H2A Ubiquitination Research Articles

Read-write mechanisms of H2A ubiquitination by Polycomb repressive complex 1.

Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication1. One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain2,3. However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read-write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1RYBP). This mechanism, which functions as a positive-feedback loop in epigenetic regulation4,5, emphasizes the pivotal role of ncPRC1RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read-write) or across nucleosomes (inter-nucleosome read-write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1RYBP in H2AK119Ub restoration to sustain repressive chromatin domains.

SOX4-BMI1 axis promotes non-small cell lung cancer progression and facilitates angiogenesis by suppressing ZNF24.

The incidence of lung cancer has become the highest among all cancer types globally, also standing as a leading cause of cancer-related deaths. Lung cancer is broadly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the latter accounting for 85% of total cases. SRY-box transcription factor 4 (SOX4), a crucial transcription factor, has been found to play a key role in the development of various cancers. However, the association between SOX4 and NSCLC is still unclear. This study investigated the clinical relevance of SOX4 and its potential mechanisms in the progression of NSCLC. Analysis of our NSCLC patient cohort revealed a significant increase in SOX4 levels in cancerous tissues, indicating its role as an independent prognostic indicator for NSCLC. In vitro experiments demonstrated that elevated SOX4 expression facilitated NSCLC cell migration, invasion, and EMT. Functionally, SOX4 drives NSCLC progression by enhancing the transcription and expression of B-cell-specific moloney leukemia virus insertion site 1 (BMI1). The oncogenic impact of SOX4-induced BMI1 expression on NSCLC advancement was validated through both in vivo and in vitro studies. In addition, our findings showed that BMI1 promoted the ubiquitination of histone H2A (H2Aub), leading to decreased zinc finger protein 24 (ZNF24) expression, which subsequently triggered vascular endothelial growth factor A (VEGF-A) secretion in NSCLC cells, thereby promoting NSCLC angiogenesis. Moreover, we evaluated the therapeutic potential of a BMI1 inhibitor in combination with Bevacizumab for NSCLC treatment using orthotopic models. The data presented in our study reveal a previously unrecognized role of the SOX4-BMI1 axis in promoting NSCLC progression and angiogenesis. This research significantly contributes to our knowledge of the interplay between SOX4 and BMI1 in NSCLC, potentially paving the way for the development of targeted therapies for this disease.

Gambogic acid impairs the maintenance and therapeutic resistance of glioma stem cells by targeting B-cell-specific Moloney leukemia virus insert site 1

BackgroundGlioblastoma (GBM) is the most common and lethal primary brain tumor with low effectiveness of available treatments. The tumor heterogeneity and therapeutic resistance are largely due to the presence of glioma stem cells (GSCs). Therefore, eliminating GSCs can overcome the progression, relapse, and resistance of GBM. Previous studies have shown that gambogic acid (GA), a natural active ingredient, has anti-glioma properties. Nonetheless, it is still unclear whether it has an inhibitory effect on GSCs and what its target might be. This study aimed to investigate the anti-tumor effects of GA on GSCs. In addition, this study found the target of GA in GSCs and elucidated the potential specific mechanisms by conducting both in vitro and in vivo experiments.B-cell-specific Moloney leukemia virus insert site 1 (BMI1) is a key stem cell factor of the polycomb group (PcG) family with important effects on the development, recurrence, and chemoresistance of several cancers. In both normal and cancer stem cells, BMI1 maintains stem cell self-renewal by regulating the cell cycle, cellular immortalization, and senescence. Its high expression in a variety of cancers correlates with poor clinical prognosis and chemoresistance. These mechanisms of BMI1 make it a potential therapeutic target for cancer therapy, and future studies may further reveal the specific roles of BMI1 mechanism and provide a basis for the development of new cancer therapeutic strategies. PurposeThis study investigated the in vitro and in vivo effects of GA in inducing apoptosis in GSCs and inhibiting GSCs self-renewal, as well as its underlying mechanisms. MethodsThis study synthesized biotinylated gambogic acid for the first time and angled for the target of gambogic acid using LC-MS/MS analysis, which has not been reported previously. Human-derived glioma stem cells GSC123 and GSC111 were used for in vitro studies, analyzing functions and mechanisms via microscale thermophoresis (MST), Annexin V/PI staining, Western blotting, immunofluorescence, and co-immunoprecipitation. The orthotopic glioma mouse model was used to assess the anti-tumor effects of GA in vivo. ResultsThis study demonstrated that GA is a specific inhibitor of BMI1, a key regulator controlling stem cell growth and self-renewal. GA binds to BMI1′s RING domain, accelerating K51-dependent degradation and suppressing H2A ubiquitination. Importantly, GA induces apoptosis, and inhibits GSC self-renewal, but minimally impacts neural progenitor cells (NPCs). GA can also be combined effectively with temozolomide and radiotherapy to increase their sensitivities in resistant cells. Furthermore, exogenous induction of BMI1 expression significantly hinders the disruption of GSCs by GA. In vivo, GA inhibits tumorigenicity, enhances the effect of temozolomide, and reduces BMI1 expression. ConclusionThese findings suggest that GA is a potential candidate for targeting GSCs and therefore be used to treat GBM.

