNA pathway and suggest that it may operate through dual mechanisms in mammalian spermatogenesis.

Three-dimensional (3D) genome architecture shapes gene regulation, yet the folding principles of compact unicellular genomes remain unclear. Among unicellular eukaryotes, the ciliate Tetrahymena thermophila provides a distinctive model, harboring a transcriptionally active somatic macronucleus (MAC) with a genome fragmented into gene-dense minichromosomes and a silent, intact germline micronucleus. To delineate macronuclear chromatin organization, we integrated nucleosome-resolution Micro-C, ATAC-seq, and RNA-seq across the Tetrahymena life cycle. We find that macronuclear chromosomes form chromosome-sized interaction domains rather than canonical A/B compartments or internal TAD-like hierarchical structures. Each macronuclear chromosome behaves as a telomere-bounded structural unit organized by two major features: Highly accessible telomere-capped ends form stable end–end interaction hubs, and promoter-proximal open chromatin sites anchor long-range internal promoter-centered loops whose strength correlates with transcriptional activity. During conjugation, the sexual life cycle of Tetrahymena , long-range internal loops, and promoter–promoter contacts are transiently diminished and subsequently restored in later conjugation stages, whereas chromosome end–end contacts remain relatively stable. A similar architecture is observed in the related ciliate Tetrahymena pyriformis , indicating conservation within the genus. Together, our results define a compact, end-anchored, and promoter-centric genome-folding strategy that organizes a fragmented, gene-dense, transcriptionally active genome without the canonical compartment/TAD hierarchy seen in metazoan genomes. These findings expand the known repertoire of eukaryotic 3D genome architectures and suggest that promoter-associated transcription hubs can evolve independently in divergent eukaryotic lineages.

Chromatin accessibility is a key determinant of transcriptional regulation across eukaryotes, yet its organization and functional significance in ciliated protists remain poorly defined. Here, we optimized ATAC-seq to delineate the chromatin landscape in the transcriptionally active macronucleus of Tetrahymena thermophila. Our analyses reveal that accessible chromatin is predominantly enriched upstream of transcription start sites (TSSs), in stark contrast to the promoter-proximal accessibility observed in human embryonic stem cells. Dynamic profiling across different life cycle stages of T. thermophila (vegetative growth, starvation, conjugation, and refeeding) uncovers coordinated shifts in chromatin architecture that closely mirror extensive transcriptional reprogramming. Moreover, transcriptional inhibition by flavopiridol reduces chromatin openness upstream of TSSs while enhancing nucleosome organization downstream of TSSs, underscoring the direct impact of transcription on shaping chromatin structure. Comparative analyses across 13 eukaryotic species further indicate that upstream-biased accessibility is an ancestral trait in unicellular eukaryotes, whereas higher vertebrates have evolved more complex promoter architectures. These findings not only establish T. thermophila as a powerful model for dissecting chromatin-dependent gene regulation but also provide novel insights into the evolutionary divergence of chromatin organization across the eukaryotic lineage.

DNA methylation is a critical epigenetic modification that functions in diverse biological processes. High-throughput sequencing has revealed substantial variations in DNA methylation patterns across species. While extensively studied in vertebrates, knowledge of invertebrate DNA methylation remains limited. We examined the DNA methylation landscape of the ancient-looking marine mollusk, Nautilus pompilius, a “living fossil”. We identified key DNA methyltransferase genes (DNMT1 and DNMT3) by homologous searches against the N. pompilius genome and employed whole-genome bisulfite sequencing (WGBS) to characterize methylation patterns. Despite generally similar gene body methylation (gbM) and genome-wide methylation levels to other invertebrates, the N. pompilius exhibits a characteristic low methylation bias specifically in the promoter and first exon regions. N. pompilius has a typical gene body methylation pattern. Both promoter and gene body methylation may work together to regulate gene expression in N. pompilius. Our research provides valuable information to the ever-expanding methylation map of invertebrates.

[This corrects the article DOI: 10.1016/j.ncrna.2022.12.004.].

Lung cancer is a major cause accounting for cancer-related mortalities, with lung adenocarcinoma (LUAD) being the most prevalent subtype. Given the high clinical and cellular heterogeneities of LUAD, accurate diagnosis and prognosis are crucial to avoid overdiagnosis and overtreatment. Taking full advantage of scRNA-Seq data to resolve the tumor heterogeneities, we explored the overall landscape of LUAD microenvironment. Utilizing the stage-specific tumor cell markers, we have developed highly accurate diagnostic and prognostic models with elevated sensitivity and specificity. The diagnostic model, developed through random forest algorithms with a thirteen-gene signature, achieved an accuracy of 96.4% and an AUC of 0.993. These metrics were further demonstrated by benchmarking with available models and scoring systems in independent cohorts. Concurrently, the prognostic model, formulated via Cox regression with a six-gene signature, effectively predicted overall survival, with elevated risk scores associated with increased fractions of cancer-associated fibroblasts, and higher likelihood of immune escape and T-cell exclusion. Subsequently, two nomograms were developed to predict survival and drug responses, facilitating their integration into clinical practice. Overall, this study underscores the potential of our models for efficient, rapid, and cost-effective diagnosis and prognosis of LUAD, adaptable to multiple expression profiling platforms and quantification methods.

Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.