Biology subjects

Liang, J.

Publications and source records attributed to Liang, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Urine Proteomics in Methamphetamine Addicted Patients

Drug addiction is a serious chronic relapsing brain disease, and methamphetamine dependence has a complex course and is difficult to treat, causing a serious public health burden. In this study, we compared and analyzed the urine proteome of methamphetamine-using patients, methamphetamine-withdrawing patients, healthy individuals. The urine proteome of methamphetamine-using patients and methamphetamine-withdrawing patients was significantly different from that of healthy individuals, and some differential proteins and their enriched biological functions showed that they were associated with addiction or neurotoxicity of methamphetamine. The fact, that the urine proteome of patients who withdrew methamphetamine for more than 3 months were significantly different from that of healthy patients, may reveal the reasons for the high rate of methamphetamine relapse and potential intervention targets. This study proved that urine proteome can reflect the effects of addiction on the body comprehensively, and has the potential to provide clues for addiction.

biochemistry↗

bioRxiv2024

Structural Basis of Differential Gene Expression at eQTLs Loci from High-Resolution Ensemble Models of 3D Single-Cell Chromatin Conformations

MotivationTechniques such as high-throughput chromosome conformation capture (Hi-C) have provided a wealth of information on the organization of the nucleus and the genome important for understanding gene expression regulation. Additionally, Genome-Wide Association Studies (GWASs) have uncovered thousands of loci related to complex traits. Expression quantitative trait loci (eQTL) studies have further linked the genetic variants to alteration in expression levels of associated target genes across individuals. However, the functional roles of many eQTLs located in non-coding regions are unclear. Current joint analyses of Hi-C and eQTLs data lack advanced computational tools, limiting what can be learned from these data. ResultIn this work, we developed a computational method for simultaneous analysis of Hi-C and eQTL data. Our method can identify a small set of non-random interactions from all Hi-C interactions. Using these non-random interactions, we reconstruct large ensemble (x105) of high-resolution single-cell 3D chromatin conformations with thorough sampling, which accurately replicate Hi-C measurements. Our results revealed the presence of many-body interactions in chromatin conformation at single-cell level in eQTL locus, offering detailed view into how three-dimensional structures of chromatin form the physical foundation for gene regulation, including how genetic variants of eQTLs affect the expression level of their associated eGenes. Furthermore, our method can deconvolve chromatin heterogeneity and investigate the spatial associations of eQTLs and eGenes at subpopulation level to reveal their regulatory impacts on gene expression. Together, ensemble modeling of thoroughly sampled single cell chromatin conformations from Hi-C, along with eQTL data, helps to decipher how chromatin 3D structures provide the physical basis for gene regulation, expression control, and aid in understanding of the overall structure-function relationships of genome organization. Availability and implementation: It is available at https://github.com/uic-liang-lab/3DChromFolding-eQTL-Loci

bioinformatics↗

bioRxiv2024

A New Approach for Discovering Functional Links Connecting Non-Coding Regulatory Variants to Gene Targets

Genome-wide association studies (GWAS) have linked thousands of genetic variants to various complex traits or diseases. However, most identified variants have weak individual effects, are correlated with nearby polymorphisms due to linkage disequilibrium (LD), and are located in non-coding cis-regulatory elements (CREs). These characteristics complicate the assessment of the direct impact of each variant on tissue specific gene expression and phenotype. To address this challenge, we have developed a novel algorithm that leverages polymer folding and 3D chromatin interactions to prioritize and identify putative causal variants and their target genes. From the millions of eQTL-Gene pairs identified by GTEx in human somatic tissues, we classify only [~]10-20% as putative functional eQTL-Gene pairs supported by phenotypic associations confirmed through CRISPR deletion experiments. Our findings show that unlike most variants, functional eQTL-Gene pairs predominantly reside within the same topologically associating domain (TAD) and have strong associations with cell-type specific cis-regulatory elements (CREs), enriched for binding sites of tissue-specific transcription factors. Unlike most approaches that rely on linear distance or other chromatin features (histone code, accessibility), our algorithm emphasizes the importance of physical interactions and 3D chromatin folding in gene regulation, as the identified eQTL-Gene pairs are all among the small fraction of physical chromatin interactions sufficient for chromatin locus folding. Overall, our algorithm reduces false positive associations between DNA variants and genes identified by eQTL analysis and uncovers novel variant-gene pair associations. These findings suggest a mechanism where a small number of regulatory variants control tissue specific gene expression via their physical association with target genes confined within the same TAD. Our approach provides new insights into the molecular mechanisms driving GWAS phenotypes.

