Biology subjects

Zhang, H.

Publications and source records attributed to Zhang, H..

At least 757 records · Page 42Linked to original sources

bioRxiv2023

Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow

The gut must perform a dual role of protecting the host against toxins and pathogens while harboring mutualistic microbiota. Previous studies suggested that the NADPH oxidase Duox contributes to intestinal homeostasis in Drosophila by producing reactive oxygen species (ROS) in the gut that stimulate epithelial renewal. We find instead that ROS generated by Duox in the Malpighian tubules leads to the production of Upd3, which enters the gut and stimulates stem cell proliferation. We describe for the first time in Drosophila the existence of a countercurrent flow system, which pushes tubule-derived Upd3 to the anterior part of the gut and stimulates epithelial renewal at a distance. Thus, our paper clarifies the role of Duox in gut homeostasis and describes the existence of retrograde fluid flow in the gut, collectively revealing a fascinating example of inter-organ communication.

immunology↗

bioRxiv2023

Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

Although Cul9 has been implicated in human carcinogenesis, its upstream regulators and roles remain unknown. Herein, we indicate that the Cul9 promoter is hypermethylated in GCs. Bioinformatics, mass spectrometry, and unbiased-kinase screen identify the tyrosine kinase Yes1 as a key regulator of Cul9. Yes1 phosphorylates Cul9 at Y1505, promoting its selective autophagy. Patient-associated mutation of Yes1 or helicobacter pylori infection induces Cul9-Y1505 phosphorylation which switches Cul9 from a tumor-suppressor to an oncogene, as evidenced by the fact that Cul9-Y1505D knockin mice are more susceptible to gastric tumorigenesis than wild-type counterparts. Metabolic profiling and ATAC sequencing reveal that Cul9-Y1505D mutant promotes pyrimidine and purine synthesis pathways in GC. DNA-demethylating drug decitabine or HG78 compound upregulates Cul9 expression and limits GC cell proliferation in a Yes1-dependent manner. The Yes1 inhibitor CH6953755 or Leflunomide and Mycophenolate mofetil (MMF) also impair the malignancy of GC with Cul9 dysregulation. Cul9 in turn binds Yes1 and disrupts Yes1 stability, establishing a feedback circuit. Collectively, our project reveals an unrecognized role of the Yes1-Cul9 loop in GC, suggesting potential therapeutic targets.

cancer biology↗

bioRxiv2023

Multicolor on-chip fluorescence microscopy for real-time surgical guidance

In curative-intent cancer surgery, intraoperative fluorescence imaging of both diseased and healthy tissue can help to ensure successful removal of all gross and microscopic disease with minimal damage to neighboring critical structures, such as nerves. Current fluorescence-guided surgery (FGS) systems, however, rely on bulky and rigid optics that incur performance-limiting trade-offs between sensitivity and maneuverability. Moreover, many FGS systems are incapable of multiplexed imaging. As a result, clinical FGS is currently limited to millimeter-scale detection of a single fluorescent target. Here we present a scalable, lens-less fluorescence imaging chip, VISION, capable of sensitive and multiplexed detection within a compact form factor. Central to VISION is a novel optical frontend design combining a low-numerical-aperture fiber optic plate (LNA-FOP) and a multi-bandpass interference filter, which is affixed to a custom CMOS image sensor. The LNA-FOP acts as a planar collimator to improve resolution and compensate for the angle-sensitivity of the interference filter, enabling high-resolution and multiplexed fluorescence imaging without lenses. We show VISION is capable of detecting tumor foci of less than 100 cells at near video framerates and, as proof of principle, can simultaneously visualize both tumor and nerves in ex vivo prostate tissue.

cancer biology↗

bioRxiv2023

Targeting CD45 by gene-edited CAR-T cells for leukemia eradication and hematopoietic stem cell transplantation preconditioning

