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Objective·To analyse the structure of non-canonical polycomb repressive complex 1.6 (PRC1.6) by negative staining and transmission electron microscopy (TEM), and obtain the three-dimensional (3D) profile information of human PRC1.6 heptameric complex.Methods·Seven PRC1.6 components, RNF2, PCGF6, RYBP, L3MBTL2, CBX3, E2F6, and TFDP1, were cloned into the pMLink vector with a 6×His-3×Flag tag at the N-terminus, respectively. The proteins were expressed in Expi293F cells grown in suspension cultures by using transfection with polyethylenimine. The tagged proteins were isolated via affinity purification with anti-DYKDDDDK G1 affinity resin, followed by gel filtration chromatography with Superdex 200 Increase 10/300 GL and glycerol density gradient centrifugation. The components of the PRC1.6 heptameric complex were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The in vitro ubiquitination activity and nucleosome-binding affinity of the purified heptameric complex were verified by the ubiquitination activity assay and the electrophoretic mobility shift assay (EMSA). The protein samples were stained by uranyl acetate and observed by TEM. The 3D information of the PRC1.6 complex was studied by single particle analysis. To predict the localization of the seven components within the structure model of PRC1.6 complex, the structure models of proteins in Protein Data Bank (PDB) were docked into the electron density map of PRC1.6 complex by using UCSF Chimera software.Results·The PRC1.6 complex with high purity and good homogeneity was obtained by eukaryotic expression, affinity purification, gel filtration chromatography and glycerol density gradient centrifugation, and confirmed as the heptameric complex by LC-MS/MS. The purified proteins showed ubiquitination activity and nucleosome-binding affinity in vitro. The 3D structure of the PRC1.6 heptameric complex with a resolution of 15.2 Å (1 Å=10-10 m) was preliminarily resolved by negative staining, TEM, and single particle analysis. The available structure models of RNF2, PCGF6, RYBP, L3MBTL2, CBX3, and DP1 proteins, as well as the predicted E2F6 structure by AlphaFold2, were docked into the reconstructed density map of PRC1.6 complex. The position of each component in the complex was preliminarily confirmed.Conclusion·The 3D structural model of the human PRC1.6 heptameric complex is obtained by negative staining, TEM, and single particle analysis.

Objective·To analyse the structure of non-canonical polycomb repressive complex 1.6 (PRC1.6) by negative staining and transmission electron microscopy (TEM), and obtain the three-dimensional (3D) profile information of human PRC1.6 heptameric complex. Methods·Seven PRC1.6 components, RNF2, PCGF6, RYBP, L3MBTL2, CBX3, E2F6, and TFDP1, were cloned into the pMLink vector with a 6×His-3×Flag tag at the N-terminus, respectively. The proteins were expressed in Expi293F cells grown in suspension cultures by using transfection with polyethylenimine. The tagged proteins were isolated via affinity purification with anti-DYKDDDDK G1 affinity resin, followed by gel filtration chromatography with Superdex 200 Increase 10/300 GL and glycerol density gradient centrifugation. The components of the PRC1.6 heptameric complex were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The in vitro ubiquitination activity and nucleosome-binding affinity of the purified heptameric complex were verified by the ubiquitination activity assay and the electrophoretic mobility shift assay (EMSA). The protein samples were stained by uranyl acetate and observed by TEM. The 3D information of the PRC1.6 complex was studied by single particle analysis. To predict the localization of the seven components within the structure model of PRC1.6 complex, the structure models of proteins in Protein Data Bank (PDB) were docked into the electron density map of PRC1.6 complex by using UCSF Chimera software. Results·The PRC1.6 complex with high purity and good homogeneity was obtained by eukaryotic expression, affinity purification, gel filtration chromatography and glycerol density gradient centrifugation, and confirmed as the heptameric complex by LC-MS/MS. The purified proteins showed ubiquitination activity and nucleosome-binding affinity in vitro. The 3D structure of the PRC1.6 heptameric complex with a resolution of 15.2 Å (1 Å=10-10 m) was preliminarily resolved by negative staining, TEM, and single particle analysis. The available structure models of RNF2, PCGF6, RYBP, L3MBTL2, CBX3, and DP1 proteins, as well as the predicted E2F6 structure by AlphaFold2, were docked into the reconstructed density map of PRC1.6 complex. The position of each component in the complex was preliminarily confirmed. Conclusion·The 3D structural model of the human PRC1.6 heptameric complex is obtained by negative staining, TEM, and single particle analysis. [ABSTRACT FROM AUTHOR]

