Biology subjects

Yanagi, T.

Publications and source records attributed to Yanagi, T..

3 recordsLinked to original sources

bioRxiv2025

Genomic REWRITE of Immune Interfaces Reveals Heritable Epigenetic Memory in Human Stem Cells

Human leukocyte antigen (HLA) polymorphism underlies antigen presentation, immune recognition, and central tolerance, yet the complex, multigenic structure of native HLA loci has precluded systematic engineering in human pluripotent stem cells (hPSCs). Existing genome engineering demonstrations in hPSCs focus on smaller integrations or at ectopic sites, leaving scar-minimized rewriting of native human loci at >100 kb scale a major technical challenge. Here, we present REWRITE, a modular platform for scar-minimized genome writing that enables engineering of large human genomic loci exceeding 100 kb at their native chromosomal sites. Using REWRITE, we deleted 105 kb spanning the dispersed HLA class-I locus and installed either compact or full-length refactored synthetic HLA haplotypes. Both refactored architectures exhibited transient activity before re-acquiring the native inactive state; upon extended culture, engineered lines yielded global transcriptomes nearly indistinguishable from parental cells. The engineered loci were genomically stable, retained interferon-responsive HLA expression, and remained compatible with differentiation into endothelial-like and thymic epithelial-like cells. Together, these results establish REWRITE as a platform for locus-scale genome engineering in hPSCs and provides a system for studying HLA haplotype structure and function in an isogenic pluripotent cell background. One sentenceA practical platform for scar-minimized rewriting of native human loci exceeding 100 kb in hPSCs, demonstrated through synthetic refactoring of the class-I HLA region.

synthetic biology↗

bioRxiv2024

Termination sequence between an inducible promoter and ubiquitous chromatin opening element (UCOE) reduces gene expression leakage and silencing

AbstractInducible gene expression circuits offer precise control over target gene activation, making them essential tools for direct reprogramming, where cells are guided to differentiate into specific cell types. However, stable circuit function and consistent expression of key inducible proteins are crucial for effective reprogramming, and DNA methylation-induced silencing hinders this stability. To address this issue, A2-ubiquitous chromatin opening elements (A2UCOE) have gained attention for their ability to maintain open chromatin and prevent methylation, thereby stabilizing gene expression. In this study, we aimed to forward program iPSCs into thymic epithelial cells (TECs) using a compact, all-in-one gene circuit composed of a doxycycline-inducible Tet-On system, 863-bp A2UCOE (0.9 UCOE), and FOXN1, a master transcription factor for TEC differentiation. This compact construct enables site-specific genome integration and stable inducible expression within iPSCs for renewably generating TECs. While the 0.9 UCOE promoted stable expression of constitutively expressed genes, it also caused unintended FOXN1 gene leakage, leading to unprogrammed gradual differentiation of the iPSCs. We generated A2UCOE fragments of varying lengths and found that gene leakage persisted regardless of fragment size. We tested spacer sequences between the A2UCOE and the Tet-On promoter consisting of varying AT-nucleotide content (35, 50, and 65%) and the 65% AT-rich SV40 poly-A terminator sequence and found that only the SV40 poly-A mitigated this leakage, and surprisingly, enhanced desired anti-silencing effects. These findings highlight the benefits and potential risks of using A2UCOE in iPSCs, and provides insights into a regulatory DNA architecture optimal for compact forward programming circuits for controlled iPSC differentiation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/629244v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@8398d7org.highwire.dtl.DTLVardef@1a75beeorg.highwire.dtl.DTLVardef@84767eorg.highwire.dtl.DTLVardef@7f5cec_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

AMP-activated protein kinase (AMPK) is a central kinase involved in energy homeostasis. Increased intracellular adenosine monophosphate (AMP) levels result in AMPK activation through the binding of AMP to the {gamma}-subunit of AMPK. Recently, we reported that AMP-induced AMPK activation is impaired in the kidneys in chronic kidney disease (CKD) despite an increase in the AMP/ATP ratio. However, the mechanisms by which AMP sensing is disrupted in CKD are unclear. In this study, we identified mechanisms of energy homeostasis in which Unc-51-like kinase 1 (ULK1)-dependent phosphorylation of AMPK{gamma}1 at Ser260/Thr262 promotes AMP sensitivity of AMPK. AMPK activation by AMP was impaired in Ulk1-/- mice despite an increased AMP/ATP ratio. We also demonstrated that MK8722, an allosteric AMPK activator, activates AMPK in the kidneys of a CKD mouse model via a pathway that is independent of AMP sensing. MK8722 treatment significantly attenuates the deterioration of renal function in CKD and is a potential therapeutic option in CKD therapeutics.

molecular biology↗