L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

Epigenetic alterations critically affect tumor development. Polycomb-group complexes constitute an evolutionarily conserved epigenetic machinery that regulates stem cell fate and development. They are implicated in tumorigenesis, primarily via histone modification. Polycomb repressive complex 1 (PRC1) complexes 1-6 (PRC1.1-6) mediate the ubiquitination of histone H2A on lysine 119 (H2AK119ub). Here, we studied the functional roles of a PRC1.6 molecule, L3MBTL2, in neuroblastoma (NB) cells. L3MBTL2-knockout and knockdown revealed that L3MBTL2 depletion suppressed NB cell proliferation via cell-cycle arrest and gamma-H2A.X upregulation. L3MBTL2-knockout profoundly suppressed xenograft tumor formation. Transcriptome analysis revealed suppressed cell-cycle-related and activated differentiation-related pathways. Break repair meiotic recombinase recruitment factor 1 (BRME1) and nuclear receptor interacting protein 3 (NRIP3) were notably de-repressed by L3MBTL2-knockout. The deletion of L3MBTL2 reduced enrichment of H2AK119ub and PCGF6 at transcriptional start site proximal regions of the targets. Add-back studies unveiled the importance of L3MBTL2-BRME1 and -NRIP3 axes for NB cell proliferation. We further manifested the association of MYCN with de-repression of NRIP3 in an L3MBTL2-deficient context. Therefore, this study first revealed the significance of L3MBTL2-mediated gene silencing in MYCN-amplified NB cells.

GGNBP2 regulates histone ubiquitination and methylation in spermatogenesis

ABSTRACT Gametogenetin binding protein 2 (GGNBP2) was indispensable in normal spermatids for transformation into mature spermatozoa in mice, and when Gametogenetin binding protein 2 is bound to BRCC36 and RAD51, the complex participates in repairing DNA double-strand breaks (DSB) during the meiotic progression of spermatocytes. Ggnbp2 knockout resulted in the up-regulation of H2AK119ubi and down-regulation of H2BK120ubi in GC-2 cells (mouse spermatogonia-derived cell line) and postnatal day 18 testis lysate. Our results also demonstrated that Gametogenetin binding protein 2 inducedASXL1 to activate the deubiquitinating enzyme BAP1 in deubiquitinating H2A, while Gametogenetin binding protein 2 knockout disrupted the interaction between ASXL1 and BAP1, resulting in BAP1 localization change. Furthermore, the Gametogenetin binding protein 2 deletion reduced H2B ubiquitination by affecting E2 enzymes and E3 ligase binding. Gametogenetin binding protein 2 regulated H2A and H2B ubiquitination levels and controlled H3K27 and H3K79 methylation by PRC2 subunits and histone H3K79 methyltransferase. Altogether, our results suggest that Ggnbp2 knockout increased DNA damage response by promoting H2A ubiquitination and H3K27trimethylation (H3K27me3) and reduced nucleosome stability by decreasing H2B ubiquitination and H3K79 dimethylation (H3K79me2), revealing new mechanisms of epigenetic phenomenon during spermatogenesis. Gametogenetin binding protein 2 seems critical in regulating histone modification and chromatin structure in spermatogenesis.

