Biology subjects

Guo, C.-J.

Publications and source records attributed to Guo, C.-J..

At least 19 recordsLinked to original sources

bioRxiv2026

Cohesin loading at regulatory elements shapes 3D genome folding during erythropoiesis

During erythropoiesis, differentiating cells silence much of the genome while maintaining high expression of select genes. It remains unclear whether this transcriptional specialization is linked to specific changes in 3D genome organization. We generated deep Micro-C maps across human erythropoiesis and identified erythroid cis-regulatory elements (CREs) that function as "matchmakers" by strengthening loops between neighboring elements. Matchmakers are associated with high H3K4me1, increase in strength during differentiation, and are linked to the expression of key erythropoiesis genes. Simulations suggest that a moderate cohesin loading bias across broad enhancer regions can explain the features of matchmakers, particularly under chromatin compaction. Targeted transcription factor and cohesin perturbations reduced matchmaker-associated structures and gene expression. We propose that matchmakers represent a subset of CREs that concentrate loop extrusion near key genes during erythroid differentiation.

genomics↗

bioRxiv2026

Mapping genetic risk mechanisms for immune-mediated diseases across human dendritic cell differentiation

Defining the cell types and mechanisms through which genetic variation operates is essential to understand the biological basis of disease. Although human dendritic cells (DCs) are crucial in regulating immunity, their rarity and limitations in available genomic data have hampered efforts to link inherited disease risk to specific DC subsets. Here, we present a single-cell multi-omic atlas of human DC differentiation from hematopoietic stem and progenitor cells (HSPCs) that includes chromatin accessibility and transcriptomic profiles to infer regulatory networks across DC subsets. By integrating this regulatory architecture with fine-mapped variants from hundreds of complex-trait genome-wide association studies, we systematically map inflammatory, autoimmune, and oncologic disease risk to DC subset-specific variant-to-gene mechanisms. For example, we identify a risk-associated variant that enhances the activity of a PLD4 regulatory element in plasmacytoid DCs, thereby increasing the risk of developing systemic lupus erythematosus and other immune disorders. Collectively, these findings enable a deeper understanding of how complex immune diseases can emerge due to the impact of genetic variation acting in specific DC subsets.

genetics↗

bioRxiv2025

Gut microbe-derived short-chain fatty acids regulate joint inflammation and activation of infiltrating and resident myeloid cells after alphavirus infection

Although oral antibiotics can predispose to joint inflammation, this phenomenon remains poorly understood. Here, we leverage mouse models of alphavirus-induced arthritis to investigate the roles of gut commensals, metabolites, and host immune mechanisms in promoting musculoskeletal inflammation. Mice treated with a short course of oral antibiotics exhibited worsened arthritis after chikungunya (CHIKV) or Mayaro virus infections. This phenotype was associated with loss of short chain fatty acids (SCFA), greater intestinal permeability, and activation of gut-associated immune cells, and required TLR4 signaling, MyD88 expression, monocytes, antigen-specific and bystander CD4+ T cells, and pro-inflammatory cytokines. Administration of exogenous SCFA or colonization of mice with bacterial species that generate SCFA mitigated CHIKV-induced joint inflammation. Single-cell RNA sequencing revealed that gut-derived SCFA ameliorate the inflammatory phenotype of synovial CD4+ T cells, infiltrating monocytes, and resident osteoclast-like cells. Thus, antibiotic-triggered gut dysbiosis exacerbates alphavirus arthritis by shaping the inflammatory profile of both infiltrating and resident immune cells in joint tissues.

immunology↗

bioRxiv2025

Genetic variation reveals a homeotic long noncoding RNA that modulates human hematopoietic stem cells