Chromatin regulation in eukaryotes plays pivotal roles in controlling the developmental regulatory gene network. This review explores the intricate interplay between chromatin regulators and environmental signals, elucidating their roles in shaping plant development. As sessile organisms, plants have evolved sophisticated mechanisms to perceive and respond to environmental cues, orchestrating developmental programs that ensure adaptability and survival. A central aspect of this dynamic response lies in the modulation of versatile gene regulatory networks, mediated in part by various chromatin regulators. Here, we summarized current understanding of the molecular mechanisms through which chromatin regulators integrate environmental signals, influencing key aspects of plant development. [ABSTRACT FROM AUTHOR]

Disulfiram (DSF) and its oxidized derivatives (DSFoxy) are currently being investigated as potential anticancer agents. We previously found that DSFoxy initiate the paraptosis-like death of tumor cells, which is of potential interest for the treatment of tumors resistant to the initiation of apoptosis. Based on bioinformatics analysis of mass spectrometric data on protein ubiquitination, we formulated a conception about the important role of disruption of the retrograde transport of damaged proteins from the endoplasmic reticulum to the cytosol in the mechanism of initiation of paraptosis-like cell death. In the present work, it has been found that DSFoxy, in the process of initiating paraptosis-like death of human adenocarcinoma HEp-2 cells, also enhances the ubiquitination of histones and histone code enzymes. In particular, this applies to the ubiquitination of histone H2BC12, histone methyltransferases responsible for transcription and repair of damaged DNA, as well as acetylating and ubiquitin-conjugating proteins. Bioinformatics analysis of changes in ubiquitination of cell nuclear proteins using the STRING database revealed during this process an increase in the occurrence of ubiquitinated proteins (functional enrichment) of cell cycle regulation, cell response to DNA damage and DNA repair, the regulation of which also depends on the histone code. This directly indicates damage to the cell nucleus and is consistent with confocal microscopy data. These results indicate that paraptosis-like death initiated by DSFoxy is accompanied, along with impairment of retrograde transport and ER stress, also by a change in regulation of the histone code, which points to a pleiotropic mechanism of cell death induction. [ABSTRACT FROM AUTHOR]

• BDE-209 suppressed meiotic promoter expression by suppressing Sohlh1 expression. • BDE-209 inhibited the chromosomal synaptonemal complex formation by L3MBTL2. • BDE-209 inhibited histone ubiquitination by L3MBTL2 affecting spermatid into sperm. • L3MBTL2 regulated spermatogenesis by affecting meiosis and spermatid deformation. The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm. [Display omitted] [ABSTRACT FROM AUTHOR]

Aging is an intricate process characterized by multiple hallmarks including stem cell exhaustion, genome instability, epigenome alteration, impaired proteostasis, and cellular senescence. Whereas each of these traits is detrimental at the cellular level, it remains unclear how they are interconnected to cause systemic organ deterioration. Here we show that abrogating Brap, a BRCA1-associated protein essential for neurogenesis, results in persistent DNA double-strand breaks and elevation of histone H2A mono- and poly-ubiquitination (H2Aub). These defects extend to cellular senescence and proteasome-mediated histone H2A proteolysis with alterations in cells' proteomic and epigenetic states. Brap deletion in the mouse brain causes neuroinflammation, impaired proteostasis, accelerated neurodegeneration, and substantially shortened the lifespan. We further show the elevation of H2Aub also occurs in human brain tissues with Alzheimer's disease. These data together suggest that chromatin aberrations mediated by H2Aub may act as a nexus of multiple aging hallmarks and promote tissue-wide degeneration.