Bmi1 facilitates the progression of cholangiocarcinoma by inhibiting Foxn2 expression dependent on a histone H2A ubiquitination manner

Cholangiocarcinoma (CCA), an exceptionally aggressive malignancy originating from the epithelium of the bile duct, poses a formidable challenge in cancer research and clinical management. Currently, attention is focused on exploring the oncogenic role and prognostic implications associated with Bmi1 in the context of CCA. In our study, we assessed the correlation of Bmi1 and Foxn2 expression across all types of CCA and evaluated their prognostic significance. Our results demonstrated that Bmi1 exhibits significantly upregulated expression in CCA tissues, while Foxn2 expression shows an inverse pattern. Simultaneously, the high expression of Bmi1, coupled with the low expression of Foxn2, indicates an unfavorable prognosis. Through in vitro and in vivo experiments, we confirmed the crucial role of Foxn2 in the proliferation, metastasis, and epithelial-mesenchymal transition (EMT) of CCA. Mechanistically, Bmi1 promotes the ubiquitination of histone H2A (H2AUb), leading to chromatin opening attenuation and a decrease in Foxn2 expression, ultimately driving CCA progression. Additionally, we described the potential value of Bmi1 and H2AUb inhibitors in treating CCA through in vitro experiments and orthotopic models. This study is of significant importance in deepening our understanding of the interaction between Bmi1 and Foxn2 in CCA and has the potential to advance the development of precision therapies for CCA.

Identification of a novel histone H2A mono-ubiquitination-inhibiting cell-active small molecule

Histone H2A mono-ubiquitination plays important roles in epigenetic gene expression and is also involved in tumorigenesis. Small molecules controlling H2A ubiquitination are of interest as potential chemical tools and anticancer drugs. To identify novel small molecule inhibitors of H2A ubiquitination, we synthesized and evaluated several compounds designed based on PRT4165 (1), which is a reported histone ubiquitin ligase RING1A inhibitor. We found that compound 11b strongly inhibited the viability and reduced histone H2A mono-ubiquitination in human osteosarcoma U2OS cells. Therefore, compound 11b is a promising lead compound for the development of H2A histone ubiquitination-inhibiting small molecules.

H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction

Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 isone of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.

Mechanisms of RNF168 nucleosome recognition and ubiquitylation

RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.

Upregulation of Hsp27 via further inhibition of histone H2A ubiquitination confers protection against myocardial ischemia/reperfusion injury by promoting glycolysis and enhancing mitochondrial function

Research suggests that ischemic glycolysis improves myocardial tolerance to anoxia and low-flow ischemia. The rate of glycolysis during ischemia reflects the severity of the injury caused by ischemia and subsequent functional recovery following reperfusion. Histone H2AK119 ubiquitination (H2Aub) is a common modification that is primarily associated with gene silencing. Recent studies have demonstrated that H2Aub contributes to the development of cardiovascular diseases. However, the underlying mechanism remains unclear. This study identified Hsp27 (heat shock protein 27) as a H2Aub binding protein and explored its involvement in mediating glycolysis and mitochondrial function. Functional studies revealed that inhibition of PRC1 (polycomb repressive complex 1) decreased H2Aub occupancy and promoted Hsp27 expression through inhibiting ubiquitination. Additionally, it increased glycolysis by activating the NF-κB/PFKFB3 signaling pathway during myocardial ischemia. Furthermore, Hsp27 reduced mitochondrial ROS production by chaperoning COQ9, and suppressed ferroptosis during reperfusion. A delivery system was developed based on PCL-PEG-MAL (PPM)-PCM-SH (CWLSEAGPVVTVRALRGTGSW) to deliver PRT4165 (PRT), a potent inhibitor of PRC1, to damaged myocardium, resulting in decreased H2Aub. These findings revealed a novel epigenetic mechanism connecting glycolysis and ferroptosis in protecting the myocardium against ischemia/reperfusion injury.

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes.

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4K20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structuraland single molecule imaging approaches. We found that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed- atomic force microscopy (HS-AFM) to monitor how the BRCA1-BARD1 complex recognises chromatin in real time, we saw a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiquitin ligase catalytic activity. Multivalent interactions across nucleosomes explain how BRCA1-BARD1 can recognise chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.

