Biology subjects

Chen, J.-X.

Publications and source records attributed to Chen, J.-X..

13 recordsLinked to original sources

bioRxiv2026

The phosphoproteomic landscape of the DNA damage response

The DNA damage response (DDR) comprises an intricate network of protein-protein interactions and signaling pathways activated by DNA lesions and genomic instability. Central to this response is protein phosphorylation, which orchestrates DNA repair, cell cycle checkpoint activation, and chromatin organization. The response of the human phosphoproteome to different DNA damage-inducing agents and the functional role of regulated phosphorylation sites remains insufficiently characterized. Here, we systematically profiled the cellular phosphoproteome following exposure to eleven DNA damage-inducing agents that humans encounter physiologically or during cancer therapy. We identified a core set of DNA damage responsive phosphorylation sites, along with DDR signatures associated with DNA double strand breaks, replication stress and a pleiotropic response. Regulated phosphorylation sites are enriched within intrinsically disordered regions (IDRs), often forming clusters of nearby modifications that can affect IDR conformations or overlap with short linear interaction motifs. We discover that the RNA damage response predominantly shapes the changes induced by reactive aldehyde formaldehyde, alkylating agent methyl methanesulfonate and oxidative stress. Finally, we demonstrate that the proteasome-associated ubiquitin E3 ligase UBE3A is targeted by ATM and ATR kinases, thus linking proteasome regulation with the DDR.

cell biology↗

bioRxiv2026

SUMO paralogues differentially affect phase separation and aggregation of intrinsically disordered proteins

SUMO, the small ubiquitin-like modifier, modulates interactions of folded proteins, but also affects the dynamics of biomolecular condensates. Moreover, SUMO can directly impinge on the biophysical properties of its targets, often increasing their solubility. TDP-43 is an RNA-binding protein containing an intrinsically disordered domain that participates in protein phase separation. Its aggregation, linked to neurodegenerative diseases, is counteracted by SUMOylation, but the underlying principles are poorly understood. We now reveal distinct mechanisms of how the two isoforms, SUMO1 and SUMO2, act on TDP-43. While SUMO1 inhibits phase separation by blocking TDP-43s self-assembly site, SUMO2 enhances phase separation via proximity-induced self-interaction involving its N-terminus and promotes a liquid-like state that prevents aggregation. The latter represents a general principle by which SUMO solubilises other targets. Our findings provide insight into how SUMOylation regulates transitions between soluble, liquid-like and aggregated states.

molecular biology↗

bioRxiv2026

The splicing paralogues SNRPB and SNRPN control differential metabolic states.

Gene duplication underlies evolutionary innovation, yet many paralogues remain highly similar, raising questions about their functional divergence and physiological relevance. The spliceosomal Sm core protein SNRPB and its mammalian-specific paralogue SNRPN share over 90% sequence identity, but their distinct expression patterns - SNRPB being ubiquitous and SNRPN confined to the brain - suggest specialized functions. Why mammals have two different spliceosomes has remained obscure. Here, we generated isogenic human cell lines expressing ectopically either SNRPB or SNRPN exclusively and found that SNRPN stabilizes transcripts involved in energy metabolism and mitochondrial function, leading to increased mitochondrial abundance and oxygen consumption. Despite similar spliceosomal interactomes, SNRPN more strongly associates with the PRMT5 methylosome complex and exhibits dynamic arginine methylation in its C-terminal region that is sensitive to translation inhibition and amino acid availability. The SNRPN-dependent transcriptome responds to translation inhibition by stabilizing long, intron-rich genes involved in amino acid and energy metabolism. Our findings reveal a nutrient-sensitive, methylation-dependent mechanism that differentiates the two paralogues. This suggests that SNRPN functions as a metabolic-specialized spliceosomal subunit thereby providing tissue-specific adaptation of RNA processing in mammals.

molecular biology↗

bioRxiv2025

Nek family members regulate Rad54 during homologous recombination in developing mice

