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Crosstalk between ubiquitin and other post-translational modifications on chromatin during DSB repair
Abstract
The cellular response to DNA double-stranded breaks (DSBs) involves a conserved mechanism of recruitment and activation of numerous proteins involved in this pathway. The events that trigger this response in mammalian cells involve several post-translational modifications, but the role of non-proteasomal ubiquitin signaling is particularly central to this pathway. Recent work has demonstrated that ubiquitination does not act alone, but in concert with other post-translational modifications, including phosphorylation, methylation, acetylation, ADP-ribosylation, and other ubiquitin-like modifiers, particularly SUMOylation. We review novel and exciting crosstalk mechanisms between ubiquitination and other post-translational modifications, many of which work synergistically with each other to activate signaling events and help recruit important DNA damage effector proteins, particularly BRCA1 (breast cancer 1, early onset) and 53BP1 (tumor protein p53 binding protein 1), to sites of DNA damage.
... The regulation of the DNA damage response requires the correct and timely coordination of a multitude of signaling events, in which PTMs, both of histones and of regulatory proteins in the pathway, play a critical role, through activation and recruitment of repair proteins [7]. For example, the recruitment of 53BP1, a mediator protein critical for the DSB repair pathway choice by directing DSB repair via nonhomologous end-joining (NHEJ), depends on the methylation of H4K20 together with the RNF8-dependent degradation of competing H4K20me readers, deacetylation of H4K16, and RNF168-mediated H2AK15 ubiquitination [8,9]. Interestingly, SETD8 and H4K20me1 were shown to accumulate at sites of DNA damage [10][11][12][13]. ...
... In addition to directing the recruitment of repair factors, PTMs, particularly ubiquitination, are also critical for controlling the levels of many proteins involved in this pathway via proteasome-dependent degradation [9,14]. SETD8 is also regulated by ubiquitin-mediated proteasomal degradation, resulting in a fluctuation of SETD8 protein levels during the cell cycle, with low levels in S phase [10,[15][16][17]. ...
SETD8 is a histone methyltransferase that plays pivotal roles in several cellular functions, including transcriptional regulation, cell cycle progression, and genome maintenance. SETD8 regulates the recruitment of 53BP1 to sites of DNA damage by controlling histone H4K20 methylation. Moreover, SETD8 levels are tightly regulated in a cell cycle-dependent manner by ubiquitin-dependent proteasomal degradation. Here, we identified ubiquitin-specific peptidase 29, USP29, as a novel regulator of SETD8. Depletion of USP29 leads to decreased SETD8 protein levels, an effect that is independent of the cell cycle. We demonstrate that SETD8 binds to USP29 in vivo, and this interaction is dependent on the catalytic activity of USP29. Wildtype USP29 can deubiquitinate SETD8 in vivo, indicating that USP29 directly regulates SETD8 protein levels. Importantly, USP29 knockdown inhibits the irradiation-induced increase in H4K20 monomethylation, thereby preventing focus formation of 53BP1 in response to DNA damage. Lastly, depletion of USP29 increases the cellular sensitivity to irradiation. These results demonstrate that USP29 is critical for the DNA damage response and cell survival, likely by controlling protein levels of SETD8.
... TOP2A organizes the genome structure, promotes chromosome segregation and is overexpressed in multiple tumors, leading to aggressive phenotypes of the disease and poor prognosis [164,165]. Post-translational modifications, such as phosphorylation, ubiquitination and SUMOylation, regulate TOP2A activity [166]. SMG1 directly phosphorylates UPF1 and possibly induces TOP2A phosphorylation through UPF1. ...
