USP14 regulates DNA damage repair by targeting RNF168-dependent ubiquitination

Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair.

Abbreviations: ATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.     

Autophagy-related protein ATG5 regulates histone H2B mono-ubiquitylation by translational control of RNF20

<正>Autophagy is an evolutionarily conserved lysosome-mediated catabolic process(Klionsky,2007).Autophagy is believed to be essential for cell survival,especially when cells were exposed to stresses,such as nutrient starvation.The activation of autophagy under starvation allows cells to survive by providing essential crudes for cell construction via degrading intracellular substrates     

RNF8-dependent histone ubiquitination during DNA damage response and spermatogenesis

Histone ubiquitination regulates the chromatin structure that is important for many biological processes. Recently, ubiquitination of histones was observed during the DNA damage response (DDR), and this modification is controlled by really interesting new gene (RING) domain E3 ligase, RNF8. Together with the E2 conjugating enzyme UBC13, RNF8 catalyzes ubiquitination of the histones H2A and H2AX during the DDR, thus facilitating downstream recruitment of DDR factors, such as p53 binding protein 1 (53BP1) and breast cancer type 1 susceptibility protein (BRCA1), to the damage site. Accordingly, the RNF8 knockout mice display phenotypes associated with failed DDR, including hypersensitivity to ionizing radiation, V(D)J recombination deficiency, and a predisposition to cancer. In addition to the DDR phenotypes, RNF8 knockout mice fail to generate mature sperm during spermatogenesis, resulting in male sterility. The RNF8 knockout mice also have a drastic reduction in histone ubiquitination in the testes. These findings indicate that the role of histone ubiquitination during chromatin remodeling in two different biological events could be linked by an RNF8-dependent mechanism. Here, we review the molecular mechanism of RNF8-dependent histone ubiquitination both in DDR and spermatogenesis.     

多巴胺通过促进RNF20降解抑制神经细胞中组蛋白H2B泛素化

表观遗传修饰参与了药物成瘾的形成过程,而在药物成瘾过程中组蛋白泛素化水平的变化仍未可知。药物成瘾过程中常表现为多巴胺(dopamine, DA)表达量的升高,因此本研究欲探讨多巴胺升高对神经细胞组蛋白泛素化的影响及其机制。Western印迹结果显示,在终浓度0.8 mmol/L的多巴胺作用8 h后,人神经母细胞瘤细胞系SH-SY5Y细胞中环指蛋白20(ring finger protein 20, RNF20)表达量降低(0.29±0.032 vs. 1.0±0.025,P<0.0001),泛素化组蛋白H2B(H2Bub1)表达量下降(0.28±0.032 vs. 1.0±0.017,P<0.0001)。但是RT-PCR结果显示,多巴胺处理SH-SY5Y细胞后,RNF20在mRNA水平的表达无明显变化。在SH-SY5Y细胞中沉默RNF20的表达,H2Bub1在蛋白质水平的表达明显降低(0.20±0.069 vs. 1.0±0.060,P=0.001)。在加入多巴胺的基础上,分别加入蛋白酶体抑制剂MG132、自噬体形成抑制剂3-MA以及空泡型H^+-ATP酶特异性抑制剂Baf-A1等药物来检测RNF20的降解途径,结果发现,加入MG132、3-MA以及Baf-A1后,RNF20表达量均比DA处理组显著上升(1.51±0.095,P=0.0003; 0.89±0.075,P=0.0021; 2.74±0.099,P<0.0001;vs. 0.27±0.044)。上述结果表明,在SH-SY5Y细胞中,RNF20对H2Bub1具有调控作用,多巴胺可通过泛素化及自噬两种途径促进RNF20降解,从而抑制组蛋白H2B泛素化。     

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.     

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.     

