Biology subjects

Chang, Y.-Y.

Publications and source records attributed to Chang, Y.-Y..

10 recordsLinked to original sources

bioRxiv2026

Identification a Compact Promoter using a New Promoter Selection Strategy and Engineering Hybrid Pol II/III Enable Efficient Genome Editing in Human Retinal Ganglion Cells

Promoters and vectors are critical components of gene therapy, enabling the delivery and expression of therapeutic genes to correct both loss- and gain-of-function mutations. Adeno-associated virus (AAV) vectors are the leading platform for in vivo gene delivery; however, the widely used Streptococcus pyogenes Cas9 (SpCas9, 4.1 kb) approaches the AAV packaging limit of 4.7 kb. This constraint often necessitates dual-vector systems, which reduce therapeutic efficiency, or the use of smaller nucleases such as SaCas9 (3.2 kb) and AacCas12b (3.4 kb), which have lower PAM site frequencies. To enhance promoter selection for gene therapy applications, we developed a strategy to identify compact, cell-preferred RNA polymerase II (Pol II) promoters. Analysis of approximately 300 compact Pol II promoters revealed that exogenous expression levels in one cell type correlate more strongly with those in other cell types than with endogenous expression, underscoring the importance of exogenous expression efficiency in promoter selection. Using this approach, we identified a compact Pol II promoter #2 (Pro2, 133 bp) that drives robust transgene expression in human retinal ganglion cells (RGCs). To enable single-AAV delivery of SpCas9, we analyzed three commonly used Pol III promoters (H1, 7SK and U6) and determined their minimal functional lengths using a CRISPR/Cas9 reporter assay. We further engineered three compact hybrid Pol II/III promoters which combined pro2 with minimal H1, 7SK and U6 (276, 294, and 323 bp) capable of co-expressing SpCas9 and gRNA, enabling efficient genome editing in both transfected HEK293 cells (approaching 100%) and human RGCs (up to 55.9%) from human stem cell-derived retinal ganglion cells (RGCs). Together, these findings establish a framework for developing single-AAV CRISPR-based gene therapy strategies. Authors contributionsPWZ and DJZ conceived the study, designed the experiments, performed data analysis and interpretation, and were the primary contributors to manuscript writing. STZ played a key role in data collection and correlation analysis. YYC, SL, LF, CJK, YD, CAB, JC, and DW contributed to the execution of essential experiments and subsequent data analysis. All authors have read and approved the final manuscript. Declaration of interestsThe authors declare no conflicts of interest.

molecular biology↗

bioRxiv2025

Dynamic changes in mRNA isoform usage during human retinal development

BackgroundAlternative mRNA splicing is a key mechanism for generating isoform diversity in eukaryotic cells. However, the extent of the splicing changes that occur during complex regulatory processes like neurodevelopment are still incompletely characterized. ResultsWe performed nanopore-based long-read RNA sequencing on differentiating human stem cell-derived retinal organoids to identify temporal patterns of isoform usage across developmental stages. We found that retinal organoids undergo dynamic shifts in isoform usage throughout differentiation, which were not necessarily accompanied with changes in overall gene expression, as was observed for many genes involved in the regulation of mRNA splicing itself. Further analysis of human stem cell-derived retinal ganglion cells uncovered neuron-specific splicing signatures. Additionally, allele-specific gene expression analysis revealed extensive allelic imbalance in induced pluripotent stem cell-derived organoid cultures. ConclusionsBy combining direct long-read RNA sequencing with human stem cell retinal models we were able to develop a comprehensive database of isoform-level changes in differentiating human retinal cells during development. These results uncovered dynamic shifts in transcript usage during retinal differentiation, adding to our knowledge base of post-transcriptional RNA processing in the developing central nervous system and human in vitro culture systems.