Verticillium dahliae, a virulent soil-borne fungus, elicits Verticillium wilt in numerous dicotyledonous plants through intricate pathogenic mechanisms. Ubiquitination, an evolutionarily conserved post-translational modification, marks and labels proteins for degradation, thereby maintaining cellular homeostasis. Within the ubiquitination cascade, ubiquitin ligase E3 demonstrates a unique capability for target protein recognition, a function often implicated in phytopathogenic virulence. Our research indicates that two ubiquitin ligase E3s, VdBre1 and VdHrd1, are intrinsically associated with virulence. Our findings demonstrate that the deletion of these two genes significantly impairs the ability of V. dahliae to colonize the vascular bundles of plants and to form typical penetration pegs. Furthermore, transcriptomic analysis suggests that VdBre1 governs the lipid metabolism pathway, while VdHrd1 participates in endoplasmic-reticulum-related processes. Western blot analyses reveal a significant decrease in histone ubiquitination and histone H3K4 trimethylation levels in the ΔVdBre1 mutant. This research illuminates the function of ubiquitin ligase E3 in V. dahliae and offers fresh theoretical perspectives. Our research identifies two novel virulence-related genes and partially explicates their roles in virulence-associated structures and gene regulatory pathways. These findings augment our understanding of the molecular mechanisms inherent to V. dahliae. [ABSTRACT FROM AUTHOR]

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.

Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity toward H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes (TSGs). Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. By contrast, disrupting H3K18ub-dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy., Competing Interests: Declaration of interests C.J.F. and J.R.H. are paid employees of Cell Signaling Technology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Verticillium dahliae, a virulent soil-borne fungus, elicits Verticillium wilt in numerous dicotyledonous plants through intricate pathogenic mechanisms. Ubiquitination, an evolutionarily conserved post-translational modification, marks and labels proteins for degradation, thereby maintaining cellular homeostasis. Within the ubiquitination cascade, ubiquitin ligase E3 demonstrates a unique capability for target protein recognition, a function often implicated in phytopathogenic virulence. Our research indicates that two ubiquitin ligase E3s, VdBre1 and VdHrd1, are intrinsically associated with virulence. Our findings demonstrate that the deletion of these two genes significantly impairs the ability of V. dahliae to colonize the vascular bundles of plants and to form typical penetration pegs. Furthermore, transcriptomic analysis suggests that VdBre1 governs the lipid metabolism pathway, while VdHrd1 participates in endoplasmic-reticulum-related processes. Western blot analyses reveal a significant decrease in histone ubiquitination and histone H3K4 trimethylation levels in the ΔVdBre1 mutant. This research illuminates the function of ubiquitin ligase E3 in V. dahliae and offers fresh theoretical perspectives. Our research identifies two novel virulence-related genes and partially explicates their roles in virulence-associated structures and gene regulatory pathways. These findings augment our understanding of the molecular mechanisms inherent to V. dahliae.

