Kwonho Hong

R-loops, three-stranded nucleic acid structures formed by an RNA-DNA hybrid, have emerged as important regulators of transcription and genome stability. Although advances in high-throughput sequencing have revealed widespread R-loop landscapes, platform-specific biases hinder the identification of conserved R-loops in specific cell types. Mouse embryonic stem cells, which are transcriptionally active, provide an ideal system for investigating the potential roles of stable R-loops in RNA biology. Here, we integrated 13 independent R-loop profiling datasets from four experimental platforms to define 27,950 Common R-loop regions in mouse embryonic stem cells and characterized their chromatin environment and associated biological functions. Common R-loop regions were reproducibly detected across methods and were preferentially localized to promoter-proximal and genic regions enriched in CpG islands. Genes associated with Common R-loops were highly and stably expressed, showing strong functional enrichment in RNA metabolic processes such as mRNA processing, RNA splicing, and ribonucleoprotein complex biogenesis. Chromatin state analysis revealed that Common R-loops are enriched in transcriptionally active and regulatory contexts. Sequence feature analysis further identified GC skew as a prominent signature of Common R-loops, particularly within transcribed chromatin states. Transcription factor motif analyses have identified distinct regulatory environments in Common R-loop regions, including pluripotency-associated OCT4-SOX2-TCF-NANOG motifs in enhancers, CTCF motifs in open chromatin, and YY1 motifs in promoters. Together, this study provides the first integrated analysis of conserved R-loop regions in mouse embryonic stem cells, revealing their preferential localization at regulatory loci linked to RNA metabolism and highlighting R-loops as structural and functional nodes in RNA biology.

R-loops, three-stranded nucleic acid structures formed by an RNA-DNA hybrid, have emerged as important regulators of transcription and genome stability. Although ad-vances in high-throughput sequencing have revealed widespread R-loop landscapes, platform-specific biases hinder the identification of conserved R-loops in specific cell types. Mouse embryonic stem cells, which are transcriptionally active, provide an ideal system for investigating the potential roles of stable R-loops in RNA biology. In this study, we integrated 13 independent R-loop profiling datasets from four experimental platforms to define 27,950 Common R-loop regions in mouse embryonic stem cells and characterized their chromatin environment and associated biological functions. Common R-loop regions were reproducibly detected across methods and were preferentially localized to pro-moter-proximal and genic regions enriched in CpG islands. Genes associated with Common R-loops were highly and stably expressed, showing strong functional en-richment in RNA metabolism process such as mRNA processing, RNA splicing, and ribonucleoprotein complex biogenesis. Chromatin state analysis revealed that Common R-loops are enriched in transcriptionally active and regulatory contexts. Transcription factor motif analyses have identified distinct regulatory environments in Common R-loop regions, including pluripotency-associated OCT4-SOX2-TCF-NANOG motifs in en-hancer, CTCF motifs in open chromatin, and YY1 motifs in promoter. Together, this study provided the first integrated analysis of conserved R-loop regions in mouse embryonic stem cells, revealing their preferential localization at regulatory loci linked to RNA metabolism and highlight R-loops as structural and functional nodes in RNA biology.

Background: Embryonic stem cells (ESCs) possess a distinctive cell cycle structure and chromatin landscape that necessitate precise coordination between transcription and DNA replication. The chromatin remodeler INO80 regulates both transcription and genome stability; however, its role in R-loop homeostasis in ESCs remains incompletely understood. R-loops, which are formed by RNA:DNA hybrids, play regulatory roles but can also induce replication stress and genomic instability if unregulated. Results: We investigated the effects of INO80 loss on R-loop dynamics, replication stress responses, and transcriptional regulation in mouse ESCs. INO80-deficient cells exhibited a global reduction in steady-state R-loops under normal conditions, with the most pronounced loss at promoters enriched for pluripotency-associated transcription factors. R-loop levels were restored under hydroxyurea-induced replication stress, indicating that INO80 is dispensable for stress-induced R-loop accumulation but is required for maintaining physiological R-loops. Cleavage Under Targets and Tagmentation and chromatin immunoprecipitation sequencing revealed that several R-loop regions overlap with INO80-binding sites and are linked to transcriptional activation and genome maintenance. Transcriptome analysis showed that INO80-stabilized R-loops are associated with genes involved in cell cycle progression, DNA repair, and chromatin organization, whereas INO80 loss results in altered transcriptional programs, G1 arrest, and increased apoptosis. These findings indicate that INO80 stabilizes functional R-loops that support transcriptional regulation and genomic integrity. Conclusions: Our study identifies a previously unrecognized role for INO80 in preserving functional R-loops in ESCs. By stabilizing promoter-associated R-loops and coordinating transcription during cell cycle progression, INO80 maintains both transcriptional regulation and genome stability. These findings underscore the importance of chromatin remodeling in maintaining R-loop homeostasis and suggest that INO80 dysfunction may have implications for stem cell identity and genomic integrity.