Homologous recombination (HR) represents an important pathway for repairing DNA double-strand breaks (DSBs) but HR factors, including RAD51, also serve to protect and restart stalled replication forks. RAD54 functions during DSB repair where it removes RAD51 from duplex DNA including heteroduplex DNA which is formed during D-loop formation. This allows subsequent DNA repair synthesis and completion of the HR process. We have previously suggested that RAD54s activity is regulated by never-in-mitosis-gene A (NIMA) related kinase 1 (NEK1) in a cell cycle-specific manner to promote RAD51 removal and HR in G2 phase without interfering with RAD51s fork stabilization role during S phase. Here, we establish that Nek1 regulates the phosphorylation of Rad54 at Ser572 (S572) to promote HR in vivo in adult mice and in vitro in fibroblasts derived from such mice. In contrast, embryonic mice and fibroblasts derived from them do not require Nek1 for HR. We further show that HR requires Rad54 phosphorylation at S572 both in embryonic and adult fibroblasts and that this is mediated in embryonic fibroblasts by Nek3 and Nek5 instead of Nek1. Thus, our work identifies a developmental change in the regulation of HR and uncovers two new factors involved in this process.

cell biology↗

bioRxiv2025

Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac inflammation and heart failure development

Inflammation promotes heart failure (HF) development, and inhibition of IL-12{beta} simultaneously attenuates interleukin-12 (IL-12) and interleukin-23 (IL-23), two important proinflammatory cytokines. In this study, we used IL-12{beta} knockout (KO) mice to test the hypothesis that genetic inhibition of IL-12{beta} would attenuate transverse aortic constriction (TAC)-induced cardiac inflammation, hypertrophy, and dysfunction, as well as the consequent lung remodeling. IL-12{beta} KO in male and female mice significantly attenuated TAC-induced cardiac dysfunction as evidenced by improved left ventricular (LV) ejection fraction and fractional shortening. IL-12{beta} KO also significantly ameliorated the TAC-induced increase of LV weight, left atrial weight, lung weight, right ventricular (RV) weight, and their ratios to body weight or tibial length in male and female mice. In addition, IL-12{beta} KO significantly attenuated TAC-induced LV leukocyte infiltration, cardiomyocyte hypertrophy, fibrosis, and the consequent lung inflammation and remodeling. Moreover, IL-12{beta} KO reduced TAC-induced alterations of LV gene profile associated with inflammation and fibrosis, as shown by bulk LV RNA sequencing. Furthermore, we found that IL-12{beta} KO significantly attenuated TAC-induced LV accumulation of multiple immune cell subsets, activation of CD4+ and CD8+ T cells, and the percentage of central memory CD4+ and CD8+ T cells in the cardiac drainage lymph nodes. Finally, IL-12{beta} KO mice showed significantly reduced IFN{gamma}+CD8+ and CXCR3+CD8+ T cells in the drainage lymph nodes as compared with WT after TAC. These findings collectively demonstrate that IL-12{beta} plays a critical role in systolic overload-induced LV inflammation, remodeling, and dysfunction, likely through cardiac immune cell infiltration.

physiology↗

bioRxiv2025

Bidirectional control of a metabolic transition by the GID ubiquitin ligase

The GID/CTLH ubiquitin ligase is a multisubunit E3 conserved across eukaryotes. GID/CTLH has been implicated in a variety of processes, including metabolic regulation, cell proliferation, embryonic development and cell differentiation. However, our understanding of substrate recognition by GID/CTLH remains incomplete. Here, we characterize the regulation, specificity and functions of the putative substrate receptor Gid11 in budding yeast. We find that upon its expression during the switch of carbon source from glucose to ethanol, Gid11 associates with the core GID complex, likely forming a large ring assembly. Through systematic mutagenesis, we identify a negatively charged pocket on Gid11 that binds substrates carrying N-terminal degrons starting with threonine and define the Thr/N-degron motif. Such Thr/N-degrons are exposed upon co-translational removal of the initiator methionine, while escaping N-terminal acetylation. Finally, we show that during the switch to ethanol, GIDGid11 targets for degradation factors linked to glycolysis, opposing the established role of GID in the degradation of gluconeogenic enzymes during the reverse switch from ethanol to glucose. Taken together, our results establish Gid11 as a bona fide GID substrate receptor and highlight GID as a regulator of metabolic transitions.

