Protein & Cell

ATAD2 possesses a C-terminal bromodomain (BRD) that plays a critical role in recognizing and binding to acetylated lysine residues. However, because the native intracellular structure of ATAD2 remains poorly defined, the mechanisms by which the ATAD2 BRD recruits acetylated histones and the regulatory pathways involved are not yet understood. In this study, we report that the ATAD2 BRD mediates the formation of liquid-liquid phase separation (LLPS) of ATAD2 in cells. This phase separation promotes the process of histone H4 acetylation, leading to the up-regulation of C-MYC, CCND3, and ATF2 gene expression and the facilitation of chromatin remodeling. Our findings elucidate a vital function of ATAD2, wherein BRD-mediated LLPS drives histone acetylation to promote cellular chromatin remodeling.

It remains unclear whether podocyte loss directly causes acute renal tubular cell (RTC) damage and interstitial fibrosis, thereby leading to renal failure. Here, we applied intermedilysin (ILY)-mediated human CD59 (hCD59) cell ablation to generate an acute, specific podocyte-ablation mouse model. Cre-induced hCD59 transgenics (ihCD59) were crossed with Nphs2Cre to generate ihCD59+/-/Nphs2Cre+/- mice. The specific and rapid podocyte-ablation mediated by ILY injection directly caused RTC necrosis, leading to renal failure and even death within 2-3 days in a dose-dependent manner. Treating mice that received an ILY lethal dose with peritoneal dialysis or administering a non-lethal dose, we extended their survival beyond six weeks and found that mice developed interstitial fibrosis and glomerulosclerosis with persistent proteinuria and tubule damage. Podocyte-ablation caused massive disruption of glomerular function at week 1, and then partial recovery by week 2. Genes and pathways of TLRs and apoptosis, and mitochondrial functions were respectively upregulated and downregulated in both ablated-podocyte mouse and biopsied-glomerulonephritis patient kidney samples. Together, this rapid podocyte-ablation causes acute RTC necrosis that progresses to interstitial fibrosis in this mouse model, which is applicable for dissecting mechanisms underlying podocyte injury-mediated tubular damage and glomerular repair, with the potential to reveal novel therapeutic targets for kidney diseases.

The cGAS-STING pathway has been widely recognized as a critical DNA-sensing pathway that plays a broad-spectrum antiviral role. Livestock, especially pigs, represents one of the most important meat sources. In this study, we identified a key lysine 61 (K61) of porcine STING (pSTING) that plays an essential role in its degradation and antiviral signaling in a species-specific manner, with K61 as the major lysine of pSTING for K48-linked ubiquitination. After virus infection, pSTING recruits the E3 ligase, RNF5, which specifically assembles a K48-linked ubiquitin chain at K61, thereby mediating pSTING proteasomal degradation and reducing its antiviral activity. Meanwhile, the deubiquitylation of K61 is mediated mainly by deubiquitinase USP20, which enhances the stability and antiviral activity of pSTING. Together, given the relatively few lysine numbers in livestock STINGs and species-specific K61 regulation of pSTING stability and antiviral function, the K61 and its specific regulatory enzymes of pSTING could serve as potential targets for breeding of antiviral pigs and design of antiviral drugs, respectively.

PUF60 is a splicing factor related to the polypyrimidine-tract binding protein U2AF2. PUF60 is deleted in developmental disorders such as Verheij syndrome and amplified in approximately 8% of cancers. Thus, both increases and decreases in PUF60 expression can have profound physiological effects. However, little is known about how changes in PUF60 expression impact global splicing patterns. Here, we created a model system of CRISPRa/i in mouse stem cells (mESCs) to transcriptionally upregulate or downregulate Puf60. Our results uncovered extensive transcriptional, post-transcriptional, and post-translational regulation of Puf60 protein expression. We observed that Puf60 protein levels in normal mESCs drop dramatically at a critical cell density, leading to cell death. Puf60 is very essential in stem cells, and its repression causes cell death and impacts specific splicing events, including its own splicing autoregulation, providing valuable insights into the functional consequences of PUF60 dysregulation. Analysis of phosphoprotein data revealed phosphorylation of threonine at the N-terminus of PUF60. Our results showed that mutating threonine to glutamate downregulates the protein and alters its localization. Thus, our study reveals a novel regulatory mechanism of Puf60 phosphorylation that mediates its function and may be related to its frequent overexpression in cancer cells.