Abstract 6286: Targeting Polycomb Repressive Complex 1 with small-molecule inhibitors as a potential therapeutic approach to combat leukemia

Abstract Background: Polycomb repressive complex 1 (PRC1) is an epigenetic regulatory complex that monoubiquitinates lysine 119 on histone H2A, resulting in the repression of target genes. The canonical core of PRC1 responsible for its E3 ligase activity is a heterodimeric unit comprised of RING1B and BMI1. Emerging data implicates RING1B in various cancers and highlights the importance of PRC1 function in the maintenance and proliferation of leukemic stem cells. Studies demonstrate that loss of PRC1 activity through RING1B knockdown impairs leukemic stem cell proliferation, causes leukemic stem cell differentiation, and enhances survival in MLL-AF9 mouse models. Therefore, development of potent small molecules targeting the activity of PRC1 might offer a new approach for generating new therapeutics for leukemia. Results: Here, we report the discovery and development of the first-in-class small-molecule inhibitors directly binding to RING1B in PRC1. We performed an NMR-based fragment screen against RING1B-BMI1, identifying fragment hit, RB-1, with low-millimolar binding affinity. RB-1 was extensively optimized into a class of inhibitors with considerably enhanced binding affinity to the low-nanomolar range. These inhibitors have been shown to diminish the H2A ubiquitination activity of PRC1 E3 ligase cores. Structural studies revealed that our PRC1 inhibitors bind directly to the RING domain of RING1B and ultimately block the protein-protein interaction between RING1B and the nucleosome. In leukemic cell lines and primary AML samples, PRC1 inhibitor treatment elicits an anti-proliferative effect on cell growth and displays on-target action seen by the reduction of the H2A ubiquitination mark. Additionally, small-molecule inhibition of PRC1 results in the induction of cellular differentiation of leukemic cells, as well as apoptosis. Gene expression analysis in leukemic cells treated with our PRC1 inhibitors reveals upregulation of multiple genes that are repressed by PRC1, which is also supported by the displacement of PRC1 from chromatin assessed by CHIP-seq studies. Furthermore, our PRC1 inhibitors are active in vivo in mouse models of leukemia, demonstrating reduction of leukemia progression upon treatment. Conclusions: We developed first-in class inhibitors directly targeting the E3 ligase core of PRC1. Treatment with our PRC1 inhibitors in leukemic cells elicit an anti-proliferative effect, blocks ubiquitination of H2A, and results in the de-repression of many genes repressed by PRC1. Additionally, our PRC1 inhibitors elicit an anti-proliferative effect on leukemia progression in vivo, demonstrating the traction for developing anti-leukemia therapeutics that inhibit PRC1. Citation Format: Miranda L. Simes, Yiwu Yao, Se Ra Park, Trupta Purohit, Hongzhi Miao, Xiaotian Zhang, Weijiang Ying, Felicia Gray, Shirish Shukla, Qingjie Zhao, Caroline Nikolaidis, Alyssa Winkler, Jolanta Grembecka, Tomasz Cierpicki. Targeting Polycomb Repressive Complex 1 with small-molecule inhibitors as a potential therapeutic approach to combat leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6286.

Radiation resistance of cancer cells caused by mitochondrial dysfunction depends on SIRT3-mediated mitophagy.

Radiation resistance is the leading cause of radiotherapy failure in patients with cancer. Enhanced DNA damage repair is the main reason for cancer cells to develop resistance to radiation. Autophagy has been widely reported to be linked to increased genome stability and radiation resistance. Mitochondria are highly involved in the cell response to radiotherapy. However, the autophagy subtype mitophagy has not been studied in terms of genome stability. We have previously demonstrated that mitochondrial dysfunction is the cause of radiation resistance in tumour cells. In the present study, we found that SIRT3 was highly expressed in colorectal cancer cells with mitochondrial dysfunction, leading to PINK1/Parkin-mediated mitophagy. Excessive activation of mitophagy enhanced DNA damage repair, therefore promoting the resistance of tumour cells to radiation. Mechanistically, mitophagy resulted in decreased RING1b expression, which led to a reduction in the ubiquitination of histone H2A at K119, thereby enhancing the repair of DNA damage caused by radiation. Additionally, high expression of SIRT3 was related to a poor tumour regression grade in rectal cancer patients treated with neoadjuvant radiotherapy. These findings suggest that restoring mitochondrial function could be an effective method for increasing the radiosensitivity of patients with colorectal cancer.