genomics↗

bioRxiv2024

A multi-gene predictive model for the radiation sensitivity of nasopharyngeal carcinoma based on machine learning

BackgroundRadiotherapy resistance is a major obstacle to the long-term survival of nasopharyngeal cancer patients, as it is a primary cause of recurrence and metastasis. Identifying radiotherapy-associated biomarkers can help improve the survival prognosis of nasopharyngeal cancer patients. Consequently, discovering biomarkers associated with radiosensitization is crucial. MethodsWe evaluated 113 combinations of machine learning algorithms and ultimately selected 48 to construct a radiotherapy sensitivity score (NPC-RSS) that can predict radiosensitivity in nasopharyngeal cancer patients. Furthermore, we analyzed the relationship between NPC-RSS and the expression of genes associated with immune and radiotherapy sensitivity profiles. We employed GSEA and ssGSEA to investigate the connection between NPC-RSS and signaling pathways. ResultsWe selected the combined model glmBoost+NaiveBayes, which had the best AUC among 48 models, for our subsequent study. The NPC-RSS, built based on the 18 genes included in this model, can predict the results of the public dataset and the in-house dataset of Zhujiang Hospital, Southern Medical University, with considerable efficiency. The key genes of NPC-RSS are closely associated with immune characteristics, including chemokine and chemokine receptor families, and histocompatibility complex (MHC), and show more active immune processes. Meanwhile, these key genes were significantly associated with the expression of radiosensitization-related genes. Furthermore, GSVA and GSEA analyses demonstrated that different expression levels of key NPC-RSS genes influenced signaling pathways, such as the Wnt/{beta}-catenin signaling pathway, JAK-STAT signaling pathway,NF-kappa B signaling pathway and T cell receptor signaling pathway, which are associated with immunity and disease progression. The consistency of the expression of key genes SMARCA2 and CD9 with NPC-RSS was validated in in-house cell lines. The radiosensitive group, classified according to NPC-RSS, exhibited a more enriched and activated state of immune infiltration compared to the radioresistant group. Moreover, in single-cell samples, NPC-RSS was higher in the radiotherapy-sensitive group, with immune cells playing a predominant role. ConclusionsIn this study, we used machine learning to construct a predictive score, called NPC-RSS, associated with radiosensitivity in nasopharyngeal carcinoma patients; moreover, NPC-RSS is strongly associated with immune characteristics, expression of radiosensitivity-related genes, and signaling pathways related to disease progression. We hope that the NPC-RCC will enable more precise selection of the NPC population of potential beneficiaries of radiation therapy.

bioinformatics↗

bioRxiv2024

Horizontally acquired CSP genes contribute to wheat adaptation and improvement

Although horizontal gene transfer (HGT) often facilitates environmental adaptation of recipient organisms, whether and how they might affect crop evolution and domestication is unclear. Here we show that three genes encoding cold shock proteins (CSPs) were transferred from bacteria to the last common ancestor of Triticeae, a tribe of the grass family that includes several major staple crops such as wheat, barley, and rye. The acquired CSP genes in wheat (TaCSPs) are functionally conserved with their bacterial homologs by encoding a nucleic acid binding protein. Experimental evidence indicates that TaCSP genes positively regulate drought response and improve photosynthetic efficiency under water deficit conditions, by directly targeting a type 1 metallothionein gene to increase ROS scavenging, which in turn contributed to the geographic expansion of wheat. We identified an elite CSP haplotype in Aegilops-tauschii, introduction of which to wheat significantly increased drought tolerance, photosynthetic efficiency and grain yields. These findings not only provide major insights into the role of HGT in crop adaptation and domestication, but also demonstrate that novel microbial genes introduced through HGT offer a stable and naturally optimized resource for transgenic crop breeding and improvement.