The maintenance of protein homeostasis is vital for all cells. Alteration in protein handling underlies several diseases. The small molecule sephin1 is a promising clinical candidate against proteostasis disruption, but its mechanism of action is still uncertain. Our experimental evidence shows that sephin1 binds G-actin and drives actin cytoskeleton misfolding, and eventually, Golgi disintegration. At first, sephin1 impairs the autophagic flux and elicits the phosphorylation of the subunit of eIF2 and the ER-stress independent expression of CHOP via GCN2 kinase. Sephin1 also inhibits the mammalian target of rapamycin (mTORC1), activates the transcription Factor EB (TFEB), drives the expression of TFEB-direct target genes, and eventually stimulates the autophagy lysosomal pathway. Our results reveal that the actin cytoskeleton may regulate autophagy via mTORC1-TFEB complemented with the GCN2-eIF2-CHOP signaling pathway.

Long noncoding RNAs (LncRNAs) have emerged as a class of important regulatory ncRNAs and are known to fine-tune numerous cellular processes including proliferation, differentiation and development; however, their role in quiescence still remains largely unexplored. A miRNA host gene lncRNA, MIR503HG, has been reported to play important role in cancer development. Here, we demonstrate the role of MIR503HG lncRNA in regulating cellular quiescence. MIR503HG displays elevated levels in human diploid fibroblasts induced to undergo quiescence. Depletion of MIR503HG in HDFs affects the entry of cells into quiescence but has no effect on cell cycle progression, suggesting its role in quiescence attainment and/or maintenance. Additionally, MIR503HG depletion led to a drastic decrease in the levels of miR508 target, PTEN with a concomitant increase in pAkt levels, indicating its role in negative regulation of miR508. Further, we demonstrate that the lncRNA MIR503HG regulates PTEN levels by acting as a ceRNA for miR508 to maintain cellular quiescence. Our studies illustrate that MIR503HG can function synergistically with miR503 to maintain cells under quiescence and both the miRNA-HG and the miRNA encoded by its gene locus synergistically control the same biological process in different ways by regulating different downstream genes.

Immediate early genes (IEGs) encode master transcription factors, including EGR1, FOS, JUN, and MYC genes that drive robust transcription in the G1 phase of cell cycle. Our previous studies indicate that topoisomerase II{beta} (TOP2B) critically regulates IEG transcription and that the activities of TOP2B are dynamically controlled through post-translational modifications by BRCA1-BARD1 and ERKs. Here, we show that two E2 enzymes, UBCH5b and UBC13/MMS2, differentiate the effects and functions of TOP2B ubiquitination by BRCA1-BARD1. Comprehensive transcriptomics, proteomics, and biochemical and molecular cellular analyses revealed a close relationship between BARD1 and key mitogen-activated protein kinase pathway genes and identified activated ERK2 as a novel kinase that phosphorylates BARD1 at S391, a previously reported mitotic phosphorylation site, whose genetic mutation has been linked to tumorigenesis. Mechanistically, the catalytic activity of ERK2 stimulates TOP2B ubiquitination mediated by BRCA1-BARD1 in complex with UBCH5b and UBC13/MMS2, which controls the binding and function of TOP2B and BARD1 for transcriptional activation at representative IEGs. Taken together, our data propose that there is a functional regulatory circuit involving TOP2B, BARD1, and ERK2, three key transcriptional activators for IEG transcription, in which the gene association and catalytic activity of TOP2B are regulated through E2-differentiated ubiquitination by BRCA1-BARD1 and the phosphorylation of BARD1 by ERK2 for productive transcription.

Oncolytic virotherapy is an innovative approach to cancer treatment that uses replication-competent viruses to selectively target and destroy cancer cells while leaving healthy tissues largely unaffected. Zika virus (ZIKV), a neurotropic orthoflavivirus, has recently gained attention as a potential oncolytic agent due to its ability to infect neural-derived cells and suppress tumor growth in preclinical models. Although existing studies have examined ZIKVs oncolytic effects, the mechanisms underlying these effects remain largely unexplored. Additionally, the roles of individual ZIKV proteins and their interactions with host factors remain incompletely understood. Here, we used RNA sequencing, affinity purification-mass spectrometry, and functional assays to uncover previously unidentified mechanisms underlying ZIKVs oncolytic activity in pediatric neural tumors. We found that the ZIKV non-structural proteins NS4A and NS5 exert oncolytic effects, reducing tumorsphere size. ZIKV-host protein-protein interaction networks were characterized and showed that integrin 3 (gene: ITGA3), a mediator of cell-matrix adhesion, interacts with ZIKV NS2B and NS4A. Integrin 3 was further shown to be involved in ZIKV- and NS4A-induced tumorsphere size reduction, while ITGA3 knockdown and ZIKV infection additively inhibited 3D invasion. These findings provide critical mechanistic insights that could inform the rational design of ZIKV-based virotherapies and highlight opportunities for combination treatment strategies.