Hematopoietic stem cell transplantation (HSCT) is widely used to treat patients with life-threatening hematologic and immune system disorders. The currently used nontargeted chemo-/radiotherapy conditioning regimens cause tissue injury and induce an array of immediate and delayed adverse effects, which limits the use of this potentially curative treatment. The growing demand to replace canonical conditioning regimens has led to the development of alternative approaches based on antibody-drug conjugates, naked antibodies and CAR T cells. Here, we propose a preconditioning strategy based on targeting CD45 on hematopoietic cells with CAR45 T cells. To avoid fratricide of CD45 CAR T cells, targeted genomic disruption of the CD45 gene was performed in human CD45 CAR T cells in combination with dasatinib treatment. CD45{Delta}CAR45 T cells showed impressive activity in terms of target cell elimination in vitro and depletion of tumor cells in vivo or human hematopoietic cells in humanized immunodeficient mice engrafted with human blood-derived HSCs. CD45{Delta}CAR45 NK cells also exhibited potent killing activity against tumor cell lines and human hematopoietic cells. Therefore, fratricide-resistant CAR45 T and NK cells have the potential to provide the benefits of full myeloablative conditioning and therapy for hematologic malignancies. Thus, we provide the proof of concept for the generation and preclinical efficacy of CAR T cells directed against CD45-expressing cells.

molecular biology↗

bioRxiv2023

UPL3 Promotes BZR1 Degradation, Growth Arrest, and Seedling Survival under Starvation Stress in Arabidopsis

BRASSINAZONE RESISTANT 1 (BZR1) is a key transcription factor of the brassinosteroid signaling pathway but also a signaling hub that integrates diverse signals that modulate plant growth. Previous studies have shown that starvation causes BZR1 degradation, but the underlying mechanisms are not understood. Here we performed quantitative proteomic analysis of BZR1 interactome under starvation conditions and identified two BZR1-interacting ubiquitin ligases, BAF1 and UPL3. Compared to the wild type, the upl3 mutants show long hypocotyl and increased BZR1 levels when grown under sugar starvation conditions but not when grown on sugar-containing media, indicating a role of UPL3 in BZR1 degradation specifically under starvation conditions. The upl3 mutants showed a reduced survival rate after starvation treatment, supporting the importance of UPL3-mediated BZR1 degradation and growth arrest for starvation survival. Treatments with inhibitors of TARGET of RAPAMYCIN (TOR) and autophagy altered BZR1 level in the wild type but were less effective in upl3, suggesting that UPL3 mediates the TOR-regulated and autophagy-dependent degradation of BZR1. Further, the UPL3 protein level is increased posttranscriptionally by starvation but decreased by sugar treatment. Our study identifies UPL3 as a key component that mediates sugar regulation of hormone signaling pathways, important for optimal growth and survival in plants. IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSThe coordination between signaling pathways that monitor the levels of photosynthate and growth hormones is crucial for optimizing growth and survival, but the underlying mechanisms are not fully understood. When the sugar level is low, the BZR1 transcription factor of the brassinosteroid (BR) signaling pathway is degraded, and hence growth is attenuated to prevent starvation and enhance survival. When sugar is sufficient, sugar signaling inhibits BZR1 degradation and enables BR promotion of plant growth. The key component that mediates starvation-induced BZR1 degradation remains unknown. QuestionWhat proteins interact with BZR1 and mediate its degradation under sugar starvation? FindingWe performed immunoprecipitation mass spectrometry analysis of BZR1 in starvation-treated Arabidopsis and identified many BZR1-interacting proteins, including two E3 ligases UPL3 and BAF1. Genetic analysis showed that UPL3 plays a specific and prominent role in promoting autophagy-dependent BZR1 degradation and plant survival under sugar-starvation conditions. Next stepHow sugar-TOR signaling regulates UPL3 level remains to be studied in the future.

plant biology↗

bioRxiv2023

The role of immune inflammatory response and inflammatory infiltrates in major depression disorder and atopic dermatitis: new insights from machine learning

