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Lee, D.-S.

Publications and source records attributed to Lee, D.-S..

15 recordsLinked to original sources
bioRxiv2026

RAP2.6 negatively regulates Arabidopsis submergence tolerance through GSNOR1-dependent redox and hormonal signaling

Group VII ETHYLENE RESPONSE FACTORs (ERFVIIs) are central regulators of plant responses to oxygen deprivation. However, the contributions of many other APETALA2 (AP2)/ERF transcription factors to hypoxia and flooding responses remain poorly understood. Here, we identify the group X ERF transcription factor RAP2.6 as a negative regulator of submergence tolerance in Arabidopsis thaliana. RAP2.6 expression declined under hypoxia, and plants overexpressing RAP2.6 exhibited reduced survival following dark submergence, whereas the rap2.6-1 loss-of-function mutant showed enhanced tolerance to submergence, hypoxia, and ethanol treatment. Enhanced tolerance of rap2.6-1 was associated with reduced membrane injury, maintenance of photosynthetic pigments and soluble sugars, and extensive metabolic remodeling. RAP2.6 overexpression altered nitric oxide (NO), S-nitrosothiol (SNO), and ethylene accumulation during submergence and was associated with greater oxidative damage and enhanced abscisic acid (ABA)-responsive gene expression. Genetic analyses using S-NITROSOGLUTATHIONE (GSNO) REDUCTASE 1 (GSNOR1) gain- and loss-of-function backgrounds demonstrated that GSNOR1-dependent GSNO homeostasis strongly modified RAP2.6-dependent submergence responses. Increased GSNOR1 activity suppressed the submergence-sensitive phenotype of RAP2.6-overexpressing plants, reduced nitro-oxidative stress, and restored carbohydrate reserves, whereas loss of GSNOR1 activity abolished the enhanced tolerance of rap2.6-1. GSNOR1 also modified RAP2.6-dependent expression of genes associated with oxygen sensing and ABA signaling, while its protective effect was not explained solely by changes in ethylene accumulation. Together, these findings identify RAP2.6 as a regulatory component of the low-oxygen response and reveal a functional interaction between RAP2.6 and GSNOR1-dependent redox signaling that coordinates plant hormonal, metabolic, and oxidative responses during submergence.

plant biology↗
bioRxiv2026

Integrative genetic and structural analyses reveal nitric oxide-driven regulation of ERFVII stability in Arabidopsis under low O2

Nitric oxide (NO) is a key signaling molecule that regulates diverse physiological responses, including adaptation to hypoxia in plants. Although stabilization of group-VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors under low O2 is known to be facilitated by the N-dragon pathway, whether NO directly mediates N-terminal cysteine (Cys) oxidation to regulate ERFVII stability remains unresolved. Here, we combined genetic, computational, and structural approaches to investigate NOs role in proteasomal degradation of ERFVII during flooding stress. Arabidopsis mutants with elevated S-Nitrosoglutathione (GSNO) levels compromised ERFVII activation during dark submergence but increased expression of genes encoding N-degron pathway enzymes. Although direct detection of in vivo NO-mediated S-nitrosylated proteins remains technically challenging, the GPS-SNO 1.0 tool predicted the conserved N-terminal second Cys residue as a high-confidence S-nitrosylation site. Structural modeling using the Schrodinger Suite 2024-4 further revealed that conversion of the Cys thiol (Cys-SH) to S-nitrosothiol (Cys-SNO) induced notable conformational changes in ERFVII TFs. Molecular docking further demonstrated that Cys-SNO-modified ERFVII peptides exhibited stronger binding affinities and altered interaction networks with N-degron pathway enzymes, supporting a role for NO-mediated structural remodeling in ERFVII degradation. Elevated GSNO also disrupted energy balance efficiency for overcoming O2 deficiency, altered the expression of sugar starvation-responsive genes, and impaired ATP binding capacity of Cys-SNO ERFVII proteins, as evidenced by docking and molecular dynamics simulations. Collectively, these results support a model in which NO-mediated modification of the conserved N-terminal Cys promotes ERFVII degradation, thereby linking NO signaling to hypoxia-responsive transcriptional regulation and metabolic adaptation during flooding stress.

plant biology↗
bioRxiv2026

AI-Informed neoantigen prioritization enables a multi-epitope mRNA/LNP vaccine with antigen-specific immunogenicity and antitumor activity