plant biology↗

bioRxiv2024

Contraction-induced endocardial id2b plays a dual role in regulating myocardial contractility and valve formation

Biomechanical cues play an essential role in sculpting organ formation. Comprehending how cardiac cells perceive and respond to biomechanical forces is a biological process with significant medical implications that remains poorly understood. Here we show that biomechanical forces activate endocardial id2b (inhibitor of DNA binding 2b) expression, thereby promoting cardiac contractility and valve formation. Taking advantage of the unique strengths of zebrafish, particularly the viability of embryos lacking heartbeats, we systematically compared the transcriptomes of hearts with impaired contractility to those of control hearts. This comparison identified id2b as a gene sensitive to blood flow. By generating a knockin reporter line, our results unveiled the presence of id2b in the endocardium, and its expression is sensitive to both pharmacological and genetic perturbations of contraction. Furthermore, id2b loss-of-function resulted in progressive heart malformation and early lethality. Combining RNA-seq analysis, electrophysiology, calcium imaging, and echocardiography, we discovered profound impairment in atrioventricular (AV) valve formation and defective excitation-contraction coupling in id2b mutants. Mechanistically, deletion of id2b reduced AV endocardial cell proliferation and led to a progressive increase in retrograde blood flow. In the myocardium, id2b directly interacted with the bHLH component tcf3b (transcription factor 3b) to restrict its activity. Inactivating id2b unleashed its inhibition on tcf3b, resulted in enhanced repressor activity of tcf3b, which subsequently suppressed the expression of nrg1 (neuregulin 1), an essential mitogen for heart development. Overall, our findings identify id2b as an endocardial cell-specific, biomechanical signaling-sensitive gene, which mediates intercellular communications between endocardium and myocardium to sculpt heart morphogenesis and function.

developmental biology↗

bioRxiv2024

CASTpFold: Computed Atlas of Surface Topography of the universe of protein Folds

Geometric and topological properties of protein structures, including surface pockets, interior cavities, and cross channels, are of fundamental importance for proteins to carry out their functions. Computed Atlas of Surface Topography of proteins (CASTp) is a widely used web server for locating, delineating, and measuring these geometric and topological properties of protein structures. Recent developments in AI-based protein structure prediction such as AlphaFold2 (AF2) have significantly expanded our knowledge on protein structures. Here we present CASTpFold, a continuation of CASTp that provides accurate and comprehensive identifications and quantifications of protein topography. It now provides (i) results on an expanded database of proteins, including the Protein Data Bank (PDB) and non-singleton representative structures of AlphaFold2 structures, covering 183 million AF2 structures; (ii) functional pockets prediction with corresponding Gene Ontology (GO) terms or Enzyme Commission (EC) numbers for AF2-predicted structures; and (iii) pocket similarity search function for surface and protein-protein interface pockets. The CASTpFold web server is freely accessible at https://cfold.bme.uic.edu/castpfold/.

biophysics↗

bioRxiv2024

Multi-omics delineate growth factor network underlying exercise effects in an Alzheimer's mouse model