The HOXA gene locus coordinates body patterning, hematopoiesis, and differentiation. While studying blood phenotype-associated variation within the HOXA locus, we identified a genetic variant, rs17437411, associated with globally reduced blood counts, protection from blood cancers, and variation in anthropometric phenotypes. We find that this variant disrupts the activity of a previously unstudied antisense long non-coding RNA (lncRNA) located between HOXA7 and HOXA9, which we have named HOTSCRAMBL. The HOTSCRAMBL variant disrupts lncRNA function and reduces human hematopoietic stem cell (HSC) self-renewal. Mechanistically, HOTSCRAMBL enables appropriate expression and splicing of HOXA genes in HSCs, most notably HOXA9, in an SRSF2-dependent manner. Given the critical role of HOXA gene expression in some blood cancers, we also demonstrate that HOTSCRAMBL variation or deletion compromises HOXA-dependent acute myeloid leukemias. Collectively, we show how insights from human genetic variation can uncover critical regulatory processes required for effective developmental gene expression.

genetics↗

bioRxiv2025

Inherited resilience to clonal hematopoiesis by modifying stem cell RNA regulation

Somatic mutations that increase hematopoietic stem cell (HSC) fitness drive their expansion in clonal hematopoiesis (CH) and predispose to blood cancers. Although CH frequently occurs with aging, it rarely progresses to overt malignancy. Population variation in the growth rate and potential of mutant clones suggests the presence of genetic factors protecting against CH, but these remain largely undefined. Here, we identify a non-coding regulatory variant, rs17834140-T, that significantly protects against CH and myeloid malignancies by downregulating HSC-selective expression and function of the RNA-binding protein MSI2. By modeling variant effects and mapping MSI2 binding targets, we uncover an RNA network that maintains human HSCs and influences CH risk. Importantly, rs17834140-T is associated with slower CH expansion rates in humans, and stem cell MSI2 levels modify ASXL1-mutant HSC clonal dominance in experimental models. These findings leverage natural resilience to highlight a key role for post-transcriptional regulation in human HSCs, and offer genetic evidence supporting inhibition of MSI2 or its downstream targets as rational strategies for blood cancer prevention.

genetics↗

bioRxiv2025

FMR1 KH0-KH1 Domains Coordinate m6A Binding and Phase Separation in Fragile X Syndrome

The Fragile X Mental Retardation Protein (FMR1) regulates neurodevelopment via m6A RNA interactions, but domain-specific roles of KH0 and KH1 in RNA binding and pathology remain unclear. Through mutagenesis and AlphaFold3 modeling, we demonstrate that KH1 serves as the primary m6A-binding interface, whereas the KH0 domain (e.g., Arg138) modulates liquid-liquid phase separation (LLPS). Pathogenic mutations in KH0 disrupt RNA binding and enhance LLPS aggregation, while m6A RNA suppresses LLPS at KH0. Structural simulations reveal synergistic cooperation between KH0 and KH1 through hydrophobic and electrostatic networks. These dual-domain interactions establish a mechanistic link between m6A dysregulation, aberrant phase separation, and Fragile X syndrome pathogenesis. Our findings propose KH0 as a therapeutic target for RNA-driven neurodevelopmental disorders.

cell biology↗

bioRxiv2025

CTP Drives Human CTPS1 Filamentation

CTP synthase (CTPS) is a key enzyme in de novo CTP synthesis, playing a critical role in nucleotide metabolism and cellular proliferation. Human CTPS1 (hCTPS1), one of the two CTPS isoforms, is essential for immune responses and is highly expressed in proliferating cells, making it a promising therapeutic target for immune-related diseases and cancer. Despite its importance, the regulatory mechanisms governing hCTPS1 activity remain poorly understood. Here, we reveal that CTP, the product of CTPS, acts as a key regulator for hCTPS1 filamentation. Using cryo-electron microscopy (cryo-EM), we resolve the high-resolution structure of CTP-bound hCTPS1 filaments, uncovering the molecular details of CTP binding and its role in filament assembly. Importantly, we demonstrate that CTP generated from the enzymatic reaction does not trigger filament disassembly, suggesting a conserved regulatory pattern. Furthermore, by analyzing the binding modes of two distinct CTP-binding pockets, we provide evidence that this filamentation mechanism is evolutionarily conserved across species, particularly in eukaryotic CTPS. Our findings not only elucidate a novel regulatory mechanism of hCTPS1 activity but also deepen the understanding of how metabolic enzymes utilize filamentation as a conserved strategy for functional regulation. This study opens new avenues for targeting hCTPS1 in therapeutic interventions.