Objective To investigate the effect of cyclic finger protein-20 (RNF20) regulating histone H2B ubiquitination (H2Bub) on macrophage migration through the NF-κB pathway. Methods The cells were divided into seven groups: group A (control group, the cells were cultured for 48 h), group B (ox-LDL stimulation group, using 50 mg/mL ox-LDL to stimulate macrophages for 48 h), group C (ox-LDL+oe-RNF20 transfection group, macrophages transfected with oe-RNF20 were stimulated with ox-LDL at a dose of 50 mg/mL for 48 h), group D (ox-LDL+pcDNA transfection control group, macrophages were transfected with pcDNA and stimulated with 50 mg/mL ox-LDL for 48 h), group E (oxLDL+SN50 intervention group, after using 50 mg/mL ox-LDL to stimulate macrophages for 24 h, followed by 10 μg/mL SN50 treatment for 24 h, and a total of 48 h), group F (ox-LDL+LY294002 intervention group, after using 50 mg/mL oxLDL to stimulate macrophages for 24 h, rats were treated with 10 μg/mL LY294002 for 24 h, and a total of 48 h) and group G (ox-LDL+p38 intervention group, using 50 mg/mL ox-LDL to stimulate for 24 h, followed by 10 μg/mL p38 for 24 h, and a total of 48 h) . Transwell assay was used to observe the migration number of macrophages, and Western blot⁃ ting was used to detect the expression levels of RNF20, H2B, H2Bub, nuclear factor-κB (NF-κB) and interleukin 6 (IL6) . Results In the ox-LDL stimulated macrophages, compared with group A, the expression of RNF20 protein and H2Bub/H2B ratio decreased, the number of migration macrophage increased, and the expression of NF-κB and IL-6 protein increased in the group B (all P<0. 01) . In the RNF20 overexpression cell model, compared with group B, the expression of RNF20 protein and H2Bub/H2B ratio increased, while the expression levels of NF-κB and IL-6 protein decreased, and the number of migration macrophage decreased in the group C (all P<0. 01) . In the group D, the expression of RNF20 protein, the ratio of H2Bub to H2B, NF-κB, IL-6 and the number of migration macrophage did not significantly change (all P>0. 05) . After treatment with SN50 (NF-κB inhibitor), SB203580 (p38 inhibitor), and LY294002 (PI3K inhibitor), the expression of RNF20 protein and the ratio of H2Bub/H2B in the groups E, F, and G did not significantly change as compared with those of the group B (all P>0. 05) . In the group E, the expression levels of NF-κB and IL-6 proteins and the number of migration macrophage decreased (all P<0. 01) . The expression levels of NF-κB and IL-6 proteins and the number of migration macrophage in the groups F and G did not significantly change (all P>0. 05) . Conclusion Decreased RNF20 and H2Bub aggravated macrophage migration and inflammatory reaction, and promoted the formation of atherosclerosis, which worked through the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Neural tube defects (NTDs) are common congenital malformations resulting in failure of the neural tube closure during early embryonic development. Although it is known that maternal folate deficiency increases the risk of NTDs, the mechanism remains elusive. Results Herein, we report that histone H2A monoubiquitination (H2AK119ub1) plays a role in neural tube closure. We found that the folate antagonist methotrexate induced H2AK119ub1 in mouse embryonic stem cells. We demonstrated that an increase in H2AK119ub1 downregulated expression of the neural tube closure-related genes Cdx2, Nes, Pax6, and Gata4 in mouse embryonic stem cells under folate deficiency conditions. We also determined that the E3 ligase Mdm2 was responsible for the methotrexate-induced increase in H2AK119ub1 and downregulation of neural tube closure-related genes. Surprisingly, we found that Mdm2 is required for MTX-induced H2A ubiquitination and is recruited to the sites of DSB, which is dependent on DNA damage signaling kinase ATM. Furthermore, folic acid supplementation restored H2AK119ub1 binding to neural tube closure-related genes. Downregulation of these genes was also observed in both brain tissue of mouse and human NTD cases, and high levels of H2AK119ub1 were found in the corresponding NTDs samples with their maternal serum folate under low levels. Pearson correlation analysis showed a significant negative correlation between expression of the neural precursor genes and H2AK119ub1. Conclusion Our results indicate that folate deficiency contributes to the onset of NTDs by altering H2AK119ub1 and subsequently affecting expression of neural tube closure-related genes. This may be a potential risk factor for NTDs in response to folate deficiency.

Abstract Background Neural tube defects (NTDs) are common birth defects involving the central nervous system. Recent studies on the etiology of human NTDs have raised the possibility that epigenetic regulation could be involved in determining susceptibility to them. Results Here, we show that the H2AK119ub1 E3 ligase CUL4B is required for the activation of retinoic acid (RA)-inducible developmentally critical homeobox (HOX) genes in NT2/D1 embryonal carcinoma cells. RA treatment led to attenuation of H2AK119ub1 due to decrease in CUL4B, further affecting HOX gene regulation. Furthermore, we found that CUL4B interacted directly with RORγ and negatively regulated its transcriptional activity. Interestingly, knockdown of RORγ decreased the expression of HOX genes along with increased H2AK119ub1 occupancy levels, at HOX gene sites in N2/D1 cells. In addition, upregulation of HOX genes was observed along with lower levels of CUL4B-mediated H2AK119ub1 in both mouse and human anencephaly NTD cases. Notably, the expression of HOXA10 genes was negatively correlated with CUL4B levels in human anencephaly NTD cases. Conclusions Our results indicate that abnormal HOX gene expression induced by aberrant CUL4B-mediated H2AK119ub1 levels may be a risk factor for NTDs, and highlight the need for further analysis of genome-wide epigenetic modifications in NTDs.