The molecular mechanisms driving SLFN11 chromatin recruitment remain partially elucidated. Using high-throughput imaging of 162 oncology-focused compounds in U2OS cells with inducible SLFN11 expression, we discovered that deubiquitinase (DUB) inhibitors drive massive SLFN11 recruitment to chromatin, preferentially at promoter regions while concurrently suppressing transcription. DUB inhibitors such as VLX-1570 promote ubiquitin-dependent enrichment of SLFN11 without detectable DNA damage, distinct from the camptothecin-induced RPA-associated SLFN11 foci formed at stressed replication forks. Yet, SLFN11 chromatin recruitment both by DUB inhibitors and DNA damage are suppressed by TAK243 demonstrating their ubiquitylation dependency. RNF168 is required for SLFN11 ubiquitylation and its subsequent chromatin association, and ubiquitylation within SLFN11s middle linker domain (lysines 390, 391, and 429) with K27-linked polyubiquitin chains is essential for the chromatin recruitment of SLFN11. These findings suggest the importance of SLFN11 ubiquitylation by RNF168 for SLFN11 chromatin recruitment and SLFN11 transcriptional regulatory role at promoter regions.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected illness with outdated treatments. Bromodomain factors (BDFs) are essential proteins that recognize acetylated lysines and have strong therapeutic potential. They form part of epigenetic complexes that regulate chromatin accessibility and, therefore, gene expression. However, little is known about their structure in trypanosomatids. Here, we used a combination of experimental and bioinformatic approaches to infer the stoichiometry and structure of T. cruzi bromodomain-containing complexes. By reconstructing the proximity networks of five BDFs using TurboID-directed proximity labeling, we identified highly interconnected components that assemble into the CRKT and NuA4 complexes. Using novel structure prediction strategies that systematically explore the stoichiometric space, we inferred that CRKT assembles into three distinct modules and NuA4 in two, with different degrees of interaction dynamics. The core module of CRKT contains two copies of each component, including BDF3, BDF5, and BDF8, arranged in a subcomplex with central symmetry. The catalytic module of CRKT has three subunits, including the histone acetyltransferase 2 (HAT2), while the BET (bromodomain and extra-terminal) module has one unit of both BDF4 and BDF1. The catalytic module of NuA4 closely resembles the yeast piccolo-NuA4 module and contains HAT1, while the TINTIN module associates with the catalytic module via the C-terminal domain of BDF6. These insights shed light on the structure and composition of epigenetic complexes in trypanosomatids, opening new avenues for rational drug design aimed at disrupting their function.

Cajal bodies (CBs) are nuclear membrane-less organelles that accumulate various short non-coding RNAs and facilitate their biogenesis. They also function in quality control during the assembly of small nuclear ribonucleoprotein particles (snRNPs), sequestering immature or defective complexes. In this paper, we show that coilin, the key scaffolding protein of CBs, is the factor that discriminates between mature and immature snRNPs. We provide evidence that the C-terminus of coilin contains a bipartite snRNP-interaction module composed of a nonspecific RNA-binding region formed by RG repeats and a Tudor-like domain that interacts specifically with Sm proteins. The Tudor-like domain contains two conserved loops that protrude from the core barrel and bind the Sm proteins E, F, and G. Both the RNA-binding and Sm-binding regions are essential for productive interactions between coilin and core snRNPs, providing a molecular explanation for the specificity of coilin-mediated sequestration of immature snRNPs in Cajal bodies.

The biosynthetic pathway of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is a universal and essential metabolic process in many organisms, providing the activated sulfate donor required for the synthesis of diverse sulfated metabolites. However, this pathway has undergone substantial evolutionary diversification among species. In Entamoeba histolytica, PAPS biosynthesis occurs within the mitosomes, mitochondrion-related organelles (MROs), representing a distinctive example of lineage-specific evolutionary adaptation. PAPS synthesis proceeds through a conserved two-step, which is sequentially catalyzed by ATP sulfurylase (AS) and adenosine 5'-phosphosulfate (APS) kinase (APSK). In this study, we focused on E. histolytica APSK (EhAPSK). EhAPSK contains an additional AS-like domain (SLD), although its functional role remains unclear. Here, we determined the crystal structure of full-length EhAPSK at 2.60 [A] resolution and the structure of the truncated EhAPSK lacking APS kinase domain (KD) (EhAPSK{Delta}KD) at 2.10 [A] resolution. Structural analyses revealed that the SLD engages in dynamic contacts with the KD. Furthermore, deletion of the domain and mutational analyses indicated that the SLD significantly influences the catalytic activity of the KD. Based on these findings, we propose a new regulatory mechanism in which transient interdomain interactions modulate APS kinase activity, representing an unique evolutionary adaptation of E. histolytica.