molecular biology↗

bioRxiv2025

Structural basis for DNA double-strand break sensing by human MRE11-RAD50-NBS1 and its TRF2 complex

The MRE11-RAD50-NBS1 (MRN) complex is a central, multifunctional factor in the detection, signaling and nucleolytic processing of DNA double-strand breaks (DSBs). To clarify how human MRN binds generic and telomeric DNA ends and can separate DNA end sensing from nuclease activities, we determined cryo-electron microscopy structures of human MRN bound to DNA and to DNA and the telomere protection factor TRF2. MRN senses DSBs through a tight clamp-like sensing state with closed coiled-coil domains, but auto-inhibited MRE11 nuclease. NBS1 wraps around the MRE11 dimer, with NBS1s ATM recruitment motif sequestered by binding to the regulatory RAD50 S site, necessitating an allosteric switch for ATM activation. At telomeric DNA, TRF2 blocks the second S site via the iDDR motif to prevent nuclease and ATM activation. Our results provide a structural framework for topological DNA sensing and separation of sensing, signaling and processing activities of mammalian MRN. Highlights- Human MRN senses DNA ends with an autoinhibited nuclease - NBS1s C-terminus binds one RAD50 S site in the sensing state - TRF2 binds MRNs second S site at telomeres - RAD50 and ATM compete for the NBS1 C-terminus

biochemistry↗

bioRxiv2024

A long non-coding RNA LINC00094 regulates the transcriptional expression of lipid metabolism-related genes as a new member of core regulatory circuitry in esophageal squamous cell carcinoma

LINC00094 as a new supper-enhancer (SE)-related long non-coding RNA is associated with poor overall survival of patients with esophageal squamous cell carcinoma (ESCC). However, the transcriptional regulatory mechanism of LINC00094 and the molecular mechanisms by which LINC00094 affects the phenotype of ESCC remains unclear. Here, we found that LINC00094 promoted the proliferation of ESCC cells both in vitro and in vivo. LINC00094 knockdown significantly reduced the expression profiles of transcription activators including transcription factor 3 (TCF3) and Kruppel like factor 5 (KLF5) and lipid metabolism-related genes. Mechanically, TCF3 and KLF5 formed a core regulatory circuitry (CRC) that bound to the SEs of LINC00094 and to their own SEs to regulate the transcriptional expression in a positive feedback loop. LINC00094 recruited TCF3 and KLF5 to form a ternary complex, which forms a new CRC with TCF3 and KLF5 that regulated its own transcription as well as lipid metabolism-related genes. Knockdown of any or all three genes inhibited the expression of genes related to lipid synthesis and consistently reduced total lipid droplet levels. Treatment with SEs inhibitors (THZ1 and JQ1) effectively inhibited the formation of this CRC and the production of lipid droplets in ESCC cells. The high-risk group of CRC-associated signatures were closely associated with poor prognosis in patients with ESCC. Our findings suggest that LINC00094 is involved in the CRC by forming a complex with TCF3 and KLF5, and this regulation model can affect the phenotype of ESCC cells by controlling the expression of lipid metabolism-related genes. HighlightsO_LIWe identified a novel functional lncRNA-LINC00094 for esophageal squamous cell carcinoma. C_LIO_LILINC00094 forms a complex with the core transcription factors TCF3 and KLF5, thereby forming a core regulatory circuitry to participate in transcriptional regulation in ESCC. C_LIO_LIA core regulatory circuitry mediated by LINC00094 regulates lipid metabolism in ESCC. C_LI

cancer biology↗

bioRxiv2023

P53 Acetylation Exerts Critical Roles In Pressure Overload Induced Coronary Microvascular Dysfunction and Heart Failure