Up-frameshift protein 1 (UPF1) plays the role of a vital controller for transcripts, ready to react in the event of an incorrect translation mechanism. It is well known as one of the key elements involved in mRNA decay pathways and participates in transcript and protein quality control in several different aspects. Firstly, UPF1 specifically degrades premature termination codon (PTC)-containing products in a nonsense-mediated mRNA decay (NMD)-coupled manner. Additionally, UPF1 can potentially act as an E3 ligase and degrade target proteins independently from mRNA decay pathways. Thus, UPF1 protects cells against the accumulation of misfolded polypeptides. However, this multitasking protein may still hide many of its functions and abilities. In this article, we summarize important discoveries in the context of UPF1, its involvement in various cellular pathways, as well as its structural importance and mutational changes related to the emergence of various pathologies and disease states. Even though the state of knowledge about this protein has significantly increased over the years, there are still many intriguing aspects that remain unresolved.
... Second, among multiple PTMs cooperate in spatio-temporal regulation of DSB repair, non-proteasomal ubiquitin signaling is central to cellular response to DNA damage. ADP-ribosylation acts early signaling and helps to initiate ubiquitination events during the DSB response by recruiting E3 ligases Ubc13-RNF8 indirectly or BRCA1/BARD1 directly [388,392,393]. Additionally, a study showed that Parp9, a mono-ADP-ribosyltransferase, forms heterodimers with the histone E3 ligase Dtx3L, and mediates NAD + -dependent mono-ADP-ribosylation of ubiquitin, which inhibits substrate modification or the synthesis of poly-Ub chains, suggesting potentially complex regulation between them [394]. ...
Posttranslational modifications (PTMs) of proteins, the major mechanism of protein function regulation, play important roles in regulating a variety of cellular physiological and pathological processes. Although the classical PTMs, such as phosphorylation, acetylation, ubiquitination and methylation, have been well studied, the emergence of many new modifications, such as succinylation, hydroxybutyrylation, and lactylation, introduces a new layer to protein regulation, leaving much more to be explored and wide application prospects. In this review, we will provide a broad overview of the significant roles of PTMs in regulating human cancer hallmarks through selecting a diverse set of examples, and update the current advances in the therapeutic implications of these PTMs in human cancer.
... The improvement of mass spectrometry (MS) technologies has largely increased the number of identified PTMs, which now accounts for around 400 [3], including acetylation, propionylation, methylation, phosphorylation, sumoylation, and ubiquitination, which have been proven to be involved in the regulation of cellular processes at diverse subcellular compartments. Of note, a growing body of literature has also outlined the interplay between the different PTMs [4][5][6], suggesting that the crosstalk is compelling for the right fulfillment of such diverse cell functions as, for example, gene expression, genome organization, cell division and DNA damage response. Given the fundamental roles exerted DNA damage repair, [56] Chromatin regulation, [57] Gene expression, [ Inactive Y-Y-T ND Immune response [79,80] Stress granule function, [76] cytoskeleton regulation [70] Immune response [81] PARP14 ...
Cellular functions are regulated through the gene expression program by the transcription of new messenger RNAs (mRNAs), alternative RNA splicing, and protein synthesis. To this end, the post-translational modifications (PTMs) of proteins add another layer of complexity, creating a continuously fine-tuned regulatory network. ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules, regulating a multitude of key functional processes as diverse as DNA damage repair (DDR), transcriptional regulation, intracellular transport, immune and stress responses, and cell survival. Additionally, due to the emerging role of ADP-ribosylation in pathological processes, ADP-ribosyltransferases (ARTs), the enzymes involved in ADPr, are attracting growing interest as new drug targets. In this review, an overview of human ARTs and their related biological functions is provided, mainly focusing on the regulation of ADP-ribosyltransferase Diphtheria toxin-like enzymes (ARTD)-dependent RNA functions. Finally, in order to unravel novel gene functional relationships, we propose the analysis of an inventory of human gene clusters, including ARTDs, which share conserved sequences at 3′ untranslated regions (UTRs).
... These modifications can target lysine residues in competition with other PTMs, including concurrent PTMs such as methylation or acetylation. One molecular role of both ubiquitination and sumoylation will be to antagonize another PTM effect [371], as it is reflected in the fine tuning of transcription factors [372,373]. ...