Removal of H2A.Z by INO80 promotes homologous recombination

The mammalian INO80 remodelling complex facilitates homologous recombination (HR), but the mechanism by which it does this is unclear. Budding yeast INO80 can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers. H2A.Z is actively incorporated at sites of damage in mammalian cells, raising the possibility that H2A.Z may need to be subsequently removed for resolution of repair. Here, we show that H2A.Z in human cells is indeed rapidly removed from chromatin flanking DNA damage by INO80. We also report that the histone chaperone ANP32E, which is implicated in removing H2AZ from chromatin, similarly promotes HR and appears to work on the same pathway as INO80 in these assays. Importantly, we demonstrate that the HR defect in cells depleted of INO80 or ANP32E can be rescued by H2A.Z co‐depletion, suggesting that H2A.Z removal from chromatin is the primary function of INO80 and ANP32E in promoting homologous recombination.     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6

The DNA of eukaryotes is wrapped around nucleosomes and packaged into chromatin. Covalent modifications of the histone proteins that comprise the nucleosome alter chromatin structure and have major effects on gene expression. Methylation of lysine 4 of histone H3 by COMPASS is required for silencing of genes located near chromosome telomeres and within the rDNA (Krogan, N. J, Dover, J., Khorrami, S., Greenblatt, J. F., Schneider, J., Johnston, M., and Shilatifard, A. (2002) J. Biol. Chem. 277, 10753-10755; Briggs, S. D., Bryk, M., Strahl, B. D., Cheung, W. L., Davie, J. K., Dent, S. Y., Winston, F., and Allis, C. D. (2001) Genes. Dev. 15, 3286-3295). To learn about the mechanism of histone methylation, we surveyed the genome of the yeast Saccharomyces cerevisiae for genes necessary for this process. By analyzing approximately 4800 mutant strains, each deleted for a different non-essential gene, we discovered that the ubiquitin-conjugating enzyme Rad6 is required for methylation of lysine 4 of histone H3. Ubiquitination of histone H2B on lysine 123 is the signal for the methylation of histone H3, which leads to silencing of genes located near telomeres.     

Innovation by homologous recombination

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Regulation of homologous recombination at telomeres in budding yeast

Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae.     

Regulation of LC3B levels by ubiquitination and proteasomal degradation

ABSTRACT

Like other biological processes, macroautophagy/autophagy must be tightly controlled for maintenance of cellular homeostasis and for proper response to changing cellular conditions. To gain insights into the regulation of autophagy, we recently conducted a genome-wide CRISPR-Cas9 knockout screen using cells expressing endogenous LC3B tagged with GFP-mCherry as a reporter. This approach allowed us to identify the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as novel autophagy regulators. We found that these enzymes cooperate to mediate monoubiquitination and proteasomal degradation of LC3B, thus limiting the pool of LC3B available for autophagy. Depletion of UBA6 or BIRC6 increased the level of cytosolic LC3B, enhancing the degradation of autophagy adaptors and the clearance of intracellular proteins aggregates. This finding could be the basis for the development of pharmacological inhibitors of UBA6 or BIRC6 for the treatment of protein aggregation disorders. Recent work by another group showed that BIRC6 itself is subject to ubiquitination and proteasomal degradation, highlighting the existence of a complex regulatory network for the control of LC3B levels.     

BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells

Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 µM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers.     

组蛋白H2B单泛素化参与DNA损伤修复的调控机制

作为一种重要的组蛋白修饰形式,H2B的单泛素化(uH2B)广泛地参与DNA复制、基因的表达与转录、DNA损伤修复及异染色质维持等生物学事件.在裂殖酵母中,H2B的单泛素化发生在其羧基端的119位赖氨酸(K119),并依赖于Rhp6/Bre1泛素连接酶复合体.研究表明,uH2B通过破坏H2A/H2B二聚体的结构促进mRNA在转录过程中的延伸,同时促进H3K4的三甲基化激活基因的表达及参与DNA损伤修复.本研究发现,Rhp6能够对核糖核苷酸还原酶抑制基因(Spd1)位点进行活跃的染色质修饰,促进H2B的单泛素化并抑制基因表达,从而促进dNTP的合成并调控DNA复制及损伤修复.重要的是,本研究发现,该过程不依赖于H3K4而决定于H3K9的三甲基化.同时uH2B直接在DNA双链断裂位点富集,通过改变染色质的结构参与DNA损伤修复,该过程中可能存在其他更为复杂的分子机制.     

Histone crosstalk directed by H2B ubiquitination is required for chromatin boundary integrity

Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3' boundary of a heterochromatin region located upstream of the chicken β-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5' boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.