Personalized neoantigen vaccines are an emerging strategy for cancer immunotherapy, but their effectiveness depends on selecting tumor-specific antigens capable of inducing functional T-cell responses. The VACINUS AI-informed neoantigen prioritization framework previously identified and peptide-validated three immunogenic Tier 1 neoantigens in the B16F10 melanoma model. In this study, we extended that framework by translating these validated neoantigens into a multi-epitope messenger RNA vaccine formulated with lipid nanoparticles and evaluating its preclinical immunogenicity and antitumor activity. The three VACINUS-prioritized B16F10 neoantigens were encoded within a single multi-epitope construct, BF-V1_27-Ser, and formulated to generate BF-RNA-P. In B16F10 tumor-bearing mice, BF-RNA-P induced neoantigen-specific CD8+T-cell responses, with the strongest response directed against the B16F10-1-4 epitope. Combination with anti-PD-1 further enhanced vaccine-induced CD44+IFN-{gamma}+ CD8+ T-cell activation, whereas anti-PD-1 alone did not induce detectable peptide-specific responses. BF-RNA-P also suppressed tumor growth in vivo, and combination treatment produced the strongest antitumor effect, reflected by reduced tumor volume and lower endpoint tumor burden. Together, these findings demonstrate that VACINUS-prioritized and peptide-validated neoantigens can be reformatted into a multi-epitope messenger RNA/lipid nanoparticle vaccine while retaining antigen-specific immunogenicity and antitumor activity. This study provides preclinical proof-of-concept for integrating AI-informed, TCR-aware neoantigen prioritization with messenger RNA/lipid nanoparticle delivery as a translational strategy for personalized cancer vaccine development.

immunology↗
bioRxiv2026

AP2/ERF transcription factor RAP2.6 regulates early flowering in Arabidopsis thaliana by altering S-nitrosothiol levels and cytokinin responses

RAP2.6, an AP2/ERF transcription factor (TF), regulates plant stress responses; however, its role in floral transition remains unexplored. Here, we evaluated RAP2.6s role in flowering and the associated transcriptional changes in Arabidopsis thaliana under long-day conditions. RAP2.6-overexpressing line showed early flowering with fewer rosette leaves, whereas rap2.6-1 mutant flowered later, had more rosette leaves, and higher expression of the floral repressor FLOWERING LOCUS C (FLC). Early flowering in the overexpressing line was accompanied by transcriptional activation of the floral integrators GIGANTEA (GI), FLOWERING LOCUS T (FT), and COSTANS (CO), potentially through RAP2.6 interaction with GCC/DRE cis-regulatory elements. RAP2.6-mediated floral transition depended on nitric oxide (NO), with flowering time largely varying based on NO bioactivity. RAP2.6 was found to be a downstream regulator of Arabidopsis S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1) in controlling S-nitrosothiol (SNO) levels, flowering time, and silique formation. The NITRIC OXIDE-ASSOCIATED 1 (NOA1)-dependent reduction in NO levels abolished early flowering in 35S::RAP2.6 plants without affecting silique formation. Furthermore, enhanced cytokinin sensitivity and upregulation of cytokinin biosynthetic genes suggest cytokinin involvement in RAP2.6-mediated flowering. Together, these findings highlight the crucial role of RAP2.6 in regulating flowering time by integrating redox and hormonal signaling to coordinate reproductive development in A. thaliana.

plant biology↗
bioRxiv2023

Foxp3 Orchestrates Reorganization of Chromatin Architecture to Establish Regulatory T Cell Identity

Chromatin conformation reorganization is emerging as an important layer of regulation for gene expression and lineage specification. Yet, how lineage-specific transcription factors contribute to the establishment of cell type-specific 3D chromatin architecture in the immune cells remains unclear, especially for the late stages of T cell subset differentiation and maturation. Regulatory T cells (Treg) are mainly generated in the thymus as a subpopulation of T cells specializing in suppressing excessive immune responses. Here, by comprehensively mapping 3D chromatin organization during Treg cell differentiation, we show that Treg-specific chromatin structures were progressively established during its lineage specification, and highly associated with Treg signature gene expression. Additionally, the binding sites of Foxp3, a Treg lineage specifying transcription factor, were highly enriched at Treg-specific chromatin loop anchors. Further comparison of the chromatin interactions between wide-type Tregs versus Treg cells from Foxp3 knock-in/knockout or newly-generated Foxp3 domain-swap mutant mouse revealed that Foxp3 was essential for the establishment of Treg-specific 3D chromatin architecture, although it was not dependent on the formation of the Foxp3 domain-swapped dimer. These results highlighted an underappreciated role of Foxp3 in modulating Treg-specific 3D chromatin structure formation.