The repair of double-stranded DNA breaks (DSBs) by homologous recombination involves the formation of branched intermediates that can lead to crossovers following nucleolytic resolution. Ubiquitin and SUMO modification is commonplace amongst the DNA damage repair proteins. What is more, a number of DSB repair factors interact with each other when sumoylated, making use of SUMO interaction motifs (SIMs). The nuclease Yen1 is tightly controlled during the cell cycle to limit the extent of crossover formation and preserve genome integrity. In this manuscript we describe further regulation of Yen1 by ubiquitination, sumoylation and non-covalent interaction with SUMO through its newly characterized SIMs. Yen1 is sumoylated by Siz1 and Siz2 SUMO ligases, especially in conditions of DNA damage. Furthermore, Yen1 is a substrate of the Slx5-Slx8 ubiquitin ligase. Loss of Slx5-Slx8 stabilizes the sumoylated fraction of Yen1, and results in persistent localization of Yen1 in nuclear foci. Slx5-Slx8-dependent ubiquitination of Yen1 occurs mainly at K714 and mutation of this lysine increases crossover formation during DSB repair and suppresses chromosome segregation defects when other nucleases are unavailable. In addition, proper and timely nucleolytic processing from Yen1 is dependent on interactions mediated by non-covalent binding to sumoylated partners. Mutations in the motifs that allow SUMO-mediated recruitment of Yen1 leads to its mis-localization, decreasing Yen1’s ability to resolve DNA joint-molecule intermediates and resulting in increased genome instability and chromosome mis-segregation.
... In assessing enriched GO biological processes in the SSC population, terms relating to translation and ribosomal assembly were downregulated ( Figure 4F), aligning with the defined role of CHD proteins in driving ribosomal assembly in other cell types (Shimono et al., 2005;Zentner et al., 2010). Other downregulated processes included transcription, mRNA splicing, and protein stability, with the latter likely being intertwined with the known interaction of CHD4/NuRD with ubiquitination machinery within the cell (Zhao et al., 2014). Osteoblast differentiation and spermatogenesis were identified as upregulated processes (Figure 4G), with ''spermatogenesis''-related genes, including Dazl and testis-expressed protein 15 (Tex15), which is involved in meiotic recombination (Yang et al., 2008). ...
Maintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. Here, we have identified a key role for the nucleosome remodeling protein CHD4 in regulating SSC function. Gene expression analyses revealed that CHD4 expression is highly enriched in the SSC population in the mouse testis. Using spermatogonial transplantation techniques it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, causing a ∼50% reduction in colonization of recipient testes. An scRNA-seq comparison revealed reduced expression of "self-renewal" genes following Chd4 knockdown, along with increased expression of signature progenitor genes. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only through its traditional association with the remodeling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated role for CHD4 in controlling fate decisions in the spermatogonial pool.
... Expression and activity of DNA repair genes are regulated at several levels (85,86): given the cell cycle-phase specificity, it is not surprising that multiple enzymes required for efficient recognition, resection and repair are controlled by CDK-cyclin phosphorylation (87,88). PTMs have long been known to play a major role in the recruitment and activation of the repair factors, i.e. protein kinase ataxia telangiectasia mutated (ATM) phosphorylates key players in DDR network (78). ...
During S phase, the cooperation between the macromolecular complexes regulating DNA synthesis, epigenetic information maintenance and DNA repair is advantageous for cells, as they can rapidly detect DNA damage and initiate the DNA damage response (DDR). UHRF1 is a fundamental epigenetic regulator; its ability to coordinate DNA methylation and histone code is unique across proteomes of different species. Recently, UHRF1’s role in DNA damage repair has been explored and recognized to be as important as its role in maintaining the epigenome. UHRF1 is a sensor for interstrand crosslinks and a determinant for the switch towards homologous recombination in the repair of double-strand breaks; its loss results in enhanced sensitivity to DNA damage. These functions are finely regulated by specific post-translational modifications and are mediated by the SRA domain, which binds to damaged DNA, and the RING domain. Here, we review recent studies on the role of UHRF1 in DDR focusing on how it recognizes DNA damage and cooperates with other proteins in its repair. We then discuss how UHRF1’s epigenetic abilities in reading and writing histone modifications, or its interactions with ncRNAs, could interlace with its role in DDR.
