Biology subjects

Luong, C.

Publications and source records attributed to Luong, C..

8 recordsLinked to original sources

bioRxiv2025

DNA Partitioning Modulates Liquid-to-Solid Transitions and the Internal Microstructure of FUS Condensates

Protein liquid-liquid phase separation has recently been recognized as an essential process involved in cellular functions, including transcription, translation, and DNA damage repair. However, a further liquid-to-solid transition (LST) of condensates due to mutation or external stimuli can result in aggregation, sometimes pathological. The unique ability of protein condensates to concentrate and sequester biomolecules is at the heart of the regulatory mechanism, controlling the dynamics and function of the condensates. While the recruitment of essential biomolecules, such as RNA has been studied to have the impact to the condensate formation, how DNA partition can affect the phase behaviour and dynamics of protein condensates is not fully elucidated. In this study, we investigate both the short-term and long-term kinetics of double-stranded DNA partitioning into preformed fresh and aged FUS protein condensates. Confocal imaging shows that DNA partition follows the core-shell diffusion pattern within the condensates. LST slows down and reduces FUS condensates ability to recruit DNA but stabilizes the DNA-FUS condensate complex due to the heterogeneous solid network formation. Using the optical technique of Spatial Dynamic Mapping (SDM), we find that DNA partition promotes coalescence and alters the characteristics of the condensates. The partition made the condensates more dynamic in the short term (within minutes) but accelerated LST in long-term incubation (within hours), ultimately leading to an irreversible porous core-shell structure of FUS condensates. Our findings reveal the kinetics of DNA partition during aging and its impact on LST, underlining the modulation of condensate properties by molecule sequestration, shedding light on possible regulation of disease-related LST of biomolecular condensates.

biophysics↗

bioRxiv2025

The BRD4-nucleosome interaction is enhanced modestly and non-selectively by histone acetylation

BRD4 regulates gene transcription in complex eukaryotes, in part through the binding of its tandem bromodomains to acetylated lysine residues found in histones and transcription factors. Despite pharmacological inhibition of these domains showing promise in preclinical studies, clinical trial data have been less encouraging so far. A stronger understanding of BRD4 biochemistry could provide a route to better outcomes. To advance on prior work, which has focused almost entirely on the binding of isolated bromodomains and acetylated peptides, we have sought the preferred nucleosomal binding partner of full-length BRD4. We demonstrate that BRD4 binds with sub-micromolar affinity to both unmodified nucleosomes and to DNA alone. In strong contrast to BRD4-peptide interactions, we also find that the affinity of BRD4 for nucleosomes is increased only 2-4-fold by histone acetylation and that this affinity has little dependence on the acetylation pattern. Despite this modest effect of acetylation, binding of BRD4 to acetyllysine in the nucleosome was more resistant to perturbation by mutation or small-molecule inhibition than BRD4-peptide interactions. Our work helps bridge the gap between cellular and prior in vitro work and provides clues to explain the in vivo chromatin occupancy profile of BRD4 and how it changes upon therapeutic inhibition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/656505v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1aa532aorg.highwire.dtl.DTLVardef@13a154forg.highwire.dtl.DTLVardef@4d2039org.highwire.dtl.DTLVardef@17e8377_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Enhancer activation from transposable elements in extrachromosomal DNA

Extrachromosomal DNA (ecDNA) drives oncogene amplification and intratumoral heterogeneity in aggressive cancers. While transposable element (TE) reactivation is common in cancer, its role on ecDNA remains unexplored. Here, we map the 3D architecture of MYC-amplified ecDNA in colorectal cancer cells and identify 68 ecDNA-interacting elements (EIEs)--genomic loci enriched for TEs that are frequently integrated onto ecDNA. We focus on an L1M4a1#LINE/L1 fragment co-amplified with MYC, which functions only in the ecDNA amplified context. Using CRISPR-CATCH, CRISPR interference, and reporter assays, we confirm its presence on ecDNA, enhancer activity, and essentiality for cancer cell fitness. These findings reveal that repetitive elements can be reactivated and co-opted as functional rather than inactive sequences on ecDNA, potentially driving oncogene expression and tumor evolution. Our study uncovers a mechanism by which ecDNA harnesses repetitive elements to shape cancer phenotypes, with implications for diagnosis and therapy.