H2A Ubiquitination Alters H3-tail Dynamics on Linker-DNA to Enhance H3K27 Methylation

Polycomb repressive complex 1 (PRC1) and PRC2 are responsible for epigenetic gene regulation. PRC1 ubiquitinates histone H2A (H2Aub), which subsequently promotes PRC2 to introduce the H3 lysine 27 tri-methyl (H3K27me3) repressive chromatin mark. Although this mechanism provides a link between the two key transcriptional repressors, PRC1 and PRC2, it is unknown how histone-tail dynamics contribute to this process. Here, we have examined the effect of H2A ubiquitination and linker-DNA on H3-tail dynamics and H3K27 methylation by PRC2. In naïve nucleosomes, the H3-tail dynamically contacts linker DNA in addition to core DNA, and the linker-DNA is as important for H3K27 methylation as H2A ubiquitination. H2A ubiquitination alters contacts between the H3-tail and DNA to improve the methyltransferase activity of the PRC2–AEBP2–JARID2 complex. Collectively, our data support a model in which H2A ubiquitination by PRC1 synergizes with linker-DNA to hold H3 histone tails poised for their methylation by PRC2–AEBP2–JARID2.

Histone tail electrostatics modulate E2-E3 enzyme dynamics: a gateway to regulate ubiquitination machinery.

BRCA1 (Breast Cancer-Associated Protein 1) is a human tumor suppressor that functions as an ubiquitin (Ub) ligase enzyme (E3) and plays a key role in genomic stability and DNA repair. Heterodimerization of BRCA1 with BARD1 (BRCA1-associated RING domain protein 1) is known to increase its Ub ligase activity and is important for its stability, and cooperative activation of UbcH5c (Ub conjugating enzyme (E2)). Recent studies demonstrate the importance of ubiquitination of the nucleosomal H2A C-terminal tail by BRCA1/BARD1-UbcH5c in which its mutations inhibit ubiquitination, predispose cells to chromosomal instability and greatly increase the likelihood of breast and ovarian cancer development. Due to the lack of molecular-level insight on the flexible and dis-ordered H2A C-tail, its ubiquitination mechanism by BRCA1/BARD1-UbcH5c and its function and relationship to cancer susceptibility remain elusive. Here, we use molecular dynamics simulations to provide molecular-level insights into the dynamics of the less-studied H2A C-tail and BRCA1/BARD1-UbcH5c on the nucleosome surface and their effect on ubiquitination. Our results precisely identify the key interactions and residues that trigger conformational transitions of BRCA1/BARD1-UbcH5c, and characterize the important role of histone electrostatics in their dynamics. We provide a mechanistic basis for the H2A C-tail lysine approach to UbcH5c and show the role of H2A C-tail and UbcH5c dynamics in lysine ubiquitination. Furthermore, our data demonstrate the potential for ubiquitination based on the lysine position of the C-tail. Altogether, the findings of this study provide unrevealed insights into the mechanism of H2A C-tail ubiquitination and help us understand the communication between Ub ligase/Ub conjugating enzymes (E3/E2) and nucleosome to regulate ubiquitination machinery, paving the way for the development of effective treatments for cancer and chronic pain.

RINGs, DUBs and Abnormal Brain Growth-Histone H2A Ubiquitination in Brain Development and Disease.