... As a result of the upregulation of MMP14 gene expression, more MMP14 could be synthesized. However, excessive accumulation of MMP14 could trigger posttranslational modifications such as the ubiquitindependent proteasome system [41,42] for clearance of these excessive MMP14 molecules and hence reduces translational MMP14 (protein) expression. Secondly, upon siCTTN treatment, both the gene (Fig. 4A) and the protein (Fig. 4D,IV) expression of CTTN was significantly reduced. ...
Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cyto-skeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.
... Protein ubiquitination is often preceded by a priming removal or addition of other modifications on the same protein, such as phosphorylation, acetylation, methylation, and SUMOylation [59][60][61]. Phosphorylation provides substantial specificity for targeting distinct pools of histones to degradation. For example, phosphorylation of tyrosine 99 (Y99) in H3 by Rad53, which is required for efficient degradation of the H3 protein, serves to target only the nonchromatin-bound excess H3 for degradation, while the nucleosomal H3 is spared from any inadvertent degradation [33]. ...
Histones constitute the primary protein building blocks of the chromatin and play key roles in the dynamic control of chromatin compaction and epigenetic regulation. Histones are regulated by intricate mechanisms that alter their functionality and stability, thereby expanding the regulation of chromatin‐transacting processes. As such, histone degradation is tightly regulated to provide spatiotemporal control of cellular histone abundance. While several mechanisms have been implicated in controlling histone stability, here, we discuss proteasome‐dependent degradation of histones and the protein modifications that are associated with it. We then highlight specific cellular and physiological states that are associated with altered histone degradation by cellular proteasomes.
... Ubiquitination is a reversible reaction that depends on adenosine triphosphate (ATP). After a multistep reaction, ubiquitin molecules are bound to the target protein [19]. This process involves the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E2, and the ubiquitin ligase E3 (Fig. 2). ...
The ubiquitin proteasome system (UPS) is a highly conserved way to regulate protein turnover in cells. The UPS hydrolyzes and destroys variant or misfolded proteins and finely regulates proteins involved in differentiation, apoptosis, and other biological processes. This system is a key regulatory factor in the proliferation, differentiation, and collagen secretion of skin fibroblasts. E3 ubiquitin protein ligases Parkin and NEDD4 regulate multiple signaling pathways in keloid. Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) binding with deubiquitinase USP10 can induce p53 destabilization and promote keloid-derived fibroblast proliferation. The UPS participates in the occurrence and development of hypertrophic scars by regulating the transforming growth factor (TGF)-β/Smad signaling pathway. An initial study suggests that TNFα-induced protein 3 (TNFAIP3) polymorphisms may be significantly associated with scleroderma susceptibility in individuals of Caucasian descent. Sumoylation and multiple ubiquitin ligases, including Smurfs, UFD2, and KLHL42, play vital roles in scleroderma by targeting the TGF-β/Smad signaling pathway. In the future, drugs targeting E3 ligases and deubiquitinating enzymes have great potential for the treatment of skin fibrosis.
... Consistent with these reports, we found that phosphorylation by GSK3b decreased the expression of cugWT1 followed by the downregulation of EGFR, cyclin D1 and Bcl2 expression levels in HCT15 and A549 cancer cells (Fig. 5). Crosstalk between phosphorylation and the ubiquitin machinery is important for regulating protein activity, stability, and interactions (47,48). Our results using an inactive mutant cugWT1-S64A indicated that GSK3b promotes cugWT1 turnover through its phosphorylation on S64 (Fig. 6). ...
