Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A.

Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.

H2B Lys34 Ubiquitination Induces Nucleosome Distortion to Stimulate Dot1L Activity.

Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme. We determined the cryo-electron microscopy structures of Dot1L-H2BK34ub nucleosome complex and the H2BK34ub nucleosome alone. The structures reveal that H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on nucleosome as H2BK120ub, which positions Dot1L for the productive conformation through direct ubiquitin-enzyme contacts. However, H2BK34-anchored ubiquitin does not directly interact with Dot1L as occurs in the case of H2BK120ub, but rather induces DNA and histone distortion around the modified site. Our findings establish the structural framework for understanding the H2BK34ub-H3K79me trans-crosstalk and highlight the diversity of mechanisms for histone ubiquitination to activate chromatin-modifying enzymes.

Chemical Synthesis of Post-Translationally Modified H2AX Reveals Redundancy in Interplay between Histone Phosphorylation, Ubiquitination, and Methylation on the Binding of 53BP1 with Nucleosomes.

The chemical synthesis of homogeneously modified histones is a powerful approach to quantitatively decipher how post-translational modifications (PTMs) modulate epigenetic events. Herein, we describe the expedient syntheses of a selection of phosphorylated and ubiquitinated H2AX proteins in a strategy integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation. These modified H2AX proteins were then used to discover that although H2AXS139 phosphorylation can enhance the binding of the DNA damage repair factor 53BP1 to either an unmodified nucleosome or that bearing a single H2AXK15ub or H4K20me2 modification, it augments 53BP1's binding only weakly to nucleosomes bearing both H2AXK15ub and H4K20me2. To better understand why such a trivalent additive effect is lacking, we solved the cryo-EM structure (3.38 Å) of the complex of 53BP1 with the H2AXK15ub/S139ph_H4K20me2 nucleosome, which showed that H2AXS139 phosphorylation distorts the interaction interface between ubiquitin and 53BP1's UDR motif. Our study revealed that there is redundancy in the interplay of multiple histone PTMs, which may be useful for controlling the dynamic distribution of effector proteins onto nucleosomes bearing different histone variants and PTMs in a time-dependent fashion, through specific cellular biochemical events.

The Bre1/Rad6 machinery: writing the central histone ubiquitin mark on H2B and beyond.

Mono-ubiquitination on H2B (H2Bub1) is an evolutionarily conserved histone post-translational modification implicated in various important physiological processes including DNA replication, transcription activation, and DNA damage repair. The Bre1/Rad6 ubiquitination machinery is currently considered to be the sole writer of H2Bub1, but the mechanistic basis by which it operates is unclear. Recently, the RING-type E3 ligase Bre1 was proposed to associate with the E2 enzyme Rad6 through a novel interaction between Bre1 RBD (Rad6 binding domain) and Rad6; and the RING domain of Bre1 that is responsible for the nucleosomal acidic patch binding also interacts with Rad6 to stimulate its catalytic activity. Recent discoveries have yielded evidence for the phenomenon of liquid-liquid phase separation in the context of H2Bub1, and its regulation by other histone post-translational modifications. This review summarizes current knowledge about Bre1/Rad6-mediated H2B ubiquitination, including the physiological functions and the molecular basis for writing and regulation of this central histone ubiquitin mark. Possible models for the Bre1/Rad6 machinery bound to nucleosomes bearing different modifications in the writing step are also disclosed.

Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16.

Epigenetic regulators have a crucial effect on gene expression based on their manipulation of histone modifications. Histone H2AK119 monoubiquitination (H2AK119Ub), a well-established hallmark in transcription repression, is dynamically regulated by the opposing activities of Polycomb repressive complex 1 (PRC1) and nucleosome deubiquitinases including the primary human USP16 and Polycomb repressive deubiquitinase (PR-DUB) complex. Recently, the catalytic mechanism for the multi-subunit PR-DUB complex has been described, but how the single-subunit USP16 recognizes the H2AK119Ub nucleosome and cleaves the ubiquitin (Ub) remains unknown. Here we report the cryo-EM structure of USP16-H2AK119Ub nucleosome complex, which unveils a fundamentally distinct mode of H2AK119Ub deubiquitination compared to PR-DUB, encompassing the nucleosome recognition pattern independent of the H2A-H2B acidic patch and the conformational heterogeneity in the Ub motif and the histone H2A C-terminal tail. Our work highlights the mechanism diversity of H2AK119Ub deubiquitination and provides a structural framework for understanding the disease-causing mutations of USP16.

Synovial sarcoma X breakpoint 1 protein uses a cryptic groove to selectively recognize H2AK119Ub nucleosomes.

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 synovial sarcoma X breakpoint 1 (SSX1). However, the mechanism underlying the selective recognition of H2AK119Ub nucleosomes by SSX1 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-Å 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.

Flexural Behavior of Damaged Hollow RC Box Girders Repaired with Prestressed CFRP.