During mammalian neurodevelopment, signaling pathways converge upon transcription factors (TFs) to establish appropriate gene expression programmes leading to the production of distinct neural and glial cell types. This process is partially regulated by the dynamic modulation of chromatin states by epigenetic systems, including the polycomb group (PcG) family of co-repressors. PcG proteins form multi-subunit assemblies that sub-divide into distinct, yet functionally related families. Polycomb repressive complexes 1 and 2 (PRC1 and 2) modify the chemical properties of chromatin by covalently modifying histone tails via H2A ubiquitination (H2AK119ub1) and H3 methylation, respectively. In contrast to the PRCs, the Polycomb repressive deubiquitinase (PR-DUB) complex removes H2AK119ub1 from chromatin through the action of the C-terminal hydrolase BAP1. Genetic screening has identified several PcG mutations that are causally associated with a range of congenital neuropathologies associated with both localised and/or systemic growth abnormalities. As PRC1 and PR-DUB hold opposing functions to control H2AK119ub1 levels across the genome, it is plausible that such neurodevelopmental disorders arise through a common mechanism. In this review, we will focus on advancements regarding the composition and opposing molecular functions of mammalian PRC1 and PR-DUB, and explore how their dysfunction contributes to the emergence of neurodevelopmental disorders.

Mechanisms of Polycomb Group Protein-Mediated Fetal Hemoglobin Silencing

Reactivation of fetal hemoglobin (HbF) is beneficial for patients with β-globin gene disorders, such as sickle cell disease and β-thalassemia. Polycomb repressive complex 1 (PRC1) and 2 (PRC2) are multi-subunit protein complexes that function as transcriptional repressors to maintain lineage-specific gene expression programs. Prior works showed that genetic or pharmacologic manipulation of select PRC2 subunits can induce HbF expression in adult erythroid cells, and an inhibitor targeting the EED subunit of PRC2 has entered a clinical trial. Yet the molecular basis underlying HbF regulation by PRC1/2 complexes is unknown. Using a CRISPR-Cas9-based genetic screen, we identified BMI1, a subunit of the PRC1 complex as novel HbF repressor. Disrupting BMI1 in the adult-type erythroid cell line HUDEP2 or primary human erythroblast cells robustly increased HbF production. We failed to detect BMI1 binding at the β-globin gene locus by CUT&RUN, indicating that BMI1 functions via an indirect mechanism. RNA-seq analysis of BMI1-deficient HUDEP2 cells uncovered upregulation of two known HbF regulators: IGF2BP1 and LIN28B, both of which induce HbF when forcibly expressed in adult erythroid cells. Depletion of either IGF2BP1 or LIN28B partially restored HbF silencing in BMI1-deficient HUDEP2 cells, whereas simultaneous depletion of IGF2BP1 and LIN28B lowered HbF production virtually to basal levels in these cells. In primary erythroid cells, IGF2BP3, which is functionally related to IGF2BP1, also contributes to the effects of BMI1 depletion. Together, these data indicate that IGF2BP1, IGF2BP3, and LIN28B are the critical BMI1 targets in human erythroid cells. Since BMI1 functions in two distinct PRC1 complexes (cPRC1 and ncPRC1), we aimed to define which of these is critical for HbF regulation. cPRC1 is defined by the presence of CBX proteins 2, 4, 6, 7, and 8, while ncPRC1 contains RYBP or its homolog YAF2. ncPRC1 and cPRC1 deposit the histone mark H2AK119ub1, and facilitate chromatin compaction, respectively. Disruption of CBX2/4/7/8 proteins but not RYAP/YAF2 in HUDEP2 cells activated HbF to levels similar to those observed upon BMI1 depletion, implicating cPRC1 in HbF control. Accordingly, CUT&RUN analysis in adult erythroid cells revealed a strong genome-wide co-localization of BMI1 and CBX2/4/7/8, including at the IGF2BP1, IGF2BP3, and LIN28B genes. Finally, BMI1 depletion does not lead to significant changes in H2AK119ub1 at these genes, in line with the fact that the repressive activity of the cPRC1 complex does not rely on its H2A ubiquitination activity. Together these results indicate that BMI1 represses HbF via the cPRC1 complex in adult erythroid cells. The BMI1-containing cPRC1 complex is known to repress transcription in conjunction with the PRC2 complex which deposits the histone mark H3K27me3. CUT&RUN profiling studies revealed focal loss of H3K27me3 in BMI1-deficient HUDEP2 cells, including at the CG-rich regions near the promoters of the IGF2BP1 and LIN28B genes. Therefore, we tested whether cPRC1 and PRC2 complexes converge on HbF regulation by regulating IGF2BP1 and LIN28B. Disruption in HUDEP2 cells of EZH2, the catalytic component of PRC2, led to global depletion of H3K27me3 along with upregulation of IGF2BP1 and LIN28B. Co-depletion of IGF2BP1 and LIN28B largely restored the EZH2 depletion-induced HbF upregulation. In summary, our studies further define the nature of PRC complexes involved in HbF regulation and identify the IGF2BP1, IGF2BP3, and LIN28B genes as critical links between PcG activity and HbF silencing. Our results may guide the development of improved PRC inhibitors for the treatment of β-globin disorders.