The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (males absent on the first) acetylates UHRF1 at K670 in the pre-RING linker region, whereas HDAC1 deacetylates UHRF1 at the same site. We also identify that K667 and K668 can also be acetylated by MOF when K670 is mutated. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity. Abolishing UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs DNMT1 recruitment and DNA methylation. Taken together, these findings identify MOF as an acetyltransferase for UHRF1 and define a mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation. [Display omitted] • MOF acetylates UHRF1 at K670, which is counteracted by HDAC1 • Acetylation by MOF enhances UHRF1 E3 ubiquitin ligase activity • Abolishment of UHRF1 acetylation impairs histone H3 ubiquitination and DNMT1 recruitment • K670 acetylation of UHRF1 is required for proper function of DNA methylation maintenance Wang et al. describe a regulatory mechanism of UHRF1 in DNA methylation maintenance. UHRF1 acetylation by MOF at lysine 670 is essential for efficient DNMT1 recruitment by facilitating histone H3 ubiquitination. These findings suggest that UHRF1 acetylation is important for its E3 ubiquitin ligase function in various biological processes. [ABSTRACT FROM AUTHOR]

Single-molecule experiments reveal the dynamics of transcription through a nucleosome with single-base-pair accuracy.

RAD6 participates in DNA double-strand breaks (DSBs) repair by ubiquitinating histone H2B in mitotic cells. In terminally differentiated cells, however, the mechanisms of DNA damage repair are less well known. In this study, we investigate whether RAD6B is involved in DSBs repair in neurons and effects of RAD6B deficiency on neuronal survival. We compared neurons of RAD6B-deficient mice with those of littermate wild type (WT) mice and induced DNA damage by X-ray irradiation. We provide evidence that RAD6B is essential for neural DDR and RAD6B deficiency results in increased genomic instability and neurodegeneration. Moreover, higher levels of p53 and p21 are present in the brains of RAD6B-deficient mice, which may be responsible for neuronal senescence, and degeneration. In addition, behavioral experiments show that RAD6B-deficient mice exhibit marked learning and memory deficits. In conclusion, these findings suggest that RAD6B is critical for neural integrity and that the absence of RAD6B accelerates neurodegeneration in mice.

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.

RAD6 participates in DNA double-strand breaks (DSBs) repair by ubiquitinating histone H2B in mitotic cells. In terminally differentiated cells, however, the mechanisms of DNA damage repair are less well known. In this study, we investigate whether RAD6B is involved in DSBs repair in neurons and effects of RAD6B deficiency on neuronal survival. We compared neurons of RAD6B-deficient mice with those of littermate wild type (WT) mice and induced DNA damage by X-ray irradiation. We provide evidence that RAD6B is essential for neural DDR and RAD6B deficiency results in increased genomic instability and neurodegeneration. Moreover, higher levels of p53 and p21 are present in the brains of RAD6B-deficient mice, which may be responsible for neuronal senescence, and degeneration. In addition, behavioral experiments show that RAD6B-deficient mice exhibit marked learning and memory deficits. In conclusion, these findings suggest that RAD6B is critical for neural integrity and that the absence of RAD6B accelerates neurodegeneration in mice. [ABSTRACT FROM AUTHOR]