biochemistry↗

bioRxiv2024

Coupling CRISPR Scanning with Targeted Chromatin Accessibility Profiling using a Double-Stranded DNA Deaminase

Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms and the development of gene therapies. However, these approaches cannot be combined with direct scalable readouts of chromatin structure and accessibility across long single-molecule chromatin fibers. Here we leverage a double-stranded DNA cytosine deaminase to profile chromatin accessibility at high depth and resolution at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). Powered by high sequence coverage at targeted loci of interest, TDAC-seq can be uniquely integrated with CRISPR perturbations to enable the functional dissection of cis-regulatory elements, where genetic perturbations and their effects on chromatin accessibility are superimposed on the same single chromatin fiber and resolved at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34+ hematopoietic stem and progenitor cells during erythroid differentiation as well as in pooled CRISPR and base editing screens tiling an enhancer controlling the globin locus. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.

molecular biology↗

bioRxiv2024

Gut bacteria-derived succinate induces enteric nervous system regeneration

Enteric neurons control gut physiology by regulating peristalsis, nutrient absorption, and secretion1. Disruptions in microbial communities caused by antibiotics or enteric infections result in the loss of enteric neurons and long-term motility disorders2-5. However, the signals and underlying mechanisms of this microbiota-neuron communication are unknown. We studied the effects of microbiota on the recovery of the enteric nervous system after microbial dysbiosis caused by antibiotics. We found that both enteric neurons and glia are lost after antibiotic exposure, but recover when the pre-treatment microbiota is restored. Using murine gnotobiotic models and fecal metabolomics, we identified neurogenic bacterial species and their derived metabolite succinate as sufficient to rescue enteric neurons and glia. Unbiased single-nuclei RNA-seq analysis uncovered a novel neural precursor-like population marked by the expression of the neuronal gene Nav2. Genetic fate-mapping showed that Plp1+ enteric glia differentiate into neurons following antibiotic exposure. In contrast, Nav2+ neurons expand upon succinate treatment and indicate an alternative mode of neuronal regeneration under recovery conditions. Our findings highlight specific microbial species, metabolites, and the underlying cellular mechanisms involved in neuronal regeneration, with potential therapeutic implications for peripheral neuropathies.

neuroscience↗

bioRxiv2023

Filamentation and inhibition of prokaryotic CTP synthase

CTP synthase (CTPS) plays a pivotal role in the de novo synthesis of CTP, a fundamental building block for RNA and DNA, which is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: ATP, UTP, CTP, and GTP. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by virus or prokaryotic pathogens. Utilizing cryo-electron microscopy, we have determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, NADH, and the covalent inhibitor DON, achieving a resolution of 2.8[A]. We construct a phylogenetic tree based on differences in filament-forming interfaces and design a variant to validate our hypothesis, providing an evolutionary perspective on the CTPS filament formation. Our computational analysis reveals a solvent-accessible ammonia tunnel upon DON binding. By comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS, differing from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determine and validate the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

biochemistry↗

bioRxiv2023

Dynamic atP5CS2 Filament Facilitates Substrate Channeling

In plants, the rapid accumulation of proline is a common response to combat abiotic stress. Delta-1-pyrroline-5-carboxylate synthase (P5CS) is a rate-limiting enzyme in proline synthesis, catalyzing the initial two-step conversion from glutamate to proline. Here, we determine the first structure of plant P5CS. Our results show that Arabidopsis thaliana P5CS2 (atP5CS2) can form enzymatic filaments in a substrate-sensitive manner. The destruction of atP5CS filaments by mutagenesis leads to a significant reduction in enzymatic activity. Furthermore, separate activity tests on two domains reveals that filament-based substrate channeling is essential for maintaining the high catalytic efficiency of atP5CS. Our study demonstrates the unique mechanism for the efficient catalysis of P5CS, shedding light on the intricate mechanisms underlying plant proline metabolism and stress response. Therefore these findings provide potential avenues for crop genetically modified breeding.