The extracellular matrix (ECM) supports spermatogonial stem cell (SSC) function by mimicking biochemical and structural features of the native niche. However, optimal feeder systems and ECM materials remain key limitations in porcine SSC (pSSC) cultures. We developed a porcine-derived ECM (pECM) from porcine feet and evaluated its effectiveness in supporting pSSC maintenance and proliferation under feeder-dependent conditions. We examined protein molecular weight distribution and pECM extract composition. Surface characterization was performed using scanning electron microscopy and atomic force microscopy. We compared pECM with conventional coatings, including gelatin and non-coated controls, using morphological analysis, WST-1 assay, cell cycle analysis, and gene/protein expression of SSC markers. pECM promoted larger, well-defined pSSC colonies and enhanced stemness-related marker expression, including PGP9.5, Thy-1, PLZF, GFRA1, NANOG, and VASA. Additionally, pECM facilitated active pSSC proliferation while suppressing feeder overgrowth, contributing to a stable and functional co-culture environment. Conversely, gelatin supported early feeder proliferation but led to growth saturation, whereas N/C showed delayed attachment and reduced viability. These findings suggest that pECM mimics the native SSC niche and improves pSSC culture. The dual function of pECM in regulating feeder behavior and enhancing pSSC maintenance highlights its potential as a biomaterial for species lacking established feeder-free protocols.

Objective: Spermatogenesis is a complex biological process that encompasses meiosis in spermatocytes and the dynamic epigenetic alterations that ensure the inheritance of genetic traits. CXXC finger protein 1 (CFP1, Cfp1, CXXC1, Cxxc1) is a critical component of the SET domain-containing 1A histone lysine methyltransferase complex that catalyzes histone H3K4 methylation and has a specific binding domain for unmethylated CpG DNA. However, our current understanding of CFP1’s role in the genome-wide regulation of DNA and H3K4 methylation remains limited.Methods: We performed genome-wide methylation analysis using reduced-representation bisulfite sequencing on spermatocytes isolated from Cfp1 knockout and wild-type mice. Promoter methylation changes were integrated with publicly available microarray and ChIP-seq data to identify genes regulated by CFP1.Results: CFP1 depletion led to significant alterations in DNA methylation, particularly in promoter regions of genes associated with meiosis, transcription regulation, and chromatin remodeling. A total of 21 genes were identified as direct targets of CFP1, exhibiting reduced promoter methylation and CFP1 binding.Conclusion: Our study findings contribute to elucidating the regulatory mechanisms of CFP1 in spermatocytes, providing valuable insights into the reproductive process and advancing our understanding of the underlying causes of infertility.

Objective: Expanded potential stem cells (EPSCs) are stem cells that can differentiate into embryonic and extraembryonic lineages, including extraembryonic endoderm and trophoblast lineages. Therefore, EPSCs have great potential in advancing regenerative medicine, elucidating disease mechanisms, and exploring early embryonic development. However, the generation and characterization of EPSCs in pigs have not been thoroughly explored. In this study, we successfully generated porcine EPSCs (pEPSCs).Methods: We reprogrammed porcine fetal fibroblasts (PFFs) using an integration-free method with Sendai virus vectors.Results: The resulting pEPSCs expressed key pluripotency markers and demonstrated the ability to differentiate between embryonic and extraembryonic lineages. Notably, reprogramming into pEPSCs was associated with a transformation of mitochondrial morphology from the elongated form observed in PFFs to a globular shape, reflecting potential alterations in energy metabolism. We observed significant remodeling of mitochondrial morphology and a subsequent shift towards glycolytic energy dependence during the reprogramming of PFFs into pEPSCs.Conclusion: Our findings provide valuable insights into the characteristics of EPSCs in pigs and highlight their potential applications in regenerative medicine, disease modeling, and emerging fields such as cell-based meat production.

Turner syndrome (TS) is an X monosomy-related disorder caused by X chromosome nondisjunction during embryonic development. Patients with TS have only one intact X chromosome, with the other either completely or partially lost. TS affects various tissues, including the liver, kidneys, brain, cardiovascular system, and ovaries. These abnormalities are suggested to involve an altered dosage of escape genes that evade X chromosome inactivation. However, the mechanisms and roles of these escape genes in the TS phenotype remain unclear. We hypothesized that the expression levels of escape genes differ between wild-type (WT) and TS cell lines. In this study, we generated induced pluripotent stem cell (iPSC) lines from WT and TS fibroblasts and examined the expression levels of escape genes in both undifferentiated fibroblasts and reprogrammed iPSCs from WT and TS samples. The reprogrammed WT and TS iPSCs exhibited general characteristics of pluripotency, including the expression of pluripotency markers and the potential to differentiate into all three germ layers. Forty-five escape genes were differentially expressed between the WT and TS cell lines. Among these, five genes (ATP7A, PHKA1, EBP, ZFX, and SMC1A) were suggested to be implicated in the TS phenotype. However, further studies using additional cell lines are necessary to clarify the correlation between TS and escape genes.