Coronary microvascular dysfunction (CMD) has been shown to contribute to cardiac hypertrophy and heart failure with preserved ejection fraction. At this point, there are no proven treatments for CMD. We have shown that histone acetylation may play a critical role in the regulation of CMD. By using a mouse model that replaces lysine with arginine at residues K98/117/161/162R of p53 (p534KR), preventing acetylation at these sites, we test the hypothesis that acetylation-deficient p534KR could improve coronary microvascular dysfunction and prevent the progression of hypertensive cardiac hypertrophy and heart failure. Wild-type (WT) and p534KR mice were subjected to pressure overload (PO) by transverse aortic constriction to induce cardiac hypertrophy and heart failure (HF). Echocardiography measurements revealed improved cardiac function together with reduction of apoptosis and fibrosis in p534KR mice. Importantly, myocardial capillary density and coronary flow reserve (CFR) were significantly improved in p534KR mice. Moreover, p534KR upregulated the expression of cardiac glycolytic enzymes and glucose transporters, as well as the level of fructose-2,6-biphosphate; increased PFK-1 activity; and attenuated cardiac hypertrophy. These changes were accompanied by increased expression of HIF-1 and proangiogenic growth factors. Additionally, the levels of SERCA-2 were significantly upregulated in sham p534KR mice as well as in p534KR mice after TAC. In vitro, p534KR significantly improved endothelial cell (EC) glycolytic function and mitochondrial respiration, and enhanced EC proliferation and angiogenesis. Similarly, acetylation-deficient p534KR significantly improved CFR and rescued cardiac dysfunction in SIRT3 KO mice. Our data reveal the importance of p53 acetylation in coronary microvascular function, cardiac function, and remodeling, and may provide a promising approach to improve hypertension-induced coronary microvascular dysfunction (CMD) and to prevent the transition of cardiac hypertrophy to heart failure.

pathology↗

bioRxiv2022

Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration

Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N6-methyladenosine (m6A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays important roles in various physiological and pathological bioprocesses. However, the role of m6A in heart regeneration remains largely unclear. To study m6A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m6A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m6A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of {Delta}Fgf16, in which the m6A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m6A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.

cell biology↗

bioRxiv2020

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system of Caenorhabditis elegans to dissect the action of two virulence factors from its natural fungal pathogen Drechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second, potentially as a consequence of a host surveillance mechanism, increases STA-2 levels in the nucleus, modifies the nucleolus, and causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.

microbiology↗

bioRxiv2019

CsHscB as a novel TLR2 agonist from carcinogenic liver fluke Clonorchis sinensis modulates host immune response

Clonorchis sinensis-a fluke dwelling on the intrahepatic bile ducts causes clonorchiasis. During C. sinensis infection, worm-host interaction results in activation of PRRs and further triggers immune responses which determine the outcome of infection. However, the mechanisms by which pathogen-associated molecules patterns from C. sinensis interacted with TLRs were poorly understood. In the present study, we identified a [~]34 kDa lipoprotein CsHscB from C. sinensis which physically bound with TLR2. We also found that recombinant CsHscB (rCsHscB) potently activated macrophage to express various proteins including TLR2, CD80, MHCII, and cytokines like IL-6, TNF-, and IL-10 in a TLR2-dependent manner but rCsHscB failed to induce IL-10 in macrophages from Tlr2-/- mice. Moreover, ERK1/2 activation was required for rCsHscB-induced IL-10 production in macrophages. In vivo study revealed that rCsHscB triggered a high induction of IL-10 in the wild-type (WT) but not in Tlr2-/- mice. Our data thus demonstrate that rCsHscB from C. sinensis is an unidentified TLR2 agonist with immune regulatory activities, and may have some therapeutic implications in future beyond parasitology.

immunology↗

bioRxiv2017

Evolutionary plasticity in the innate immune function of Akirin

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NF{kappa}B. Although nematodes lack NF{kappa}B, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

immunology↗