biochemistry↗

bioRxiv2023

Functional characterization of helminth-associated Clostridia reveals covariates of Treg differentiation

Parasitic helminths influence the composition of the gut microbiome. However, the microbiomes of individuals living in helminth-endemic regions are understudied. The Orang Asli, an indigenous population in Malaysia with high burdens of the helminth Trichuris trichiura, displayed microbiotas enriched in Clostridiales, an order of spore-forming obligate anaerobes previously shown to have immunogenic properties. We previously isolated novel Clostridiales that were enriched in these individuals and found that a subset promoted the Trichuris life cycle. Here, we further characterized the functional properties of these bacteria. Enzymatic and metabolomic profiling revealed a range of activities associated with metabolism and host response. Consistent with this finding, monocolonization of mice with individual isolates identified bacteria that were potent inducers of regulatory T cell (Treg) differentiation in the colon. Comparisons between variables revealed by these studies identified enzymatic properties correlated with Treg induction and Trichuris egg hatching. These results provide functional insights into the microbiotas of an understudied population.

microbiology↗

bioRxiv2023

Structural basis for activation of glutaminase

Glutaminase is a rate-limiting enzyme in glutaminolysis, which produces glutamate from glutamine and enters the TCA cycle[1]. In addition, it plays a key role in redox homeostasis[2], autophagy[3], immune system regulation[4], central nervous system maintenance[5], and senolysis[6]. Therefore, the allosteric regulation of glutaminase is a fascinating topic that has broad implications for our understanding of glutamine metabolism and related diseases[7-9]. Phosphate was discovered as a natural agonist for glutaminase in 1947[10], but the structural basis and mechanism for this regulation remains unclear. Using cryo-electron microscopy, here we determine the structure of human glutaminase with phosphate. This structure allows us to capture phosphate binding at the dimer-dimer interface at near atomic resolution, revealing an allosteric activation mechanism by remodelling the catalytic pocket. Surprisingly, we find that phosphate antagonizes BPTES (a classical antagonist) and CB-839 (the current subject of several phase II clinical trials). Accurate identification of phosphate binding sites lays the foundation for the design of glutaminase agonists and antagonists with broad pharmaceutical significance.

biochemistry↗

bioRxiv2023

Structural basis of bifunctional CTP/dCTP synthase

Nucleotides are important molecules of life. According to the sugar, nucleotides can be divided into nucleotides and deoxynucleotides, which are the basic components of RNA and DNA respectively. CTP synthase (CTPS) catalyzes the last step in the de novo synthesis of CTP, which can form cytoophidia in cells from all three domains of life. We have recently found that CTPS combines with NTPs to form filaments, and solved the structures of CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can also serve as dCTPS, i.e. CTPS/dCTPS can not only bind UTP, ATP and GTP to generate CTP, but also bind deoxynucleotides to generate dCTP. However, the structural basis of the bifunctional enzyme CTPS/dCTPS binding deoxynucleotide is not clear. In this study, we find that CTPS/dCTPS can form filaments bound with deoxynucleotides. Biochemically, we compare the binding and reaction characteristics of the corresponding nucleotides/deoxynucleotides and CTPS/dCTPS. Using cryo-electron microscopy, we solve the the structure of CTPS/dCTPS bound with deoxynucleotides at near-atomic resolution. This study not only provides a structural basis for understanding the catalysis and regulation of bifunctional CTPS/dCTPS, but also opens a door for further exploration the compartmentation of CTPS/dCTPS inside a cell.