molecular biology↗

bioRxiv2024

Multi-Omics Mapping of Gut Microbiota's Role in Progesterone Metabolism

5-neurosteroids such as allopregnanolone and isopregnanolone play critical roles in neurological health and mood regulation, yet current therapeutic production faces significant limitations. We demonstrate that specific gut microbes represent a previously unrecognized source of bioavailable 5-neurosteroids that reach the central nervous system via the gut-brain axis. Through integrated metabolomic and genomic analyses of progesterone-amended fecal cultures, we identified Holdemania as a major producer of isopregnanolone via microbial steroid 5-reductase (BaiJ type 2) and 3{beta}-hydroxysteroid dehydrogenase/reductase. Phylogenetic analysis revealed that BaiJ-like sequences cluster predominantly within Firmicutes, with Holdemania species forming a distinct clade. In female C57BL/6 mice administered progesterone and H. filiformis, 5-neurosteroids including isopregnanolone predominated in gut tissues while allopregnanolone was the major hepatic neurosteroid. Critically, using stable isotope tracing with [3,4-{superscript 1}3C2]progesterone, we detected {superscript 1}3C-labeled isopregnanolone in brain tissue, providing direct evidence for gut-to-brain transport of microbiota-derived neurosteroids. High-fat diet significantly enhanced brain 5-neurosteroid accumulation. Global meta-analysis reveals reduced Holdemania abundance in PCOS patients (n = 346) compared to healthy women (n = 321). These findings identify gut microbiota as pharmacologically relevant neurosteroid producers and position H. filiformis as a promising probiotic candidate for enhancing endogenous neurosteroid production to treat mood disorders and other neuropsychiatric conditions. HighlightsO_LIHoldemania was identified as a major producer of 5-neurosteroids, particularly isopregnanolone, in the intestinal tract C_LIO_LIHoldemania 5-reductase (BaiJ type 2) belongs to a distinct phylogenetic clade compared to characterized Clostridium BaiJ (type 1) C_LIO_LI5-neurosteroids occurred predominantly in the cecum of female mice administered H. filiformis, progesterone, and high-fat diet C_LIO_LI{superscript 1}3C-labeled 5-neurosteroids were detected in brain tissue of female mice orally administered [3,4-{superscript 1}3C2]progesterone and H. filiformis, demonstrating gut-to-brain transport C_LIO_LIGut microbes such as H. filiformis represent promising probiotic candidates for enhancing 5-neurosteroid production and circulation via the gut-brain axis C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/628284v3_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1b85caaorg.highwire.dtl.DTLVardef@cbb352org.highwire.dtl.DTLVardef@169f7fcorg.highwire.dtl.DTLVardef@17c638_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Broadly conserved FlgV controls flagellar assembly and Borrelia burgdorferi dissemination in mice

Flagella propel pathogens through their environments yet are expensive to synthesize and are immunogenic. Thus, complex hierarchical regulatory networks control flagellar gene expression. Spirochetes are highly motile bacteria, but peculiarly in the Lyme spirochete Borrelia burgdorferi, the archetypal flagellar regulator {sigma}28 is absent. We rediscovered gene bb0268 in B. burgdorferi as flgV, a broadly-conserved gene in the flagellar superoperon alongside {sigma}28 in many Spirochaetes, Firmicutes and other phyla, with distant homologs in Epsilonproteobacteria. We found that B. burgdorferi FlgV is localized within flagellar motors. B. burgdorferi lacking flgV construct fewer and shorter flagellar filaments and are defective in cell division and motility. During the enzootic cycle, B. burgdorferi lacking flgV survive and replicate in Ixodes ticks but are attenuated for dissemination and infection in mice. Our work defines infection timepoints when spirochete motility is most crucial and implicates FlgV as a broadly distributed structural flagellar component that modulates flagellar assembly.

microbiology↗

bioRxiv2022

Computer simulations reveal pathogenicity and inheritance modes of hearing loss-causing germinal variants

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In the present study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal "plugs" aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homomeric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable heteromeric channels did not. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus cells and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.

biophysics↗

bioRxiv2021

A phosphoinositide and RAB switch controls early macropinocytosis

Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluids into giant vesicles known as macropinosomes. This mechanism is used by immune cells to sample the surroundings for antigens and can be exploited by cancer cells for nutrient uptake. What determines the fate of macropinosomes after they have been internalized is largely unknown. Here we investigate the role of the phosphatidylinositol 3-kinase VPS34/PIK3C3 and its product phosphatidylinositol 3-phosphate (PtdIns3P) in macropinosome fate determination. Inhibition of VPS34 led to a decrease in macropinosome survival and fluid phase uptake as well as preventing recruitment of early endosomal factors, including the small GTPase RAB5 and its effectors, to the forming macropinosomes. Instead, forming macropinosomes under VPS34 inhibition accumulated regulators of endocytic recycling, including RAB8A, RAB10, RAB11A, and PtdIns4P, which led to fusion of macropinosomes with the plasma membrane. Whereas RAB5 was critical for macropinosome formation, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a PtdIns3P-controlled switch that balances macropinosome fate between the default, endolysosomal maturation and an alternative, secretory route.

cell biology↗

bioRxiv2020

MAP kinase Slt2p attenuates cell wall mRNA decay by downregulating the RNA-binding protein Rbp1p in response to stress