A 1,2,3-Triazole Derivative of Quinazoline Exhibits Antitumor Activity by Tethering RNF168 to SQSTM1/P62.

Quinazoline and its derivatives have drawn much attention in the development of potential antitumor agents. Here, we synthesized a series of 1,2,3-triazole derivatives of quinazoline at the C6 position and evaluated for their cytotoxic activity in various human cancer cell lines. We found that compound 5a was the most cytotoxic to HCT-116 cells (IC50, 0.36 μM). Target profiling found that 5a directly binds to both the autophagy-associated protein SQSTM1/P62 and the E3 ligase RNF168, promoting their interaction. Consistently, 5a treatment induces a decrease in RNF168-mediated H2A ubiquitination and compromises homologous recombination-mediated DNA repair, thus increasing the sensitivity of HCT-116 to X-ray radiation. Moreover, 5a suppressed xenografted tumor growth in mice in a dose-dependent manner. Taken together, the 1,2,3-triazole derivative of quinazoline 5a may serve as a novel compound for tumor therapy based on its role in promoting a P62/RNF168 interaction.

Dynamic switching of crotonylation to ubiquitination of H2A at lysine 119 attenuates transcription-replication conflicts caused by replication stress.

The reversible post-translational modification (PTM) of proteins plays an important role in many cellular processes. Lysine crotonylation (Kcr) is a newly identified PTM, but its functional significance remains unclear. Here, we found that Kcr is involved in the replication stress response. We show that crotonylation of histone H2A at lysine 119 (H2AK119) and ubiquitination of H2AK119 are reversibly regulated by replication stress. Decrotonylation of H2AK119 by SIRT1 is a prerequisite for subsequent ubiquitination of H2AK119 by BMI1. Accumulation of ubiquitinated H2AK119 at reversed replication forks leads to the release of RNA Polymerase II and transcription repression in the vicinity of stalled replication forks. These effects attenuate transcription–replication conflicts (TRCs) and TRC-associated R-loop formation and DNA double-strand breaks. These findings suggest that decrotonylation and ubiquitination of H2A at lysine 119 act together to resolve replication stress-induced TRCs and protect genome stability.

BMI1 promotes cholangiocarcinoma progression and correlates with antitumor immunity in an exosome-dependent manner.

Cholangiocarcinoma (CCA) is a class of malignant tumors originating from bile duct epithelial cells. Due to difficult early diagnosis and limited treatment, the prognosis of CCA is extremely poor. BMI1 is dysregulated in many human malignancies. However, the prognostic significance and oncogenic role of BMI1 in cholangiocarcinoma (CCA) are not well elucidated. In the present study, we investigated its clinical importance and the potential mechanisms in the progression of CCA. We detected BMI1 expression in a large CCA cohort. We demonstrated that BMI1 was substantially upregulated in CCA tissues and was identified as an independent prognostic biomarker of CCA. Moreover, overexpression of BMI1 promoted CCA proliferation, migration, and invasion. And BMI1 knockdown could inhibit proliferation and metastases of CCA in vitro and in vitro/vivo validation. Interestingly, we found that CCA-derived exosomes contain BMI1 proteins, which can transfer BMI1 between CCA cells. The unique BMI1-containing exosomes promote CCA proliferation and metastasis through autocrine/paracrine mechanisms. In addition, we demonstrated that BMI1 inhibits CD8+T cell-recruiting chemokines by promoting repressive H2A ubiquitination in CCA cells. BMI1 is an unfavorable prognostic biomarker of CCA. Our data depict a novel function of BMI1 in CCA tumorigenesis and metastasis mediated by exosomes. Besides, BMI1 inhibition may augment immune checkpoint blockade to inhibit tumor progression by activating cell-intrinsic immunity of CCA.