Physical exercise represents a primary defense against age-related cognitive decline and neurodegenerative disorders like Alzheimers disease (AD). To impartially investigate the underlying mechanisms, we conducted single-nucleus transcriptomic and chromatin accessibility analyses (snRNA-seq and ATAC-seq) on the hippocampus of mice carrying AD-linked NL-G-F mutations in the amyloid precursor protein gene (APPNL-G-F) following prolonged voluntary wheel-running exercise. Our study reveals that exercise mitigates amyloid-induced changes in both transcriptomic expression and chromatin accessibility through cell type-specific transcriptional regulatory networks. These networks converge on the activation of growth factor signaling pathways, particularly the epidermal growth factor receptor (EGFR) and insulin signaling, correlating with an increased proportion of immature dentate granule cells and oligodendrocytes. Notably, the beneficial effects of exercise on neurocognitive functions can be blocked by pharmacological inhibition of EGFR and the downstream phosphoinositide 3-kinases (PI3K). Furthermore, exercise leads to elevated levels of heparin-binding EGF (HB-EGF) in the blood, and intranasal administration of HB-EGF enhances memory function in sedentary APPNL-G-F mice. These findings offer a panoramic delineation of cell type-specific hippocampal transcriptional networks activated by exercise and suggest EGF-related growth factor signaling as a druggable contributor to exercise-induced memory enhancement, thereby suggesting therapeutic avenues for combatting AD-related cognitive decline.

animal behavior and cognition↗

bioRxiv2024

The transcription factor ZNF469 regulates collagen production in liver fibrosis

Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the worlds population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.

molecular biology↗

bioRxiv2024

Evolution of a Eukaryotic Transcription Factor's co-TF Dependence Involves Multiple Intrinsically Disordered Regions Affecting Activation and Autoinhibition

Combinatorial control by transcription factors (TFs) is central to eukaryotic gene regulation, yet its mechanism, evolution, and regulatory impact are not well understood. Here we use natural variation in the yeast phosphate starvation (PHO) response to examine the genetic basis and species variation in TF interdependence. In Saccharomyces cerevisiae, main TF Pho4 relies on the co-TF Pho2 to regulate [~]28 genes, whereas in the related pathogen Candida glabrata, Pho4 has reduced Pho2 dependence and regulates [~]70 genes. We found C. glabrata Pho4 (CgPho4) binds the same motif with 3-4 fold higher affinity. Machine learning and yeast one-hybrid assay identify two intrinsically disordered regions (IDRs) in CgPho4 that boost its activation domains activity. In ScPho4, an IDR next to the DNA binding domain both allows for enhanced activity with Pho2 and inhibits activity without Pho2. This study reveals how IDR divergence drives TF interdependence evolution by influencing activation potential and autoinhibition.

evolutionary biology↗

bioRxiv2024

Phaeocystis blooms caused carbon drawdown during the Antarctic Cold Reversal from sedimentary ancient DNA

The Southern Ocean plays a crucial role in the global carbon budget. Modeling studies propose that the atmospheric CO2 plateau during the Antarctic Cold Reversal (ACR; 14,700 to 12,700 calibrated years before present (cal yr BP)) is related to increased marine productivity. However, proxy evidence relating environmental conditions as well as primary community composition and productivity to carbon drawdown is missing. Our ancient DNA shotgun metagenomic analysis of marine sediments revealed Phaeocystis antarctica (haptophyte) as a key element of the primary producer community. Independent proxy evidence (blooming-related bacteria, Ba/Fe ratio) from the same sediment record point to high productivity in response to enhanced sea-ice seasonality caused by ACR cooling. Post ACR, abrupt Phaeocystis community loss shows how sensitive this ecosystem is to warming, potentially representing a key tipping element that may be further enhanced by the Phaeocystis-related sulfur cycle-climate feedback. As an analogy for present warming, it highlights the importance of regions with high seasonal sea-ice variability and Phaeocystis-dominance, such as the Ross Sea, for stabilizing atmospheric CO2 content. Additionally, our shotgun metagenomic data portray complex Holocene ecosystem establishment including key Antarctic taxa such as penguins, whales, and Antarctic fishes with implications for ongoing conservation efforts.

ecology↗

bioRxiv2024

Transcriptional dynamics of the murine heart during perinatal development at single-cell resolution