The Wilms' tumor 1 (WT1) gene is well known as a chameleon gene. It plays a role as a tumor suppressor in Wilms' tumor but also acts as an oncogene in other cancers. Previously, our group reported that a canonical AUG starting site for the WT1 protein (augWT1) acts as a tumor suppressor, whereas a CUG starting site for the WT1 protein (cugWT1) functions as an oncogene. In this study, we report an oncogenic role of cugWT1 in the AOM/DSS-induced colon cancer mouse model and in a urethane-induced lung cancer model in mice lacking cugWT1. Development of chemically-induced tumors was significantly depressed in cugWT1-deficient mice. Moreover, glycogen synthase kinase 3β promoted phosphorylation of cugWT1 at S64, resulting in ubiquitination and degradation of the cugWT1 associated with the F-box−/− WD repeat-containing protein 8. Overall, our findings suggest that inhibition of cugWT1 expression provides a potential candidate target for therapy.
Significance
These findings demonstrate that CUG-translated WT1 plays an oncogenic role in vivo, and GSK3β-mediated phosphorylation of cugWT1 induces its ubiquitination and degradation in concert with FBXW8.
... Among these are 53BP1 and BRCA1, critical for the DSB repair pathway choice. In fact, the recruitment of 53BP1 to the site of the DNA lesion depends on the methylation of histone H4 on Lys20 (H4K20), the degradation of competing H4K20me readers, deacetylation of H4K16 and ubiquitination of H2AK15 [24]. Such modified histones are read and interpreted by specific domains. ...
Chromatin plays a pivotal role in regulating the DNA damage response and during DNA double-strand break repair. Upon the generation of DNA breaks, the chromatin structure is altered by post-translational modifications of histones and chromatin remodeling. How the chromatin structure, and the epigenetic information that it carries, is reestablished after the completion of DNA break repair remains unclear though. Also, how these processes influence recovery of the cell cycle remains poorly understood. We recently performed a reverse genetic screen for novel chromatin regulators that control checkpoint recovery after DNA damage. Here we discuss the implications of PHD finger protein 6 (PHF6) and additional candidates from the NuA4 ATPase-dependent chromatin-remodeling complex and the Cohesin complex, required for sister chromatid cohesion, in DNA repair and checkpoint recovery in more detail. In addition, the potential role of this novel function of PHF6 in cancer development and treatment is reviewed.
https://www.tandfonline.com/eprint/2EE232NIZEJF4JH95ZJK/full?target=10.1080/15384101.2020.1796037
... BBAP-mediated monoubiquitination of H4K91 may also prime histones for additional PTMs including H4K20 methylation [104]. A more comprehensive overview of these modifications is presented elsewhere (see [179,180]). ...
Eukaryotic cells are constantly exposed to both endogenous and exogenous stressors that promote the induction of DNA damage. Of this damage, double strand breaks (DSBs) are the most lethal and must be efficiently repaired in order to maintain genomic integrity. Repair of DSBs occurs primarily through one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these pathways is in part regulated by histone post-translational modifications (PTMs) including ubiquitination. Ubiquitinated histones not only influence transcription and chromatin architecture at sites neighboring DSBs but serve as critical recruitment platforms for repair machinery as well. The reversal of these modifications by deubiquitinating enzymes (DUBs) is increasingly being recognized in a number of cellular processes including DSB repair. In this context, DUBs ensure proper levels of ubiquitin, regulate recruitment of downstream effectors, dictate repair pathway choice, and facilitate appropriate termination of the repair response. This review outlines the current understanding of histone ubiquitination in response to DSBs, followed by a comprehensive overview of the DUBs that catalyze the removal of these marks.
... Cross-talk between ubiquitination and other post-translational modifications such as phosphorylation have also been described [233][234][235]. While the SM N100 degron does not contain known phosphorylation sites, there is evidence of phosphorylated residues outside this domain (Supplementary Table 2) [226]. ...