BackgroundMajor depression disorder (MDD) and atopic dermatitis (AD) are distinct disorders involving immune inflammatory responses. This study aimed to investigate the comorbid relationship between AD and MDD and to identify possible common mechanisms. MethodsWe obtained AD and MDD data from the Gene Expression Omnibus (GEO) database. Differential expression analysis and the Genecard database were employed to identify shared genes associated with inflammatory diseases. These shared genes were then subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Hub genes were selected based on the protein-protein interactions using CytoHubba, and key regulatory genes were identified through enrichment analysis. Subsequently, we conducted immune infiltration and correlation analyses of the shared genes in AD. Finally, we employed three machine learning models to predict the significance of shared genes. ResultsA total of 17 shared genes were identified in the AD_Inflammatory_MDD dataset (S100A9, PTGER2, PI3, SNCA, DAB2, PDGFA, FSTL1, CALD1, XK, UTS2, DHRS9, PARD3, NFIB,TMEM158, LIPH, RAB27B, and SH3BRL2). These genes were associated with biological processes such as the regulation of mesenchymal cell proliferation, mesenchymal cell proliferation, and glial cell differentiation. The neuroactive ligand-receptor interaction, IL-17 signaling, and Rap1 signaling pathways were significantly enriched in KEGG analysis. SNCA, S100A9, SH3BGRL2, RAB27B, TMEM158, DAB2, FSTL1, CALD1, and XK were identified as hub genes contributing to comorbid AD and MDD development. The three machine learning models consistently identified SNCA and PARD3 as important biomarkers. ConclusionSNCA, S100A9, SH3BGRL2, RAB27B, TMEM158, DAB2, FSTL1, CALD1, and XK were identified as significant genes contributing to the development of AD and MDD comorbidities. Immune infiltration analysis showed a notable increase in the infiltration of various subtypes of CD4+ T cells, suggesting a potential association between the development of skin inflammation and the immune response. Across different machine learning models, SNCA and PARD3 consistently emerged as important biomarkers.

genetics↗

bioRxiv2023

Title: Abrogation and Homeostatic Restoration of IgE Responses by a Universal IgE Allergy CTL Vaccine:The Three Signal Self/Non-Self/Self (S/NS/S) Model

Natural IgE cytotoxic peptides (nECPs), which are derived from the constant domain of the heavy chain of human IgE producing B cells via endoplasmic reticulum (ER) stress, are decorated onto MHC class 1a molecules (MHCIa) as unique biomarkers for CTL (cytotoxic T lymphocyte)-mediated immune surveillance. Human IgE exhibits only one isotype and lacks polymorphisms; IgE is pivotal in mediating diverse, allergen-specific allergies. Therefore, by disrupting self-IgE tolerance via costimulation, the cytotoxic T lymphocytes (CTLs) induced by nECPs can serve as universal allergy vaccines (UAVs) in humans to dampen IgE production mediated by diverse allergen-specific IgE- secreting B cells and plasma cells expressing surface nECP-MHCIa as targets. The study herein has enabled the identification of nECPs produced through the correspondence principle 1, 2. Furthermore, nECP-tetramer-specific CTLs were found to be converted into CD4 Tregs that restored IgE competence via the homeostatic principle, mediated by SREBP-1c suppressed DCs. Thus, nECPs showed causal efficacy and safety as UAVs for treating type I hypersensitivity IgE-mediated allergies. The applied vaccination concept presented provides the foundation to unify, integrate through a singular class of tetramer-specific TCR clonotypes. The three signal model is proposed on the mechanisms underlying central tolerance, breaking tolerance and regaining peripheral tolerance via homeostasis concerning nECP as an efficacious and safe UAV to treat type I IgE-mediated hypersensitivity. One Sentence SummaryHuman IgE self-peptides are identified as universal allergy vaccines that inhibit IgE synthesis while allowing homeostatic IgE recovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/561777v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18cb814org.highwire.dtl.DTLVardef@1893312org.highwire.dtl.DTLVardef@78221aorg.highwire.dtl.DTLVardef@1071098_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphic abstract text Three cell S/NS/S model of Universal Allergy Vaccines (UAV): Natural IgE peptides (nECPs) presented by enabler DCs break central IgE tolerance (Self), leading to CTLs that inhibit IgE production (Non-self). Generative DCs converted by the metabolic milieu transform the pre-existing nECP-specific CTLs into nECP-specific Tregs leading to homeostatic recovery of IgE competence (S).

