Molecular mechanisms of gene amplification are diverse, and most remain elusive. Accordingly, the application of gene amplification to construct stable gene expression systems is insufficient. We have developed a novel gene amplification method “BiTREx” based on a defined molecular mechanism that is controllable and does not require selection pressure, using budding yeast as a model. BiTREx stems from the fact that a nick introduced by Cas9 nickase causes replisome disassembly to generate a single-end double-strand break to be repaired by break-induced replication. Suppose the replisome that has just replicated the target tandem repeat encounters a nick at its flanking site. In that case, BIR should frequently initiate thorough non-allelic single-strand DNA invasion to expand the tandem repeat. Indeed, BiTREx successfully expanded a CUP1 array consisting of 16 copies of 2 kb repeat units to ~500 repeat units (~1 Mb). Furthermore, BiTREx successfully expanded not only another natural tandem gene array (ENA1 array) but artificial ones consisting of ~10-kb repeat units inserted at the CUP1 locus. Thus, BiTREx is a versatile method to enable various novel applications in synthetic genomics. We will also show a seamless system for gene integration followed by BiTREx in this symposium, and present the possibility of applying gene amplification by BiTREx to various researches.

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cell Genomics  

Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here, we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has just replicated the array, forming a single-ended double-strand break. This break is subsequently end-resected to become single stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching ∼1 Mb. Furthermore, appropriate splint DNAs allow BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.

DOI： 10.1016/j.xgen.2025.100811

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PubMed

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.17912/micropub.biology.001582

Repository Public URL： https://hdl.handle.net/2324/7358365

Language：English  

The lung airways are characterized by long and wide proximal branches and short and thin distal branches. In this study, we investigated the emergence of this hierarchical structure through experimental observations and computational models. We focused on the branch formation in the pseudoglandular stage and examined the response of mouse lung epithelium to fibroblast growth factor 10 (FGF10) by monitoring extracellular signal-regulated kinase (ERK) activity. The ERK activity increased depending on the epithelial tissue curvature. This curvature-dependent response decreased as the development progressed. Therefore, to understand how these epithelial changes affect branching morphology, we constructed a computational model of curvature-dependent epithelial growth. We demonstrated that branch length was controlled by the curvature dependence of growth that was consistent with the experimental observations and lung morphology. However, the branching of the thin branches is suppressed in this model, which is inconsistent with the fact that thin branches in the lung are short. Thus, we introduced branch formation by apical constriction, which was shown to be regulated by Wnt signaling in our previous studies. Mathematical analysis indicated that the effect of apical constriction is cell shape-dependent, suggesting that apical constriction ameliorates the branching of thin branches. Finally, we were able to provide clarity on the hierarchical branching structure through an integrated computational model of curvature-dependent growth and cell shape regulation. We proposed that curvature-dependent growth involving FGF and Wnt-mediated cell shape regulation coordinate to control the spatial scale and frequency of branch formation.

DOI： 10.1101/2023.03.23.533381

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-023-44083-4

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1038/s41598-021-87912-6

Event date： 2023.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：神戸ポートアイランド   Country：Japan  

Event date： 2025.3

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2024.11

Language：Japanese   Presentation type：Poster presentation  

Venue：福岡国際会議場、マリンメッセA館・B館   Country：Japan  

Event date： 2024.10 - 2024.11

Language：English  

Event date： 2024.10 - 2024.11

Language：English  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Introduction: Drug transporters are expressed in a number of tissues such as the intestine, liver, and kidney, and play key roles in drug absorption, distribution, and excretion. Variations in drug transporter gene expression significantly contribute to interindividual differences in drug responses. Epigenetic regulation of drug transporter genes has recently emerged as an important mechanism. Epigenetic regulation alters the expression of genes without changing DNA sequences. Epigenetic control mechanisms are associated with DNA methylation, histone modifications, and microRNAs. Herein we discuss recent advances in the study of the transcriptional and post-transcriptional mechanisms of drug transporters with a focus on epigenetic regulation. Areas covered: This review summarizes recent research on the epigenetic regulation of drug transporter genes, and highlights the importance of identifying novel biomarkers based on epigenetics for use in individualized drug therapy. Expert opinion: Researchers are actively attempting to elucidate the epigenetic mechanisms that control the expression of drug transporters, which affect the pharmacokinetics of drugs. Current evidence suggests that epigenetic changes play an important role in drug transporter function. A clearer understanding of epigenetic regulation in drug transporter genes will provide an insight into novel approaches to individualized drug therapy.

DOI： https://doi.org/10.1080/17425255.2017.1230199

Type：Academic society, research group, etc. 

Type：Academic society, research group, etc. 

Type：Competition, symposium, etc. 

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Author：Myself