Squalene monooxygenase (SM) is a vital sterol synthesis enzyme across eukaryotic life. In yeast, it is a therapeutic target for treating certain fungal infections, and in mammals it is a rate-limiting enzyme that represents a key control point in the cholesterol synthesis pathway. SM introduces an oxygen atom to squalene, which becomes the signature oxygen of the hydroxyl group in cholesterol. Our knowledge of SM has advanced tremendously since its initial cloning and characterization. Early research developed mammalian SM inhibitors to target SM for cholesterol-lowering purposes. The substrate squalene has gained considerable interest for its health benefits and in nanomedicine for delivery of drugs. More recently, SM has been implicated as a key dysregulated component in certain cancers. In this review, we summarize our present knowledge of SM, focusing on the regulation of SM and the gene encoding it, SQLE. Furthermore, we offer insights into the role of SM across different organisms and its significance in human health and disease.
... Also, the modification of histone and non-histone proteins by methylation and acetylation are involved in the regulation of DNA repair. For example, the recruitment of 53BP1 depends on the methylation of H4K20, the RNF8-dependent degradation of competing H4K20me readers, deacetylation of H4K16 and RNF168-mediated H2AK15 ubiquitination (16). In addition to promoting the direct recruitment of repair proteins, chromatin modifications can physically facilitate the accessibility of regulatory proteins to the lesion. ...
Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.
... This omnipresence, together with the highly dynamic nature of many PTMs and the feature that some PTMs often serve as switchboxes for various pathways, enable additional layers of regulation, especially fine-tuning in cellular signaling. Ubiquitination is one of the PTMs that occur abundantly in eukaryotic cells and crosstalk between ubiquitination and other PTMs has been observed in various cellular and physiological processes (Hunter, 2007;Khoury et al., 2011;Zhao et al., 2014). In recent years, ubiquitination has emerged as a key regulator of plant immunity (Marino et al., 2012;Trujillo and Shirasu, 2010;Zhou and Zeng, 2017). ...
Posttranslational modifications (PTMs) are central to the modulation of protein activity, stability, subcellular localization, and interaction with partners. They greatly expand the diversity and functionality of the proteome and have taken the center stage as key players in regulating numerous cellular and physiological processes. Increasing evidence indicates that in addition to a single regulatory PTM, many proteins are modified by multiple different types of PTMs in an orchestrated manner to collectively modulate the biological outcome. Such PTM crosstalk creates a combinatorial explosion in the number of proteoforms in a cell and greatly improve the ability of plants to rapidly mount and fine tune responses to different external and internal cues. While PTM crosstalk has been investigated in depth in humans, animals and yeast, the study of interplay between different PTMs in plants is still at its infant stage. In the past decade, investigations showed that PTMs are widely involved and play critical roles in the regulation of interactions between plants and pathogens. In particular, ubiquitination has emerged as a key regulator of plant immunity. This review discusses recent studies of the crosstalk between ubiquitination and six other PTMs, i.e. phosphorylation, SUMOylation, poly(ADP-ribosyl)ation, acetylation, redox modification, and glycosylation in the regulation of plant immunity. The two basic ways by which PTMs communicate as well as the underlying mechanisms and diverse outcomes of the PTM crosstalk in plant immunity are highlighted.
... However, more recently ubiquitin has also emerged as a powerful and versatile signaling system that regulates many different biological pathways (Chen and Sun, 2009;Husnjak and Dikic, 2012;Ramanathan and Ye, 2012;Oh et al., 2018;Rape, 2018;Clague et al., 2019;Mattern et al., 2019;Spit et al., 2019). Ubiquitin can influence protein-protein interactions, protein targeting and sorting, and regulates processes such as gene expression, and DNA repair (Ranjitkar et al., 2010;Piro et al., 2012;Marteijn et al., 2014;van Cuijk et al., 2014;Zhao et al., 2014;Venkatesh and Workman, 2015). Ubiquitin impinges on many critical processes in the cell and thereby plays major roles in normal development and human aging and disease. ...
Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin−76 amino acids–complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.