immunology↗

bioRxiv2023

Exploring the bounded rationality in human decision anomalies through an assemblable computational framework

Some seemingly irrational decision behaviors (anomalies), once seen as flaws in human cognition, have recently received explanations from a rational perspective. The basic idea is that the brain has limited cognitive resources to process the quantities (e.g., value, probability, time, etc.) required for decision making, with specific biases arising as byproducts of the resource allocation that is optimized for the environment. While appealing for providing normative accounts, the existing resource-rational models have limitations such as inconsistent assumptions across models, a focus on single environmental factors, and limited coverage of decision anomalies. One challenging anomaly is the peanuts effect, a pervasive phenomenon in decision-making under risk that implies an interdependence between the processing of value and probability. To extend the resource rationality approach to explain the peanuts effect, here we develop a computational framework--the Assemblable Resource-Rational Modules (ARRM)--that integrates ideas from different lines of boundedly-rational decision models as freely assembled modules. The framework can accommodate the joint functioning of multiple environmental factors, and allow new models to be built and tested along with the existing ones, potentially opening a wider range of decision phenomena to bounded rationality modeling. For one new and three published datasets that cover two different task paradigms and both the gain and loss domains, our boundedly-rational models reproduce two characteristic features of the peanuts effect and outperform previous models in fitting human decision behaviors.

neuroscience↗

bioRxiv2023

A distinct subpopulation of adipocytes in adult perirenal adipose tissue arises through brown-to-white conversion but reverts to a thermogenic phenotype upon cold exposure

Perirenal adipose tissue (PRAT) is a unique visceral depot that contains a mixture of brown and white adipocytes. The origin and plasticity of such cellular heterogeneity remains unknown. Here, we combine single-nucleus RNA sequencing with genetic lineage tracing to reveal the existence of a distinct subpopulation of Ucp1-/Cidea+ adipocytes that arises from brown-to-white conversion during postnatal life in the peri-ureter region of mouse PRAT. Cold exposure restores Ucp1 expression and a thermogenic phenotype in this subpopulation. These cells have a transcriptome that is distinct from subcutaneous beige adipocytes and may represent a unique type of cold-recruitable adipocytes. These results pave the way for studies of PRAT physiology and mechanisms controlling the plasticity of brown/white adipocyte phenotypes.

developmental biology↗

bioRxiv2023

Integrating large-scale meta-GWAS and PigGTEx resources to decipher the genetic basis of complex traits in pig.

Understanding the molecular and cellular mechanisms that underlie complex traits in pigs is crucial for enhancing their genetic improvement program and unleashing their substantial potentials in human biomedicine research. Here, we conducted a meta-GWAS analysis for 232 complex traits with 28.3 million imputed whole-genome sequence variants in 70,328 individuals from 14 pig breeds. We identified a total of 6,878 genomic regions associated with 139 complex traits. By integrating with the Pig Genotype-Tissue Expression (PigGTEx) resource, we systemically explored the biological context and regulatory circuits through which these trait-associated variants act and finally prioritized 16,664 variant-gene-tissue-trait circuits. For instance, rs344053754 regulates the expression of UGT2B31 in the liver by affecting the activity of regulatory elements and ultimately influences litter weight at weaning. Furthermore, we investigated the conservation of genetic and regulatory mechanisms underlying 136 human traits and 232 pig traits. Overall, our multi-breed meta-GWAS in pigs provides invaluable resources and novel insights for understanding the regulatory and evolutionary mechanisms of complex traits in both pigs and humans.

genetics↗

bioRxiv2023

Lactoylglutathione promotes inflammatory signaling in macrophages

Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH, while demonstrating a potentiated inflammatory response when exposed to lipopolysaccharides, corresponding with a rise in histone lactoylation. Interestingly, our data demonstrate that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state, however, upon stimulation, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is a primary contributing factor facilitating the inflammatory response.

biochemistry↗

bioRxiv2023

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5-untranslated region (5 UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its -subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2 phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2A{Delta} mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative IRES elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

genetics↗

bioRxiv2023

Collaborative Augmented Reconstruction for Scaled Production of 3D Neuron Morphology in Mouse and Human Brains

