Biology subjects

Truong, D. M.

Publications and source records attributed to Truong, D. M..

7 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

Human macroH2A1 drives nucleosome dephasing and genome instability in histone-humanized yeast

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones providing additional layers of structural and epigenetic regulation. Here, we systematically replaced individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. Variants H2A.J, TsH2B, and H3.5 complemented for their respective replicative counterparts. However, macroH2A1 failed to complement and its expression was toxic in yeast, negatively interacting with native yeast histones and kinetochore genes. To isolate yeast with "macroH2A1 chromatin" we decoupled the effects of its macro and histone fold domains, which revealed that both domains sufficed to override native yeast nucleosome positioning. Furthermore, both modified constructs of macroH2A1 exhibited lower nucleosome occupancy that correlated with decreased short-range chromatin interactions (<20 Kb), disrupted centromeric clustering, and increased chromosome instability. While supporting viability, macroH2A1 dramatically alters chromatin organization in yeast, leading to genome instability and massive fitness defects.

genetics↗

bioRxiv2022

DASH/Dam1 complex mutants stabilize ploidy by weakening kinetochore-microtubule attachments

Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes, defined by their centromeric evolutionary origins, however, they are not adaptive. Instead we show that a set of missense mutations in outer kineto-chore proteins drive adaptation to human histones. Further, we characterize the molecular mechanism of two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. Lastly, we show that one mutant, DAD1E50D, while suppressing chromosome instability in mitosis, leads to gross defects in meiosis. In sum, our data show how a set of point mutations evolved in the histone-humanized yeasts to counterbalance human histone induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.

genetics↗

bioRxiv2019

Comprehensive scanning mutagenesis of human retrotransposon LINE-1 identifies motifs essential for function

Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. The L1-encoded proteins ORF1p and ORF2p enable the element to jump from one locus to another via a \"copy and paste\" mechanism. ORF1p is an RNA-binding protein and ORF2p has endonuclease and reverse transcriptase activities. The huge number of truncated L1 remnants in the human genome suggests that the host has likely evolved mechanisms to prevent full L1 replication and thereby decrease the proliferation of active elements and reduce the mutagenic potential of L1. In turn, L1 appears to have a minimized length to increase the probability of successful full-length replication. This streamlining would be expected to lead to high information density. Here, we describe the construction and initial characterization of a library of 538 consecutive trialanine substitutions that scan along ORF1p and ORF2p to identify functionally important regions. In accordance with the streamlining hypothesis, retrotransposition was overall very sensitive to mutations in ORF1p and ORF2p, only 16% of trialanine mutants retained near-wild-type activity. All ORF1p mutants formed near-wild-type levels of mRNA transcripts and seventy-five percent formed near-wild-type levels of protein. Two ORF1p mutants present a unique nucleolar-relocalization phenotype. Regions of ORF2p that are sensitive to mutagenesis, but lack phylogenetic conservation were also identified. We provide comprehensive information on the regions most critical to retrotransposition. This resource will guide future studies of intermolecular interactions that form with RNA, proteins and target DNA throughout the L1 life cycle.

genetics↗

bioRxiv2019

Superloser: a plasmid shuffling vector for Saccharomyces cerevisiae with exceedingly low background

Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway - even essential ones - while maintaining low false positive rates (less than 1 in 108 per cell). This plasmid, dubbed \"Superloser\", was designed with reduced sequence similarity to commonly used yeast plasmids (i.e. pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker URA3 to reduce spontaneous 5-fluoroorotic acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof of concept shuffling experiment: swapping yeasts core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4x109) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as low as 1 in 108 cells.

genetics↗

bioRxiv2017

Resetting the yeast epigenome with human nucleosomes

Humans and yeast are separated by a billion years of evolution, yet their conserved core histones retain central roles in gene regulation. Here, we \"reset\" yeast to use core human nucleosomes in lieu of their own, an exceedingly rare event which initially took twenty days. The cells adapt, however, by acquiring suppressor mutations in cell-division genes, or by acquiring certain aneuploidy states. Robust growth was also restored by converting five histone residues back to their yeast counterparts. We reveal that humanized nucleosomes in yeast are positioned according to endogenous yeast DNA sequence and chromatin-remodeling network, as judged by a yeast-like nucleosome repeat length. However, human nucleosomes have higher DNA occupancy and reduce RNA content. Adaptation to new biological conditions presented a special challenge for these cells due to slower chromatin remodeling. This humanized yeast poses many fundamental new questions about the nature of chromatin and how it is linked to many cell processes, and provides a platform to study histone variants via yeast epigenome reprogramming.\n\nHighlights- Only 1 in 107 yeast survive with fully human nucleosomes, but they rapidly evolve\n- Nucleosome positioning and nucleosome repeat length is not influenced by histone type\n- Human nucleosomes remodel slowly and delay yeast environmental adaptation\n- Human core nucleosomes are more repressive and globally reduce transcription in yeast

synthetic biology↗