Translation efficiency remains a major limitation for RNA therapeutics. Conventional optimization targets the 5 untranslated region (5 UTR), while the 3 UTR is viewed mainly as a stabilizing element. Here, we demonstrate that the 3 UTR can be rationally engineered to actively enhance translation. Using an intracellular directed-evolution platform based on the SINEB2 element, we identified RNA modules P51 and its compact variant P51t3,which markedly increased protein output without affecting mRNA levels. P51t3 consistently boosted expression two- to six-fold across plasmid, in vitro transcribed mRNA, and recombinant AAV systems. Mechanistic studies revealed that P51t3 binds ribosomal protein RPL39, recruiting 60S subunits to the initiation site through the natural closed-loop translation model. By integrating evolutionary selection with 3 UTR design, this work redefines the 3 UTR as an active translational enhancer and provides a broadly applicable regulatory element for next-generation mRNA and gene-delivery therapeutics.

H3K27ac and H3K4me3 are enriched at transcriptional start sites and have been implicated in transcription. However, how these marks concertedly regulate transcription is not fully understood. Here, we developed a dual chemically inducible CRISPR/dCas9-based epigenome editing system that enables independent, temporal and transcription stage-specific modulation of H3K27ac and H3K4me3 at a specific gene locus. Stage-specific removal of H3K4me3 impaired RNA polymerase II recruitment, increased promoter-proximal pausing, reduced productive elongation, and accelerates mRNA decay via increased m6A deposition. Losing both H3K27ac and H3K4me3 rapidly abolished transcriptional activity, while preserving H3K4me3 without H3K27ac can partially sustain transcription. These findings revealed a functional hierarchy and interdependence between H3K27ac and H3K4me3 in different transcription stages and the established versatile tool will contribute to the functional dissection of the temporal dynamics of chromatin modifications in gene regulation.

Ferroptosis is a unique form of regulated cell death characterized by iron-dependent lipid peroxidation. Although the molecular details of ferroptosis regulation have been widely explored, the contributions of short non-coding RNAs (ncRNAs) to ferroptosis regulation, other than miRNAs remain poorly understood. Here, we identified vault RNA1-1 (vtRNA1-1) as a previously unrecognized short ncRNA regulator of ferroptosis in hepatocellular carcinoma (HCC) cells. vtRNA1-1 expression was upregulated by ferroptosis inducers and exhibited strong negative correlation with ferroptosis sensitivity, thus protecting cells from ferroptosis. vtRNA1-1 levels were elevated in selected ferroptosis-resistant cells, while its depletion reversed the phenotype thus resensitizing these cells to ferroptosis. These findings suggested a contribution of vtRNA1-1 to both intrinsic and acquired ferroptosis resistance. Mechanistically, we uncovered that increased oxidative stress, which potentiates lipid peroxidation, specifically induced expression of the vtRNA1-1 paralog in an NF-{kappa}B dependent manner. Elevated vtRNA1-1 levels suppressed NF-{kappa}B-mediated pro-oxidant gene expression, thereby limiting reactive oxygen species (ROS) accumulation and alleviating oxidative stress. Taken together, oxidative stress-inducible vtRNA1-1 governs redox balance by forming a reciprocal regulatory loop with NF-{kappa}B and this loop determines ferroptosis susceptibility by adjusting basal ROS levels. Our findings provide unprecedented insights into the regulation of redox homeostasis in HCC cells mediated by a short ncRNA and uncovered vtRNA1-1 as a potential therapeutic target for overcoming ferroptosis resistance in liver cancer.