Digital reconstruction of the intricate 3D morphology of individual neurons from microscopic images is widely recognized as a crucial challenge in both individual research laboratories and large-scale scientific projects focusing on cell types and brain anatomy. This task often fails both conventional manual reconstruction and state-of-the-art automatic reconstruction algorithms, even many of which are based on artificial intelligence (AI). It is also critical but challenging to organize multiple neuroanatomists to produce and cross-validate biologically relevant and agreeable reconstructions in scaled data production. Here we propose an approach based on collaborative human intelligence augmented by AI. Specifically, we have developed a Collaborative Augmented Reconstruction (CAR) platform for neuron reconstruction at scale. This platform allows for immersive interaction and efficient collaborative-editing of neuron anatomy using a variety of client devices, such as desktop workstations, virtual reality headsets, and mobile phones, enabling users to contribute anytime and anywhere and take advantage of several AI-based automation tools. We have tested CARs applicability for challenging mouse and human neurons towards a scaled and faithful data production. Our data indicate that the CAR platform is suitable for generating tens of thousands of neuronal reconstructions used in our companion studies.

neuroscience↗

bioRxiv2023

Conservation of the ancestral function of GTSF1 in transposon silencing in unicellular Paramecium

The PIWI-interacting RNA (piRNA) pathway is crucial for transposon repression and the maintenance of genomic integrity. Gametocyte specific factor 1 (GTSF1), an indispensable auxiliary factor of PIWI, was recently shown to potentiate the catalytic activity of PIWI in many metazoans. Whether the requirement of GTSF1 extends to PIWI proteins beyond metazoans is unknown. In this study, we identified a homolog of GTSF1 in the unicellular eukaryote Paramecium tetraurelia (PtGTSF1) and found that its role as a PIWI-cofactor is conserved. PtGTSF1 interacts with PIWI (Ptiwi09) and Polycomb Repressive Complex 2 (PRC2) and is essential for PIWI-dependent DNA elimination of transposons during sexual development. PtGTSF1 is crucial for the degradation of PIWI-bound small RNAs recognizing the organisms own genomic sequences. Without PtGTSF1, self-matching small RNAs are not degraded and results in an accumulation of H3K9me3 and H3K27me3, which disturbs transposon recognition and slows down their elimination. Our results demonstrate that the PIWI-GTSF1 interaction also exists in unicellular eukaryotes with the ancestral function of transposon silencing.

cell biology↗

bioRxiv2023

Admix-kit: An Integrated Toolkit and Pipeline for Genetic Analyses of Admixed Populations

SummaryAdmixed populations, with their unique and diverse genetic backgrounds, are often underrepresented in genetic studies. This oversight not only limits our understanding but also exacerbates existing health disparities. One major barrier has been the lack of efficient tools tailored for the special challenges of genetic study of admixed populations. Here, we present admix-kit, an integrated toolkit and pipeline for genetic analyses of admixed populations. Admix-kit implements a suite of methods to facilitate genotype and phenotype simulation, association testing, genetic architecture inference, and polygenic scoring in admixed populations. Availability and implementationAdmix-kit package is open-source and available at https://github.com/KangchengHou/admix-kit. Additionally, users can use the pipeline designed for admixed genotype simulation available at https://github.com/UW-GAC/admix-kit_workflow.

