Biology subjects

Li, J.-B.

Publications and source records attributed to Li, J.-B..

6 recordsLinked to original sources

bioRxiv2024

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally-defined K63-Polyubiquitylated Linker Histone H1

The chemical synthesis of histones with homogeneous modifications is a potent approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). Here, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine-Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on ubiquitylated chromatosomes with different ubiquitin lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally-defined polyubiquitylated H1.0 by CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

biochemistry↗

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↗

bioRxiv2023

A Cryptic Basic Groove formed by Ubiquitin and Histone H3 Mediates the Selective Recognition of H2AK119Ub Nucleosomes by Synovial Sarcoma X Breakpoint 1 Protein

The cancer-specific fusion oncoprotein SS18-SSX1 disturbs chromatin accessibility by hijacking the BAF complex from the promoters and enhancers to the polycomb-repressed chromatin regions. This process relies on the selective recognition of H2AK119Ub nucleosomes by SSX1. However, the mechanism by which SSX1 selective recognition of H2AK119Ub nucleosomes in the absence of ubiquitin (Ub)-binding capacity remains unknown. Here we report the cryo-EM structure of SSX1 bound to H2AK119Ub nucleosomes at 3.1 [A] resolution. Combined in vitro biochemical and cellular assays revealed that the Ub recognition by SSX1 is unique and depends on a cryptic basic groove formed by H3 and the Ub motif on the H2AK119 site. Moreover, this unorthodox binding mode of SSX1 induces DNA unwrapping at the entry/exit sites. Together, our results describe a unique mode of site-specific ubiquitinated nucleosome recognition that underlies the specific hijacking of the BAF complex to polycomb regions by SS18-SSX1 in synovial sarcoma.

molecular biology↗

bioRxiv2021

Geraniol Enhances Inhibitory Inputs to Paraventricular Thalamic Nucleus and Induces Sedation in Mice

Geraniol (GE), a plant-derived acyclic monoterpene, shows a wide variety of beneficial effects. Notably, recent studies have reported the potential sedative effects of GE in fish and rats. However, the mechanisms of GE in sedation remain elusive. Here, we found that GE reduced locomotion, relieved pentylenetetrazol (PTZ)-induced seizures, altered the electroencephalogram (EEG), and facilitated general anesthesia in mice. Meanwhile, GE decreased c-Fos expression and suppressed the calcium activity in the paraventricular thalamic nucleus (PVT). Microinjection of GE into the PVT reduced locomotion and facilitated propofol-induced anesthesia. Furthermore, the electrophysiology results showed that GE-induced dramatic membrane hyperpolarization and suppressed the neuronal activity of PVT neurons, mainly by prolonging spontaneous inhibitory postsynaptic currents and inducing tonic inhibitory currents via GABAA receptors. Our study revealed that GE enhances inhibitory inputs to PVT neurons and induces sedation in mice. These findings provide a potential candidate for further development of sedatives and anesthetics. Graphic Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/441289v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@fd8424org.highwire.dtl.DTLVardef@1ee77caorg.highwire.dtl.DTLVardef@faab42org.highwire.dtl.DTLVardef@101b921_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

PBN-PVT projection modulates negative emotions in mice

Long-lasting negative affections dampen enthusiasm for life, and dealing with negative affective states is essential for individual survival. The parabrachial nucleus (PBN) and the thalamic paraventricular nucleus (PVT) are critical for modulating affective states in mice. However, the functional role of the PBN-PVT projection in modulating affective states remains elusive. Here, we show that the PBN neurons send dense projection fibers to the PVT and form direct excitatory synapses with the PVT neurons. Activation of the PBN-PVT projection or PVT-projecting PBN neurons induces robust anxiety-like, aversion-like, and fear-like behaviors without affecting nociceptive behaviors. Inhibition of the PBN-PVT projection or PVT-projecting PBN neurons reduces fear-like and aversion-like behaviors. Furthermore, the PVT neurons innervated by the PBN are activated by aversive stimulation, and activation of PBN-PVT projection enhances the neuronal activity of PVT neurons in response to the aversive stimulus. Activation of these downstream PVT neurons induces anxiety-like behavior behaviors. Thus, our study indicates that the PBN-PVT projection modulates negative affective states in mice.

neuroscience↗

bioRxiv2019

Development of a Genomic DNA Reference Material Panel for Thalassemia Genetic Testing

Thalassemia is one of the most common autosomal recessive inherited diseases worldwide, and it is also highly prevalent and variable in Southern China. Various types of genetic testing technologies have been developed for diagnosis and screening of thalassemia. Characterized genomic DNA reference materials are necessary for assay development, validation, proficiency testing and quality assurance. However, there is no publicly available reference materials for thalassemia genetic testing as yet. To address the need for these materials, the National Institutes for Food and Drug Control and the China National Gene Bank established 31 new cell lines with 2 wild genotypes and 29 distinct genotypes of thalassemia which account for approximately 90% thalassemia carriers in China. The genomic DNA of 31 cell lines were characterized by four clinical genetic testing laboratories using different genetic testing methods and technology platforms. The genotyping results were concordant among four laboratories. In addition, the results of stability test demonstrated that the genotypes of these DNA samples were not influenced by preanalytical conditions such as long-term exposure to high temperature(37{square}) environment and repeated freeze-thawing. In conclusion, we developed the first national panel of 31 genomic DNA reference materials which are renewable and publicly available for the quality assurance of various genetic testing methods and will facilitate research and development in thalassemia genetic testing.

genetics↗