Heart maturation and remodelling during the foetal and early postnatal period are critical for proper survival and growth of the foetus, yet our knowledge of the molecular processes involved are lacking for many cardiac cell types. To gain a deeper understanding of the transcriptional dynamics of the heart during the perinatal period, we performed single-cell RNA-seq on E14.5, E16.5, E18.5, P0, P4 and P7 mouse hearts to establish a catalogue of 49,769 cells. Gene regulatory network and pathway activity analyses underscored that heart maturation is strongly associated with regulation of cell growth and proliferation via pathways such as TGF{beta}. We additionally identified a common, cell type-independent signature for imprinted genes over time. Surprisingly, bioinformatics analyses and confirmation with RNAscope confirmed that while lncRNA H19 expression decreased over time in multiple cardiac cell types, it remained stably expressed in endocardial cells between E14.5 and P7. This suggests a differential requirement for H19 in the endocardium, and points towards an endocardium-specific maturation process when compared to other cardiac cell types. We envision this dataset to serve as a resource for better understanding perinatal heart maturation at the transcriptomic level, and to help bridge the gap between early developmental and adult heart stages for single-cell transcriptomics.

developmental biology↗

bioRxiv2024

Unveiling neoadjuvant chemotherapy-induced immune landscape remodeling and metabolic reprogramming in lung adenocarcinoma by scRNA-sequencing

BackgroundChemotherapy is widely used to treat lung adenocarcinoma (LUAD) patients comprehensively. Considering the limitations of chemotherapy due to drug resistance and other issues, it is crucial to explore the impact of chemotherapy and immunotherapy on these aspects. Methods and MaterialsTumor samples from nine LUAD patients, of which four only received surgery and five received neoadjuvant chemotherapy, were subjected to scRNA-seq analysis. In vitro and in vivo assays, including flow cytometry, immunofluorescence, Seahorse assay, and tumor xenograft models, were carried out to validate our findings. ResultsA total of 83,622 cells were enrolled for subsequent analyses. The composition of cell types exhibited high heterogeneity across different groups. Functional enrichment analysis revealed that chemotherapy drove significant metabolic reprogramming in tumor cells and macrophages. We identified two subtypes of macrophages: Anti-mac cells (CD45+CD11b+CD86+) and Pro-mac cells (CD45+CD11b+ARG+) and sorted them by flow cytometry. The proportion of Pro-mac cells in lung adenocarcinoma tissues increased significantly after neoadjuvant chemotherapy. Pro-mac cells promote tumor growth and angiogenesis and also suppress tumor immunity. Moreover, through analyzing the remodeling of T and B cells induced by neoadjuvant therapy, we noted that chemotherapy ignited a relatively more robust immune cytotoxic response towards tumor cells. ConclusionOur study demonstrates that chemotherapy induces metabolic reprogramming within the TME of LUAD, particularly affecting the function and composition of immune cells such as macrophages and T cells. We believe our findings will offer insight into the mechanisms of drug resistance and provide novel therapeutic targets for LUAD in the future.

cancer biology↗

bioRxiv2024

Sarcomeric SRX:DRX Equilibrium in Alport and LDLR/P407 Mouse Models of HFpEF.

Cardiac myosin energetic states that regulate heart contractility define interactions of myosin cross-bridges with actin-containing thin filaments have been functionally linked with the pathology of hypertrophic cardiomyopathy (HCM). In particular, the balance between the disordered relaxed (DRX) and super relaxed (SRX) states that correlate respectively with enhanced force and energy conservation significantly determine myocardial performance and energy utilization. Compelling evidence suggests that a balanced SRX and DRX states proportion is a prerequisite for long-term cardiac health. Whereas roles for altered SRX: DRX proportions in HCM have been studied in depth, the mechanics of sarcomeric dysfunction and SRX: DRX proportions have not been reported in models of acquired heart failure (HF) including HF with preserved ejection fraction (HFpEF). Here, we quantified SRX andDRX myosin populations in two mouse models of HFpEF, including Alport and LDLR/P407 mice that represent cardiorenal/hypertensive and cardiometabolic/hyperlipidemic mouse models of HFpEF, respectively. We report significant changes in the SRX:DRX in both HFpEF mouse models, with an increased DRX state associated with Alport mice and a stabilized SRX state associated with LDLR/P407 mice. These findings correlate respectively with the hypercontractility and metabolic dysregulation with bradycardia phenotypes.