... [64] In addition to ubiquitylation, other posttranslational modifications of lysine that could also potentially affect the association of proteins with LLPS, such as methylation, sumoylation, neddylation, or acetylation, have been identified. [79,80] Indeed, acetylation of lysine reverses LLPS and diminishes colocalization of tau protein with SGs. [78] Nevertheless, it is not clear how the UPS shifts be-tween supporting the ability of RNA-binding proteins to form phase-separated RNA structures and promoting the degradation of these IDPs. ...
The fate of eukaryotic proteins, from their synthesis to destruction, is supervised by the ubiquitin–proteasome system (UPS). The UPS is the primary pathway responsible for selective proteolysis of intracellular proteins, which is guided by covalent attachment of ubiquitin to target proteins by E1 (activating), E2 (conjugating), and E3 (ligating) enzymes in a process known as ubiquitylation. The UPS can also regulate protein synthesis by influencing multiple steps of RNA (ribonucleic acid) metabolism. Here, recent publications concerning the interplay between the UPS and different types of RNA are reviewed. This interplay mainly involves specific RNA‐binding E3 ligases that link RNA‐dependent processes with protein ubiquitylation. The emerging understanding of their modes of RNA binding, their RNA targets, and their molecular and cellular functions are primarily focused on. It is discussed how the UPS adapted to interact with different types of RNA and how RNA molecules influence the ubiquitin signaling components. The interplay between RNAs and the RNA‐interacting ubiquitylation machinery. RNA‐binding ubiquitin ligases (RBULs) are the primary representative of the ubiquitylation machinery that supervises RNA‐dependent mechanisms. In turn, RNAs can modulate the activities of the ubiquitin–proteasome system in response to environmental or physiological cues.
... Because SUMO can competitively conjugate to the same lysine sites on target proteins as ubiquitin, it is well-accepted that sumoylation can stabilize target proteins, as exemplified by PCNA [43], IKBα [44] and Smad4 [45]. More recently, there is increasing evidence that sumoylation can also target some proteins for proteasomal degradation [26,46], e.g. HIF1-α [47], BMAL1 [48], EGR1 [49], PML and PML-RARA [50]. ...
Background:
Abnormal reactivation of androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) mainly results from overexpression and down-regulation of AR. Sumoylation of AR can influence its function. However, regulation of AR sumoylation by SUMO E3 ligases PIASs to modify AR distribution and stability are not well understood.
Methods:
We assessed the potential effect of SUMO3 modification on AR intracellular localization by immunostaining in AR-negative prostate cancer DU145 cells, and detected the effect of PIAS1/SUMO3 overexpression on AR sumoylation related degradation. Then we characterized AR sumoylation sites involved modified by SUMO3, and the key residue of PIAS1 involved in itself sumoylation and further mediated AR sumoylation (sumo3-conjugated), translocation and degradation. Finally we detected the recognition of PIAS1 (sumoylation ligase) to MDM2, a ubiquin ligase mediated AR degradation.
Results:
We demonstrate that SUMO E3 ligase PIAS1, along with SUMO3, mediates AR cytosolic translocation and subsequent degradation via a ubiquitin-proteasome pathway. Although AR sumoylation occurs prior to ubiquitination, the SUMO-acceptor lysine 386 on AR, together with ubiquitin-acceptor lysine 845, contribute to PIAS1/SUMO3-induced AR nuclear export, ubiquitination and subsequent degradation. Moreover, PIAS1 itself is modified by SUMO3 overexpression, and mutation of SUMO-acceptor lysine 117 on PIAS1 can impair AR cytoplasmic distribution, demonstrating the essential role of sumoylated PIAS1 in AR translocation. We further determine that sumoylated PIAS1 interacts with AR lysine 386 and 845 to form a binary complex. Consistent with the effect on AR distribution, SUMO3 modification of PIAS1 is also required for AR ubiquitination and degradation by recruiting ubiquitin E3 ligase MDM2.
Conclusion:
Taken together, SUMO3 modification of PIAS1 modulates AR cellular distribution and stability. Our study provided the evidence the crosstalk between AR sumoylation and ubquitination mediated by PIAS1 and SUMO3.