In recent years, numerous studies have explored the benefits of utilizing prestressed carbon fiber-reinforced polymer (CFRP) for strengthening concrete structures. However, research on the reinforcement of prestressed CFRP on full-scale hollow RC box girders, particularly damaged bridges, remains limited. In this study, both experiments and finite element analysis (FEA) were performed to investigate the flexural behavior of full-scale hollow RC box girders with varying degrees of damage, which were strengthened using CFRP with different levels of prestress. The adhesive behavior of the CFRP-concrete interface was considered in the FEA. Numerical simulations were conducted to assess the flexural behaviors of the girders, including failure modes, yield and ultimate loads, and deflections. The results revealed that the application of prestressed CFRP efficiently increased the yield and ultimate loads of the box girders. Specifically, when the degree of damage of the hollow box girder was less than 23%, the flexural bearing capacity of the repaired girder could be recovered after being strengthened with two prestressed CFRP strips measuring 50 mm in width and 3 mm in thickness. However, the risk of premature debonding at the CFRP-concrete interface increased when the prestressing level of CFRP and degree of damage of hollow RC box girders exceeded 35% and 40%, respectively. These findings suggest that the use of prestressed CFRP may be a promising method for repairing damaged hollow RC box girders, but careful consideration of the degree of damage and prestressing level would be necessary to ensure the effectiveness and safety of the repair.

Flexural Behavior of Full-Scale Damaged Hollow RC Beams Strengthened with Prestressed SCFRP Plate under Four-Point Bending.

The advantages of using prestressed carbon fiber reinforced polymer (CFRP) for strengthening and retrofitting structures have been reported in recent years. In this regard, most of the studies on prestressed CFRP technique have been carried out in the laboratory test with small-scale and no damage (reinforced concrete) RC beam. However, the real structures that need to be retrofitted in service are often degraded or damaged due to early cracking. This paper aims at studying the effect of prestressed CFRP method on full-scale and damaged RC beams. The damaged levels of four full-scale damaged hollow RC beams taken from an old bridge were evaluated. One damaged beam was tested to check the residual capacity, and the other three were strengthened with prestressed composite strengthened CFRP and steel-carbon fiber reinforced polymer (SCFRP). The flexural behavior of non-strengthened and prestressed strengthened beams was investigated. During the experiments, the failure modes, deflection, yield and ultimate load, strains of concrete, steel reinforcements, and SCFRP were measured and analyzed. The results showed that the stiffness at the elastic stage was increased by 64.9%, 66.9%, and 67.1% after strengthened by SCFRP with 30%, 40%, and 60% prestressing level. Moreover, the ultimate load of damaged hollow RC beams were improved by 19.53%, 21.82%, and 31.9%, respectively. The flexural behavior of the severely damaged RC beam with strength reduction coefficient of 0.65 can be recovered after being strengthened by SCFRP with 40% prestressing levels. Meanwhile, SCFRP-concrete interface debonding failure occurred when the prestressing level exceed 60%, and the characteristics of brittle failure became more evident with increased prestressing level of the SCFRP.

Met1-specific motifs conserved in OTUB subfamily of green plants enable rice OTUB1 to hydrolyse Met1 ubiquitin chains.

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1' pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.

Mechanical Performance of Steel Slag Concrete under Biaxial Compression.

The mechanical performance of steel slag concrete (SSC) under biaxial compression is investigated by a servo-controlled static-dynamic true triaxial machine (TAWZ-5000/3000). Three replacement ratios of steel slags and four kinds of stress ratio (0.25:1, 0.5:1, 0.75:1, and 1:1) are examined in this study. According to the test results, the influences of replacement ratio and stress ratio on the strength, deformation properties, stress-strain curves, and failure mode of SSC are analyzed. The results show that the failure mode of SSC under biaxial compression is plate-splitting crack. Both the strength and deformation of SSC are larger than the corresponding values of the uniaxial compression. Under the same stress ratio, the value of principal stress σ 3 f increases first and then decreases with the increase in the replacement ratio. Under the same replacement ratio, σ 3 f increases first and then decreases as the stress ratio increases, and the maximum of σ 3 f is obtained at the stress ratio of α = 0.5:1. Based on the analysis and test data, the strength failure criterion of SSC under biaxial compression stresses is proposed.

Experimental Investigation on the Impact Resistance of Carbon Fibers Reinforced Coral Concrete.

In this study, the impact resistance of coral concrete with different carbon fiber (CF) dosages subjected to drop-weight impact test was investigated. For this purpose, three concrete strength grades (C20, C30, C40) and six CF dosages (0.0%, 0.3%, 0.6%, 1.0%, 1.5%, and 2.0% by weight of the binder) were considered, and a total of 18 groups of carbon fibers reinforced coral concrete (CFRCC) were cast. For each group, eight specimens were tested following the drop-weight impact test suggested by CECS 13. Then, the two-parameter Weibull distribution theory was adopted to statistically analyze the variations in experimental results. The results indicated that the addition of CFs could transform the failure pattern from obvious brittleness to relatively good ductility and improve the impact resistance of coral concrete. Moreover, the impact resistance of CFRCC increases with the CF dosage increasing. The statistical analysis showed that the probability distribution of the blow numbers at the initial crack and final failure of CFRCC approximately follows the two-parameter Weibull distribution.