The yeast cell wall integrity (CWI) MAPK pathway is a signaling cascade function in maintaining cell wall integrity under stressful environmental conditions. Recently, the activity and signaling of Slt2p (Mpk1p) MAP kinase has been shown to control assembly of the processing body (P-body) upon cell wall stresses, implicating its posttranscriptional role in decay of cell wall mRNAs. However, how Slt2p MAP kinase directly regulates the stability of cell wall transcripts during cell wall stress remains unclear. Here, we reported that the RNA-binding protein Rbp1p (Ngr1p) is a downstream effector and target of Slt2p MAP kinase during activation of the cell wall stress signaling cascade. In addition to the well-defined target mitochondrial porin mRNA, we found that Rbp1p also negatively regulates the stability of a subset of Slt2p-regulated cell wall transcripts. Deletion of RBP1 increases the level of cell wall transcripts and partially suppresses the hypersensitivity of the slt2{Delta} deletion strain to cell wall damage. Slt2p is necessary for cell wall stress-induced stabilization of cell wall transcripts. Deletion of RBP1 compromises the destabilization of cell wall transcripts in slt2{Delta} mutants under cell wall stress. Notably, C-terminal deleted Slt2p impairs its function in promoting turnover of the Rbp1p protein and fails to stabilize cell wall transcripts, although it can complement the growth defect of the slt2{Delta} strain upon cell wall stress. Altogether, our results demonstrate that MAP kinase Slt2p attenuates CWI mRNA decay in response to cell wall damage by downregulating the activity of the RNA-binding protein Rbp1p.

cell biology↗

bioRxiv2020

Population-based Carrier Screening and Prenatal Diagnosis of Fragile X Syndrome in East Asian Populations

Identification of carriers of fragile X syndrome (FXS) with the subsequent prenatal diagnosis, and knowledge of FXS-associated genetic profiles are essential for intervention in specific populations. We report the results of carrier screening of 39,458 East Asian adult women and prenatal diagnosis from 87 FXS carriers. The prevalence of FXS carriers and incidence of full mutation fetuses in carrier pregnancies were found to be 1/556 and 11.0%, respectively. The prevalence of FXS carriers and full mutation fetuses was estimated to be 1/581 and 1/3124 in East Asian populations, respectively. We confirmed the validity of the current threshold of CGG repeats for FMR1 categorization; the integral risks of full mutation expansion were approximately 6.0%, 43.8%, and 100% for premutation alleles with 55-74, 75-89, and [≥]90 CGG repeats, respectively. The protective effect of AGG interruption in East Asian populations was validated, which is important in protecting premutation alleles with 75-89 CGG repeats from full mutation expansion. Lastly, family history was shown not an effective indicator for FXS carrier screening in East Asian populations and population-based screening was more cost-effective. This study provides an insight into the largest carrier screening and prenatal diagnosis for FXS in East Asian populations to date. The FXS-associated genetic profiles of East Asian populations are delineated and population-based carrier screening is shown to be promising for FXS intervention.

genetics↗

bioRxiv2018

An efficient timer and sizer of protein motions reveals the time scales of functional dynamics in the ribosome

The clock of life ticks as fast as how efficiently proteins could perform their functional dynamics. Protein complexes execute functions via several large-scale intrinsic motions across multiple conformational states, which occur at a timescale of nano-to milliseconds for well-folded proteins. Computationally expensive molecular dynamics (MD) simulation has been the only theoretical tool to time and size these motions, though barely to their slowest ends. Here, we convert a simple elastic network model (ENM), which takes a few seconds (ubiquitin) to hours (ribosome) for the analysis, into a molecular timer and sizer to gauge the slowest functional motions of proteins. Quasi-harmonic analysis, fluctuation-profile matching (FPM) and the Wiener-Khintchine theorem (WKT) are used to define the \"time-periods\", t, for anharmonic principal components (PCs) which are validated by NMR order parameters. The PCs with their respective \"time-periods\" are mapped to the eigenvalues ({lambda}ENM) of the corresponding ENM modes. Thus, the power laws t(ns) = 86.9{lambda}ENM-1.9 and {sigma}2([A]2) = 46.1 {lambda}ENM-2.5 are established allowing the characterization of the time scales of Nuclear Magnetic Resonance (NMR)-solved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter.\n\nAuthor SummaryThe time scale of biological processes is governed by protein functional dynamics that often corresponds to the largest conformational spread and the longest time scales among all possible motions. Current simulation methodologies cannot reach the slowest, often functional, motions especially for supramolecular machineries. Borrowing the spring-bead model used in polymer physics since 60s, the efficient elastic network model (ENM), introduced in 90s, captured all modes of protein motions but largely underestimated the time scales of slowest modes due to its harmonic approximation.\n\nHere we map water-damped modes sampled by MD simulations to corresponding ones in ENM and thereby establish 2 power laws that describe the authentic time scales and sizes for the slowest anharmonic modes as functions of ENM eigenvalues. With that, we adequately describe the sizes and time scales for three proteins, confirmed by NMR spectroscopy, and ribosome that contains ~0.2 million heavy atoms (~ 20 thousands coarse-grained nodes).

biophysics↗

bioRxiv2017

The Genomic Landscape Of Tree Rot In Phellinus noxius And Its Hymenochaetales Members

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomics analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens, and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single-nucleotide-polymorphisms (SNPs) with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.

genomics↗