While 19 S proteasome regulatory particle (RP) inhibition is a promising new avenue for treating bortezomib-resistant myeloma, the anti-tumor impact of inhibiting 19 S RP component PSMD14 could not be explained by a selective inhibition of proteasomal activity. Here, we report that PSMD14 interacts with NSD2 on chromatin, independent of 19 S RP. Functionally, PSMD14 acts as a histone H2AK119 deubiquitinase, facilitating NSD2-directed H3K36 dimethylation. Integrative genomic and epigenomic analyses revealed the functional coordination of PSMD14 and NSD2 in transcriptional activation of target genes (e.g., RELA) linked to myelomagenesis. Reciprocally, RELA transactivates PSMD14 , forming a PSMD14/NSD2-RELA positive feedback loop. Remarkably, PSMD14 inhibitors enhance bortezomib sensitivity and fosters anti-myeloma synergy. PSMD14 expression is elevated in myeloma and inversely correlated with overall survival. Our study uncovers an unappreciated function of PSMD14 as an epigenetic regulator and a myeloma driver, supporting the pursuit of PSMD14 as a therapeutic target to overcome the treatment limitation of myeloma. [Display omitted] • The proteasome component PSMD14 is a histone deubiquitinase acting on H2AK119 • PSMD14 physically interacts and functionally connects with NSD2 on chromatin • The positive feedback loop between PSMD14/NSD2 and RELA promotes myelomagenesis • Chemical inhibition of PSMD14 confers synergistic anti-MM activity with bortezomib He et al. unveil the proteasome component PSMD14 as an epigenetic modulator and myeloma oncogenic driver, revealing the PSMD14/NSD2-RELA positive feedback loop's role in myelomagenesis. This study highlights PSMD14 as a potential therapeutic target for countering multiple myeloma treatment limitations. [ABSTRACT FROM AUTHOR]

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.

BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs) for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT). Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1), and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub), BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR). In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs) through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis.

The conserved nuclear protein ENY2 (Sus1 in yeast) is involved in coupling transcription and mRNA export in yeast and metazoa, as it is a component both of the transcriptional co-activator complex SAGA and of the mRNA export complex TREX-2. Arabidopsis thaliana ENY2 is widely expressed in the plant and it localizes to the nucleoplasm, but unlike its yeast/metazoan orthologs, it is not enriched in the nuclear envelope. Affinity purification of ENY2 in combination with mass spectrometry revealed that it co-purified with SAGA components, but not with the nuclear pore-associated TREX-2. In addition, further targeted proteomics analyses by reciprocal tagging established the composition of the Arabidopsis SAGA complex consisting of the four modules HATm, SPTm, TAFm and DUBm, and that several SAGA subunits occur in alternative variants. While the HATm, SPTm and TAFm robustly co-purified with each other, the deubiquitination module (DUBm) appears to associate with the other SAGA modules more weakly/dynamically. Consistent with a homology model of the Arabidopsis DUBm, the SGF11 protein interacts directly with ENY2 and UBP22. Plants depleted in the DUBm components, SGF11 or ENY2, are phenotypically only mildly affected, but they contain increased levels of ubiquitinated histone H2B, indicating that the SAGA-DUBm has histone deubiquitination activity in plants. In addition to transcription-related proteins (i.e., transcript elongation factors, Mediator), many splicing factors were found to associate with SAGA, linking the SAGA complex and ongoing transcription with mRNA processing. [ABSTRACT FROM AUTHOR]

Rad6B, a principal component of the translesion synthesis pathway, and activator of canonical Wnt signaling, plays an essential role in cutaneous melanoma development and progression. As Rad6 is encoded by two genes, namely, UBE2A (RAD6A) and UBE2B (RAD6B), in humans, we compared their expressions in melanomas and normal melanocytes. While both genes are weakly expressed in normal melanocytes, Rad6B is more robustly expressed in melanoma lines and patient-derived metastatic melanomas than RAD6A. The characterization of RAD6B transcripts revealed coexpression of various splice variants representing truncated or modified functional versions of wild-type RAD6B in melanomas, but not in normal melanocytes. Notably, two RAD6B isoforms with intact catalytic domains, RAD6BΔexon4 and RAD6Bintron5ins, were identified. We confirmed that RAD6BΔexon4 and RAD6Bintron5ins variants are expressed as 14 and 15 kDa proteins, respectively, with functional in vivo ubiquitin conjugating activity. Whole exome sequence analysis of 30 patient-derived melanomas showed RAD6B variants coexpressed with wild-type RAD6B in all samples analyzed, and RAD6Bintron5ins variants were found in half the cases. These variants constitute the majority of the RAD6B transcriptome in contrast to RAD6A, which was predominantly wild-type. The expression of functional RAD6B variants only in melanomas reveals RAD6B’s molecular heterogeneity and its association with melanoma pathogenesis.