biochemistry↗

bioRxiv2022

Gut bacteria-derived serotonin promotes immune tolerance in early life

The gut microbiome promotes immune system development in early life, but the neonatal gut metabolome remains undefined. Here, we demonstrate that, distinct from adults, the neonatal mouse gut is enriched with neurotransmitters, and specific bacteria produce serotonin directly while downregulating monoamine oxidase A to limit serotonin breakdown. Serotonin inhibits mTOR activation to promote regulatory T cells and suppress T cell responses both ex vivo and in vivo in the neonatal intestine. Oral gavage of serotonin into neonatal mice leads to long-term immune tolerance toward both dietary antigens and commensal bacteria as well as alterations of the gut microbiome. Together, our study has uncovered unique microbiome-dependent mechanisms to maximize serotonin in the neonatal gut and a novel role for intestinal serotonin to promote immune tolerance in early life.

immunology↗

bioRxiv2022

GTP as an enzymatic synchronizer

CTP synthase (CTPS), the sole enzyme for de novo CTP biosynthesis, requires GTP as an allosteric effector for efficient catalysis1. Although the labile intermediates and chemical reaction steps have been characterized, how CTPS coordinates its structural domains through interactions with GTP to continuously catalyze thus reactions and thereby synthesize CTP correctly remains elusive. Through cryo-EM analysis of 34 structural states (2.0-3.3 [A] resolution) spanning pre-catalytic, catalytic, and post-catalytic conformations, we delineate how GTP cycling--intact binding and dissociation--directly gates three sequential activation checkpoints. 1) Catalytic initiation gate: GTP dissociation precedes substrate entry, transitioning CTPS to a substrate-competent state. 2) Intermediate-dependent GTP recruitment: 4Pi-UTP formation allosterically remodels the GAT domain, licensing GTP rebinding. 3) Ammonia transfer gate: GTP directly constitutes the transient interdomain gas tunnel for ammonia delivery while stimulating the glutamine hydrolyzing. CTP synthesis completion triggers GTP dissociation, allowing glutamate release. Each catalytic cycle couples CTP production to a single GTP-binding event, demonstrating that GTP cycling operates as a catalytic-phase timer. This work provides a comprehensive model for the efficient catalysis of CTPS and establishes effector cycling as a dynamic gating mechanism in enzymes.

biochemistry↗

bioRxiv2022

Structural basis of human PRPS2 filaments

PRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While hPRPS1 and hPRPS2 are expressed in most tissues, hPRPS3 is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming the cytoophidium is considered as a distinctive mechanism involving the polymerization of the protein. In order to investigate the function and molecular mechanism of hPRPS2 polymerization, we solve the polymer structure of hPRPS2 at 3.08[A] resolution using cryo-Electron Microscopy (cryo-EM). hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity. Our findings suggest that the regulation of hPRPS2 polymer is distinct from E. coli PRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis.

biochemistry↗

bioRxiv2022

Allosteric inhibition of PRPS is moderated by filamentous polymerization

Phosphoribosyl pyrophosphate (PRPP) is an important intermediate for the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) has been associated with human disorders including Arts syndrome, retinal dystrophy and gouty arthritis. Recent studies have revealed that PRPS can form filamentous cytoophidia in prokaryotes and eukaryotes. Here we resolve two distinct filament structures of E. coli PRPS at near-atomic resolution under Cryo-EM. Formation of two types of filaments is controlled by the binding of different ligands. While the type A filament attenuates the allosteric inhibition of PRPS by ADP, the type B filament enhances the inhibition. In addition, a novel conformation of the regulatory flexible loop of PRPS was found occupying the ATP binding site. AMP/ADP bound at a noncanonical allosteric site interacts with the regulatory flexible loop and facilitates the binding of ATP. Our findings not only reveal molecular mechanisms of the regulation of PRPS with structural basis, but also suggest a distinctive bidirectional regulatory system for PRPP production via PRPS polymerization.

biochemistry↗