cell biology↗

bioRxiv2024

Discovery and characterization of chromosomal inversions in the arboviral vector mosquito Aedes aegypti

Chromosomal inversions play a fundamental role in evolution and have been shown to regulate epidemiologically important traits in malaria mosquitoes. However, they have never been characterized in Aedes aegypti, the major vector of arboviruses, because of the poor structure of its polytene chromosomes. In this study, we applied a Hi-C proximity ligation approach to identify chromosomal inversions in 25 strains of Ae. aegypti, acquired from its worldwide distribution, as well as in one strain of Ae. mascarensis. The study identified 21 multi-megabase inversions with uneven distributions along the three chromosomes. All chromosomal inversions, including one specific for Ae. mascarensis, were polymorphic. Nevertheless, geographic origin separated the strains into two clusters carrying African and non-African inversions suggesting their potential association with Ae. aegypti subspecies. Some of the inversions colocalized with chemoreceptor genes and quantitative trait loci associated with pathogen infection, implicating the potential role of inversions in host choice and disease transmission.

evolutionary biology↗

bioRxiv2024

Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer

Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which these variants drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full-length AR (ARfl), despite its continued high-level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in VCaP16 cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression in VCaP cells increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and NFIX knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

cancer biology↗

bioRxiv2024

Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation

The spatiotemporal transition of small GTPase Rab5 to Rab7 is crucial for early-to-late endosome maturation, yet the precise mechanism governing Rab5-to-Rab7 switching remains elusive. USP8, a ubiquitin-specific protease, plays a prominent role in the endosomal sorting of a wide range of transmembrane receptors and is a promising target in cancer therapy. Here, we identified that USP8 is recruited to Rab5-positive carriers by Rabex5, a guanine nucleotide exchange factor (GEF) for Rab5. The recruitment of USP8 dissociates Rabex5 from early endosomes (EEs) and meanwhile promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In USP8-deficient cells, the level of active Rab5 is increased, while the Rab7 signal is decreased. As a result, enlarged EEs with abundant intraluminal vesicles accumulate and digestive lysosomes are rudimentary. Together, our results reveal an important and unexpected role of a deubiquitinating enzyme in endosome maturation.

cell biology↗

bioRxiv2024

Capturing Snapshots of Nucleosomal H2A K13/K15 Ubiquitination Mediated by the Monomeric E3 Ligase RNF168

AbstractsThe DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, plays a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent Lys13 and Lys15 sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to ubiquitinate H2AK13/15 site-specifically has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c[~]Ub in complex with nucleosomes. Here, we developed mechanism-based chemical trapping strategies and determined the cryo-EM structures of the RNF168/UbcH5c-Ub/NCP complex captured in transient H2AK13/15 monoubiquitination and adjacent dual-monoubiquitination reactions. Our structural analysis revealed that RNF168 stably binds to the nucleosomal H2A-H2B acidic patch through a basic helix with multiple interactions, which positions the UbcH5c active centre directly over the H2A N-terminus, providing a "helix-anchoring" mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the "compass-binding" mode of dimeric E3 ligases. Furthermore, our chemically synthesized ubiquitinated histones have enabled the elucidation of the efficiency of Ub installation and the interplay between the initial and subsequent Ub modifications on the adjacent H2A K13 and K15 sites. Overall, our work not only provides structural snapshots of H2A K13/K15 site-specific monoubiquitination and adjacent dual-monoubiquitination, but also offers a near-atomic resolution structural framework for understanding how pathogenic mutations or physiological modifications affect the molecular function of RNF168 in H2A K13/15 ubiquitination.

biochemistry↗