genetics↗

bioRxiv2023

The neurovascular coupling in the attention during visual working memory

How to focus attention during visual working memory (vWM) depends on ones ability to filter out distractors and expand the scope of targets. Although the spatiotemporal properties of attention processes in WM are well documented, it is still unclear how the mechanisms of neurovascular coupling (NVC) between electroencephalographic (EEG) signals and hemodynamic activity of attention during vWM. To investigate the NVC mechanism underlying attention during vWM, we recorded simultaneous functional near-infrared spectroscopy (fNIRS) and EEG data when humans were performing cued change-detection tasks. The multimodal data showed that the control and scope processes during vWM were involved in similar temporal profiles of frontal theta event-related synchronization (ERS) and posterior contralateral delay activities (CDA), and revealed similar distributions of hemodynamic activation within the frontal eye fields (FEF) and superior parietal lobule (SPL). These task-related features have a common NVC outcome across individuals: the higher EEG features (theta ERS or CDA amplitude), the greater the increment of local oxygenated hemoglobin (HbO) signals within the FEF and SPL. Moreover, when distractors should be filtered out, EEG-informed NVC is involved in a broader range of brain regions in the frontoparietal network (FPN). These results provided unique neurovascular evidence for the mechanisms of attention scope and control in vWM. Interestingly, there might be a negative relationship between behavioral metrics and theta-informed NVC strengths within the FEF for attention control. On a dynamic basis, the NVC features had higher discriminatory power for predicting behavior than EEG features and fNIRS features alone. Together, these results highlight what multimodal approaches can advance our understanding of the role of attention in vWM and how the fluctuations of NVC are associated with actual behavior.

neuroscience↗

bioRxiv2023

Addressing preferred orientation in single-particle cryo-EM through AI-generated auxiliary particles.

The single-particle cryo-EM field faces the persistent challenge of preferred orientation, lacking general computational solutions. We introduce cryoPROS, an AI-based approach designed to address the above issue. By generating the auxiliary particles with a conditional deep generative model, cryoPROS addresses the intrinsic bias in orientation estimation for the observed particles. We effectively employed cryoPROS in the cryo-EM single particle analysis of the hemagglutinin trimer, showing the ability to restore the near-atomic resolution structure on non-tilt data. Moreover, the enhanced version named cryoPROS-MP significantly improves the resolution of the membrane protein NaX using the no-tilted data that contains the effects of micelles. Compared to the classical approaches, cryoPROS does not need special experimental or image acquisition techniques, providing a purely computational yet effective solution for the preferred orientation problem. Finally, we conduct extensive experiments that establish the low risk of model bias and the high robustness of cryoPROS.

biophysics↗

bioRxiv2023

5-hydroxymethylcytosines regulate gene expression as a passive DNA demethylation resisting epigenetic mark in proliferative somatic cells

Enzymatic erasure of DNA methylation in mammals involves iterative 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (TET) family of DNA dioxygenase proteins. As the most abundant form of oxidized 5mC, the prevailing model considers 5-hydroxymethylcytosine (5hmC) as a key nexus in active DNA demethylation that can either indirectly facilitate replication-dependent depletion of 5mC by inhibiting maintenance DNA methylation machinery (UHRF1/DNMT1), or directly be iteratively oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) and restored to cytosine (C) through thymine DNA glycosylase (TDG)-mediated 5fC/5caC excision repair. In proliferative somatic cells, to what extent TET-dependent removal of 5mC entails indirect DNA demethylation via 5hmC-induced replication-dependent dilution or direct iterative conversion of 5hmC to 5fC/5caC is unclear. Here we leverage a catalytic processivity stalling variant of human TET1 (TET1.var: T1662E) to decouple the stepwise generation of 5hmC from subsequent 5fC/5caC generation, excision and repair. By using a CRISPR/dCas9-based epigenome-editing platform, we demonstrate that 5fC/5caC excision repair (by wild-type TET1, TET1.wt), but not 5hmC generation alone (by TET1.var), is requisite for robust restoration of unmodified cytosines and reversal of somatic silencing of the methylation-sensitive, germline-specific RHOXF2B gene promoter. Furthermore, integrated whole-genome multi-modal epigenetic sequencing reveals that hemi-hydroxymethylated CpG dyads predominantly resist replication-dependent depletion of 5mC on the opposing strand in TET1.var-expressing cells. Notably, TET1.var-mediated 5hmC generation is sufficient to induce similar levels of differential gene expression (compared to TET1.wt) without inducing major changes in unmodified cytosine profiles across the genome. Our study suggests 5hmC alone plays a limited role in driving replication-dependent DNA demethylation in the presence of functional DNMT1/UHRF1 mechanisms, but can regulate gene expression as a bona fide epigenetic mark in proliferative somatic cells.

genomics↗