DNA double strand breaks need to be repaired in an organized fashion to preserve genomic integrity. In the organization of faithful repair, histone ubiquitination plays a crucial role. Recent findings suggest an integrated model for DNA repair regulation through site-specific histone ubiquitination and crosstalk to other posttranslational modifications. Here we discuss how site-specific histone ubiquitination is achieved on a molecular level and how different multi-protein complexes work together to integrate different histone ubiquitination states. We propose a model where site-specific H2A ubiquitination organizes the spatio-temporal recruitment of DNA repair factors which will ultimately contribute to DNA repair pathway choice between homologous recombination and non-homologous end joining. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is the major cause of end stage renal failure. Although, several therapeutic targets have emerged to prevent the progression of DN, the number of people with DN still continues to rise worldwide, suggesting an urgent need of novel targets to prevent DN completely. Currently, the role of ubiquitin proteasome system (UPS) has been highlighted in the pathogenesis and progression of various diseases like obesity, insulin resistance, atherosclerosis, cancers, neurodegerative disorders and including secondary complications of diabetes. UPS mainly involves in protein homeostatis through ubiquitination (post translational modification) and proteasomal degradation of various proteins. Ubiquitination, not only involves in proteasomal degradation, but also directs the substrate proteins to participate in multitude of cell signalling pathways. However, very little is known about ubiquitination and UPS in the progression of DN. This review mainly focuses on UPS and its components including E2 conjugating enzymes, E3 ligases and deubiquitinases (DUBs) in the development of DN and thus may help us to find novel therapeutic targets with in UPS to prevent DN completely in future. [ABSTRACT FROM AUTHOR]

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study. [ABSTRACT FROM AUTHOR]

Mdm2 is the key negative regulator of the tumour suppressor p53, making it an attractive target for anti-cancer drug design. We recently identified a new role of Mdm2 in gene repression through its direct interaction with several proteins of the polycomb group (PcG) family. PcG proteins form polycomb repressive complexes PRC1 and PRC2. PRC2 (via EZH2) mediates histone 3 lysine 27 (H3K27) trimethylation, and PRC1 (via RING1B) mediates histone 2A lysine 119 (H2AK119) monoubiquitination. Both PRCs mostly support a compact and transcriptionally silent chromatin structure. We found that Mdm2 regulates a gene expression profile similar to that of PRC2 independent of p53. Moreover, Mdm2 promotes the stemness of murine induced pluripotent stem cells and human mesenchymal stem cells, and supports the survival of tumour cells. Mdm2 is recruited to target gene promoters by the PRC2 member and histone methyltransferase EZH2, and enhances PRC-dependent repressive chromatin modifications, specifically H3K27me3 and H2AK119ub1. Mdm2 also cooperates in gene repression with the PRC1 protein RING1B, a H2AK119 ubiquitin ligase. Here we discuss the possible implications of these p53-independent functions of Mdm2 in chromatin dynamics and in the stem cell phenotype. We propose that the p53-independent functions of Mdm2 should be taken into account for cancer drug design. So far, the majority of clinically tested Mdm2 inhibitors target its binding to p53 but do not affect the new functions of Mdm2 described here. However, when targeting the E3 ligase activity of Mdm2, a broader spectrum of its oncogenic activities might become druggable. [ABSTRACT FROM AUTHOR]