The circadian clock maintains temporal control of metabolic processes and exerts a key role in adipocyte development. Discovery of clock-modulatory compounds may provide new avenues for metabolic disease therapy. Here we report the identification of flavonoid compounds, Quercetin and Fisetin, as clock-activating molecules with direct inhibitory action on adipogenesis and adipocyte lipid metabolism. Quercetin and Fisetin displayed robust ROR agonism that promoted clock oscillation with induction of clock genes. Treating preadipocytes with these compounds blocked their adipogenic differentiation. In mature adipocytes, Quercetin and Fisetin suppressed lipid accumulation by inhibiting lipogenic enzymes. Furthermore, activation of ROR by a synthetic agonist or ectopic expression were sufficient to inhibit adipogenesis. In mice treated with Quercetin or Fisetin, ROR was markedly induced in adipose depots with strong suppression of the adipogenic and lipogenic programs. While quercetin significantly attenuated lipid storage in adipose tissue in vivo accompanied with lowering of free fatty acids and improved insulin sensitivity, fisetin displayed a less robust effect with differential regulation of lipolytic pathway. Collectively, these findings uncovered the clock-activating properties of quercetin and fisetin that prevent adipocyte maturation and hypertrophy to limit adipose tissue expansion. These actions contribute, at least in part, to their beneficial effects on metabolic disorders.

Dysregulated lipid metabolism drives cancer progression, yet the molecular mechanisms linking lipid accumulation to pancreatic cancer remain poorly understood. Here, we identify methyltransferase-like 7B (METTL7B) as a key regulator of lipid metabolism and tumor progression in pancreatic cancer. Integrated analysis of large-scale transcriptomic datasets revealed that METTL7B is upregulated in pancreatic cancer compared with normal tissues, and elevated METTL7B expression is associated with poor patient prognosis. Functional experiments demonstrated that METTL7B is required for pancreatic cancer cell proliferation, migration, and metastasis. We identified hepatocyte nuclear factor 4 (HNF4A) and hepatocyte nuclear factor 4{gamma} (HNF4G), well-established regulators of lipid homeostasis, as transcription factors that directly upregulate METTL7B expression in pancreatic cancer cells. Moreover, we showed that METTL7B localizes around lipid droplets and promotes their accumulation in pancreatic cancer cells. Lipidomic analyses revealed that METTL7B depletion selectively reduces long-chain triacylglycerols and increases shorter-chain ceramides, while also modulating other lipid droplet-related lipid classes in a chain-length-dependent manner. Collectively, these findings suggest that METTL7B regulates lipid metabolism and malignant behavior in pancreatic cancer and may represent a potential therapeutic target.

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

CRISPR-based epigenome editing represents a programmable strategy to precisely modulate gene expression, holding immense promise for therapeutic applications. However, the large size of the dCas proteins substantially impedes the delivery via adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions, and functional assays, we identified and characterized PmCas12m, a novel miniature subtype V-M CRISPR-Cas12m. PmCas12m exhibited flexible 5-YTN-3 PAM-dependent recognition and robust double-stranded DNA binding properties, while lacking DNA cleavage activity, thus positioning it as an ideal tool for epigenome editing. Cryogenic electron microscopy (cryo-EM) structures of PmCas12m unveiled its unique molecular mechanism of DNA binding facilitating interference. Guided by these structural insights, we employed deep mutational scanning (DMS) and protein engineering to develop xCas12m, a hypercompact variant with highly potent and specific epigenome editing capabilities in human cells. We further constructed the xCas12m-CRISPRoff platform in a single AAV vector, which achieved durable epigenetic silencing and effective inhibition of hepatitis B virus (HBV) infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome editing platform with transformative potential for treating diseases, paving the way for clinical translation of epigenetic therapies.

Silencing complexes formed by PIWI-clade Argonaute (Ago) proteins and PIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, controlling the deleterious activities of transposable elements (TEs) in animal germline. However, our understanding of PIWI-piRNA-directed TE silencing remains incomplete. Here, we systemically characterize the proximity proteome of PIWI members, Piwi, Aubergine (Aub), and Ago3 in the germline of Drosophila ovaries. Functional screening identifies previously uncharacterized factors involved in TE silencing, including H3K4me3 writer and transcriptional coactivator Set1. Transcriptome analysis reveals that Set1 acts as an indispensable repressor for TEs, particularly those forming telomeres. The involvement of Set1 in Piwi pathway is further supported by its critical role in the production of antisense, TE-targeting piRNAs. Notably, catalytic activity of Set1 is dispensable for TE silencing. Genome-wide chromatin binding analysis using CUT&Tag demonstrates that Set1 preferentially associates with TE sequences and localizes to subtelomeric piRNA cluster loci, suggesting a role in promoting piRNA precursor transcription through direct binding. Collectively, these findings uncover a noncanonical function of Set1 in Piwi-mediated TE silencing and telomere control in germline nuclei.