genetics↗

bioRxiv2024

Escape from X inactivation is directly modulated by levels of Xist non-coding RNA

In placental females, one copy of the two X chromosomes is largely silenced during a narrow developmental time window, in a process mediated by the non-coding RNA Xist 1. Here, we demonstrate that Xist can initiate X-chromosome inactivation (XCI) well beyond early embryogenesis. By modifying its endogenous level, we show that Xist has the capacity to actively silence genes that escape XCI both in neuronal progenitor cells (NPCs) and in vivo, in mouse embryos. We also show that Xist plays a direct role in eliminating TAD-like structures associated with clusters of escapee genes on the inactive X chromosome, and that this is dependent on Xists XCI initiation partner, SPEN 2. We further demonstrate that Xists function in suppressing gene expression of escapees and topological domain formation is reversible for up to seven days post-induction, but that sustained Xist up-regulation leads to progressively irreversible silencing and CpG island DNA methylation of facultative escapees. Thus, the distinctive transcriptional and regulatory topologies of the silenced X chromosome is actively, directly - and reversibly - controlled by Xist RNA throughout life.

developmental biology↗

bioRxiv2023

DNA-guided transcription factor cooperativity shapes face and limb mesenchyme

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how Coordinator, a long DNA motif comprised of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, while HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in shared regulation of genes involved in cell-type and positional identities, and ultimately shapes facial morphology and evolution.

genomics↗

bioRxiv2022

Cross-linking mass spectrometry discovers, evaluates, and validates the experimental and predicted structural proteome

Significant recent advances in structural biology, particularly in the field of cryo-electron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability or - in the case of complexes - simply not having yet been analysed. Here, we demonstrate the power of combining cross-linking mass spectrometry (XL-MS) with artificial intelligence-based structure prediction to discover and experimentally substantiate models for protein and protein complex structures at proteome scale. We present the deepest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein-protein interactions. We show that integrative models of complexes driven by AlphaFold Multimer and inspired and corroborated by the XL-MS data offer new opportunities to deeply mine the structural proteome and interactome and reveal new mechanisms underlying protein structure and function.

biochemistry↗

bioRxiv2022

Single-cell multiome of the human retina and deep learning nominate causal variants in complex eye diseases

Genome-wide association studies (GWAS) of eye disorders have identified hundreds of genetic variants associated with ocular disease. However, the vast majority of these variants are noncoding, making it challenging to interpret their function. Here, we present a joint single-cell atlas of gene expression and chromatin accessibility of the adult human retina with >50,000 cells, which we used to analyze noncoding single-nucleotide polymorphisms (SNPs) implicated by GWAS of age-related macular degeneration, glaucoma, diabetic retinopathy, myopia, and type 2 macular telangiectasia. We integrate this atlas with a HiChIP enhancer connectome, expression quantitative trait loci (eQTL) data, and base-resolution deep learning models to predict noncoding SNPs with causal roles in eye disease, assess SNP impact on transcription factor binding, and define their known and novel target genes. Our efforts nominate pathogenic SNP-target gene interactions for multiple vision disorders and provide a potentially powerful resource for interpreting noncoding variation in the eye.

genomics↗

bioRxiv2020

Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome

The long non-coding RNA Xist exploits numerous effector proteins to progressively induce gene silencing across the X chromosome and form the inactive X (Xi)-compartment. The mechanism underlying formation of the chromosome-wide Xi-compartment is poorly understood. Here, we find that formation of the Xi-compartment is induced by [~]50 locally confined granules, where two Xist RNA molecules nucleate supra-molecular complexes (SMCs) of interacting proteins. Xist-SMCs are transient structures that concentrate rapidly recycling proteins in the X by increasing protein binding affinity. We find that gene silencing originates at Xist-SMCs and propagates across the entire chromosome over time, achieved by Polycomb-mediated coalescence of chromatin regions and aggregation, via its intrinsically disordered domains, of the critical silencing factor SPEN. Our results suggest a new model for X chromosome inactivation, in which Xist RNA induces macromolecular crowding of heterochromatinizing proteins near distinct sites which ultimately increases their density throughout the chromosome. This mechanism enables deterministic gene silencing without the need for Xist ribonucleoprotein complex-chromatin interactions at each target gene.

cell biology↗