Monoubiquitinated lysine 119 of histone H2A (H2AK119ub) is a highly abundant epigenetic mark, associated with gene repression and deposited on chromatin by the polycomb repressor complex 1 (PRC1), which is an essential regulator of diverse transcriptional programs in mammalian development and tissue homeostasis. While multiple deubiquitinases (DUBs) with catalytic activity for H2AK119ub (H2A-DUBs) have been identified, we lack systematic analyses of their roles and cross-talk in transcriptional regulation. Here, we address H2A-DUB functions in epigenetic regulation of mammalian development and tissue maintenance by conducting a meta-analysis of 248 genomics datasets from 32 independent studies, focusing on the mouse model and covering embryonic stem cells (ESCs), hematopoietic, and immune cell lineages. This covers all the publicly available datasets that map genomic H2A-DUB binding and H2AK119ub distributions (ChIP-Seq), and all datasets assessing dysregulation in gene expression in the relevant H2A-DUB knockout models (RNA-Seq). Many accessory datasets for PRC1–2 and DUB-interacting proteins are also analyzed and interpreted, as well as further data assessing chromatin accessibility (ATAC-Seq) and transcriptional activity (RNA-seq). We report co-localization in the binding of H2A-DUBs BAP1, USP16, and to a lesser extent others that is conserved across different cell-types, and also the enrichment of antagonistic PRC1–2 protein complexes at the same genomic locations. Such conserved sites enriched for the H2A-DUBs and PRC1–2 are proximal to transcriptionally active genes that engage in housekeeping cellular functions. Nevertheless, they exhibit H2AK119ub levels significantly above the genomic average that can undergo further increase with H2A-DUB knockout. This indicates a cooperation between H2A-DUBs and PRC1–2 in the modulation of housekeeping transcriptional programs, conserved across many cell types, likely operating through their antagonistic effects on H2AK119ub and the regulation of local H2AK119ub turnover. Our study further highlights existing knowledge gaps and discusses important directions for future work. [ABSTRACT FROM AUTHOR]

Summary Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, and loss of autophagy has been linked to increased genome instability. Here, we report that loss of autophagy is coupled to reduced histone H2A ubiquitination after DNA damage. p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, an E3 ligase essential for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSB repair. Moreover, nuclear-localized p62 increased the sensitivity of tumor cells to radiation both in vitro and in vivo, and this required its interaction with RNF168. Our findings indicate that autophagy-deficiency-induced p62 accumulation results in inhibition of histone ubiquitination and highlight the complex relationship between autophagy and the DNA damage response. [ABSTRACT FROM AUTHOR]

Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each other's binding but suggest that the key functions of PRC1 lie beyond the enzymatic capabilities of RING1B. [ABSTRACT FROM AUTHOR]

The involvement of chromatin remodelling in dormancy cycling in the soil seed bank ( SSB) is poorly understood. Natural variation between the winter and summer annual Arabidopsis ecotypes Cvi and Bur was exploited to investigate the expression of genes involved in chromatin remodelling via histone 2 B ( H2 B) ubiquitination/de-ubiquitination and histone acetylation/deacetylation, the repressive histone methyl transferases CURLY LEAF ( CLF) and SWINGER ( SWN), and the gene silencing repressor ROS1 ( REPRESSOR OF SILENCING1) and promoter of silencing KYP/ SUVH4 ( KRYPTONITE), during dormancy cycling in the SSB. ROS1 expression was positively correlated with dormancy while the reverse was observed for CLF and KYP/ SUVH4. We propose ROS1 dependent repression of silencing and a sequential requirement of CLF and KYP/ SUVH4 dependent gene repression and silencing for the maintenance and suppression of dormancy during dormancy cycling. Seasonal expression of H2 B modifying genes was correlated negatively with temperature and positively with DOG1 expression, as were histone acetyltransferase genes, with histone deacetylases positively correlated with temperature. Changes in the histone marks H3 K4me3 and H3 K27me3 were seen on DOG1 ( DELAY OF GERMINATION1) in Cvi during dormancy cycling. H3 K4me3 activating marks remained stable along DOG1. During relief of dormancy, H3 K27me3 repressive marks slowly accumulated and accelerated on exposure to light completing dormancy loss. We propose that these marks on DOG1 serve as a thermal sensing mechanism during dormancy cycling in preparation for light repression of dormancy. Overall, chromatin remodelling plays a vital role in temporal sensing through regulation of gene expression. [ABSTRACT FROM AUTHOR]