immunology↗
bioRxiv2022

Epigenomic and chromosomal architectural reconfiguration in developing human frontal cortex and hippocampus

The human frontal cortex and hippocampus play critical roles in learning and cognition. We investigated the epigenomic and 3D chromatin conformational reorganization during the development of the frontal cortex and hippocampus, using more than 53,000 joint single-nucleus profiles of chromatin conformation and DNA methylation (sn-m3C-seq). The remodeling of DNA methylation predominantly occurs during late-gestational to early-infant development and is temporally separated from chromatin conformation dynamics. Neurons have a unique Domain-Dominant chromatin conformation that is different from the Compartment-Dominant conformation of glial cells and non-brain tissues. We reconstructed the regulatory programs of cell-type differentiation and found putatively causal common variants for schizophrenia strongly overlap with chromatin loop-connected, cell-type-specific regulatory regions. Our data demonstrate that single-cell 3D-regulome is an effective approach for dissecting neuropsychiatric risk loci.

genomics↗
bioRxiv2022

Deciphering the Evolutionary History of Complex Rearrangements in Head and Neck Cancer Patients Using Multi-Omic Approach

Despite the large efforts in international cancer genome consortium studies, there are still a large proportion of tumors with complex genomic rearrangement often remained without a clinically relevant molecular characterization. Integration of multi-omic data helps elucidating evolutionary history of such cases and identifying predictive molecular markers. Here we present the findings of our proof-of-principle study that investigated the evolutionary history of complex rearrangements in primary head and neck tumor genomes integrating long-read whole-genome, Hi-C, and RNA sequencing. We report a HPV-positive case with development of complex genomic rearrangements tracing back to HPV-mediated genomic instability and a HPV-negative case with an enhancer hi-jacking in a region of chromothripsis predicted to co-occur with a neoloop and a super-enhancer. These structural alterations resulted in overexpression of the oncogenes CCND1 and ALK, respectively, validated with immunohistochemistry assay. Furthermore, we introduce a novel analytic approach utilizing long-read whole-genome data distinguishing somatic mutations before and after structural variants. Our findings highlight the need for multi-modal sequencing strategies to increase our understanding of cancer evolution and rare biomarkers in poorly understood cancers.

genomics↗
bioRxiv2022

Extensive mosaicism by somatic L1 retrotransposition in normal human cells

Over the course of an individuals lifetime, genomic alterations accumulate in somatic cells. However, the mutational landscape by retrotranspositions of long interspersed nuclear element-1 (L1), a widespread mobile element in the human genome, is poorly understood in normal cells. Here, we explored the whole-genome sequences of 892 single-cell clones established from various tissues collected from 28 individuals. Remarkably, 88% of colorectal epithelial cells acquired somatic L1 retrotranspositions (soL1Rs), carrying [~]3 events per cell on average with substantial intra- and inter-individual variances, which was accelerated at least 10-fold during tumourigenesis. Breakpoints of soL1Rs suggested that a few variant mechanisms can be involved in the L1 retrotransposition processes. Fingerprinting of donor L1s using source-specific unique sequences revealed 34 hot L1s, 44% of which were newly discovered in this study, and many ultra-rare hot L1s in the human population showed higher retrotransposition potential in somatic lineages than common sources. Multi-dimensional analysis of soL1Rs with early embryonic developmental relationships, genome-wide methylation, and gene expression profiles of the clones demonstrated that (1) soL1Rs occur from early embryogenesis at a substantial rate, (2) epigenetic activation of hot L1s is stochastically acquired during the wave of early global epigenomic reprogramming, rather than by the sporadic loss-of-methylation at the late stage, and (3) most L1 transcripts in the cytoplasm do not generate soL1Rs in somatic lineages. In summary, this study provides insights into the retrotransposition dynamics of L1s in the human genome and the resultant somatic mosaicism in normal human cells.

genomics↗
bioRxiv2021

Highly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

biochemistry↗
bioRxiv2020

Impaired hand dexterity function in a non-human primate model with chronic Parkinson's disease

Symptoms of Parkinsons disease (PD) caused by loss of dopaminergic neurons are accompanied by movement disorders, including tremors, rigidity, bradykinesia, and akinesia. Non-human primate (NHP) models with PD play an essential role in the analysis of PD pathophysiology and behavior symptoms. As impairments of hand dexterity function can affect activities of daily living in patients with PD, research on hand dexterity function in NHP models with chronic PD is essential. Traditional rating scales previously used in the evaluation of animal spontaneous behavior were insufficient due to factors related to subjectivity and passivity. Thus, experimentally designed applications for an appropriate apparatus are necessary. In this study, we aimed to longitudinally assess hand dexterity function using hand dexterity task (HDT) in NHP-PD models. To validate this assessment, we analyzed an alteration in Parkinsonian tremor symptoms, loss of dopaminergic neuron, and positron emission tomography (PET) imaging of dopamine transporters (DAT) in these models. HDT latency significantly increased in NHP-PD models. In addition, a significant inverse correlation between HDT and DAT was identified, but no local bias was found. The correlation with intention tremor symptoms was lower than the resting tremor. In conclusion, the evaluation of HDT may reflect behavioral symptoms of NHP-PD models. Furthermore, HDT was effectively used to experimentally distinguish intention tremors from other tremors.

neuroscience↗
bioRxiv2020

Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less off-target mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable diseases caused by genetic mutation.

biochemistry↗
bioRxiv2019

Single nucleus multi-omics links human cortical cell regulatory genome diversity to disease risk variants

Single-cell technologies enable measure of unique cellular signatures, but are typically limited to a single modality. Computational approaches allow integration of diverse single-cell datasets, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells in tissues, we devised single-nucleus methylCytosine, Chromatin accessibility and Transcriptome sequencing (snmC2T-seq) and applied it to post-mortem human frontal cortex tissue. We developed a computational framework to validate fine-grained cell types using multi-modal information and assessed the effectiveness of computational integration methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling prediction of cell types with causal roles in disease.

genomics↗
bioRxiv2018

Single-cell multi-omic profiling of chromatin conformation and DNA methylome

Recent advances in the development of single cell epigenomic assays have facilitated the analysis of gene regulatory landscapes in complex biological systems. Methods for detection of single-cell epigenomic variation such as DNA methylation sequencing and ATAC-seq hold tremendous promise for delineating distinct cell types and identifying their critical cis-regulatory sequences. Emerging evidence has shown that in addition to cis-regulatory sequences, dynamic regulation of 3D chromatin conformation is a critical mechanism for the modulation of gene expression during development and disease. It remains unclear whether single-cell Chromatin Conformation Capture (3C) or Hi-C profiles are suitable for cell type identification and allow the reconstruction of cell-type specific chromatin conformation maps. To address these challenges, we have developed a multi-omic method single-nucleus methyl-3C sequencing (sn-m3C-seq) to profile chromatin conformation and DNA methylation from the same cell. We have shown that bulk m3C-seq and sn-m3C-seq accurately capture chromatin organization information and robustly separate mouse cell types. We have developed a fluorescent-activated nuclei sorting strategy based on DNA content that eliminates nuclei multiplets caused by crosslinking. The sn-m3C-seq method allows high-resolution cell-type classification using two orthogonal types of epigenomic information and the reconstruction of cell-type specific chromatin conformation maps.

genomics↗
bioRxiv2018

S100A8/A9 mediate the reprograming of normal mammary epithelial cells induced by dynamic cell-cell interactions with adjacent breast cancer cells.

To understand the potential effects of cancer cells on surrounding normal mammary epithelial cells, we performed direct co-culture of non-tumorigenic mammary epithelial MCF10A cells and various breast cancer cells. Firstly, we observed dynamic cell-cell interactions between the MCF10A cells and breast cancer cells including lamellipodia or nanotube-like contacts and transfer of extracellular vesicles. Co-cultured MCF10A cells exhibited features of epithelial-mesenchymal transition, and showed increased capacity of cell proliferation, migration, colony formation, and 3-dimensional sphere formation. Transcriptome analysis and phosphor-protein array suggested that several cancer-related pathways are significantly dysregulated in MCF10A cells after the direct co-culture with breast cancer cells. S100A8 and S100A9 showed distinct up-regulation in the co-cultured MCF10A cells and their microenvironmental upregulation was also observed in the orthotropic xenograft of syngeneic mouse mammary tumors. When S100A8/A9 overexpression was induced in MCF10A cells, the cells showed phenotypic features of directly co-cultured MCF10A cells in terms of in vitro cell behaviors and signaling activities suggesting a S100A8/A9-mediated transition program in non-tumorigenic epithelial cells. This study suggests the possibility of dynamic cell-cell interactions between non-tumorigenic mammary epithelial cells and breast cancer cells that could lead to a substantial transition in molecular and functional characteristics of mammary epithelial cells.

cancer biology↗