... The role of ubiquitin and SUMO modification in the DNA-damage response is established at the chromatin level, where upon recruitment they initiate complex signaling events for DNA repair and/or the induction of apoptosis (Jackson and Durocher, 2013;Zhao et al., 2014). Nuclear functions of NEDD8 required for DNA repair have also been reported through NEDDylation of cullins and non-cullin targets, including histones H2A, H4, and PCNA (Li et al., 2014;Ma et al., 2013;Guan et al., 2018). ...
Ubiquitin and ubiquitin-like chains are finely balanced by conjugating and de-conjugating enzymes. Alterations in this balance trigger the response to stress conditions and are often observed in pathologies. How such changes are detected is not well understood. We identify the HSP70 chaperone as a sensor of changes in the balance between mono- and poly-NEDDylation. Upon DNA damage, the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerization and apoptosis induction, a step that requires the HSP70 ATPase activity. HSP70 binds to NEDD8, and, in vitro, the conversion of NEDD8 chains into mono-NEDD8 stimulates HSP70 ATPase activity. This effect is independent of NEDD8 conjugation onto substrates. The study indicates that the NEDD8 cycle is a regulatory module of HSP70 function. These findings may be important in tumorigenesis, as we find decreased NEDP1 levels in hepatocellular carcinoma with concomitant accumulation of NEDD8 conjugates.
... Also, the modification of histone and non-histone proteins by methylation and acetylation are involved in the regulation of DNA repair. For example, the recruitment of 53BP1 depends on the methylation of H4K20, the RNF8-dependent degradation of competing H4K20me readers, deacetylation of H4K16 and RNF168mediated H2AK15 ubiquitination (16). In addition to promoting the direct recruitment of repair proteins, chromatin modifications can physically facilitate the accessibility of regulatory proteins to the lesion. ...
Post-translational histone modifications and chromatin remodelling play a critical role in the mechanisms controlling the integrity of the genome. Here we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating the balance between DNA damage-induced focus formation by 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knock down leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knock down decreases CtIP and BRCA1 protein and mRNA levels and cells depleted of PHF2 display genome instability and are sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.
... One of the most widely studied protein modifiers is ubiquitin (Ub), which targets proteins for turnover by proteasomes [3,4]. Non-proteolytic consequences of ubiquitin attachment have also been described, including vesicular protein trafficking, chromatin packing dynamics, and DNA repair [5,6]. ...
... Like ubiquitin and phosphorylation 78 , transcription factor lysine methylation is not limited to a single event. Many studies have found that lysine methylation can achieve distinct biological outcomes indirectly by acting in combination with other types of PMTs that occur at near or distant site 49 . ...
Protein lysine methylation is a critical and dynamic post-translational modification that can regulate protein stability and function. This post-translational modification is regulated by lysine methyltransferases and lysine demethylases. Recent studies using mass-spectrometric techniques have revealed that in addition to histones, a great number of transcription factors are also methylated, often at multiple sites and to different degrees (mono-, di-, trimethyl lysine). The biomedical significance of transcription factor methylation in human diseases, including cancer, has been explored recently. Some studies have demonstrated that interfering with transcription factor lysine methylation both in vitro and in vivo can inhibit cancer cell proliferation, thereby reversing tumor progression. The inhibitors targeting lysine methyltransferases and lysine demethylases have been under development for the past two decades, and may be used as potential anticancer agents in the clinic. In this review, we focus on the current findings of transcription factor lysine methylation, and the effects on both transcriptional activity and target gene expression. We outlined the biological significance of transcription factor lysine methylation on tumor progression and highlighted its clinical value in cancer therapy.
... Crosstalk between phosphorylation and the Ub machinery is important for regulating protein quantity, activity and interactions (19,20). In some contexts phosphorylation generates PTM motifs (phospho-degrons) that are recognized by receptor proteins associated with the ubiquitinproteasome degradation machinery. ...
IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3 (Glycogen Synthase Kinase 3) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7 (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elon-gation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear 'spent' molecules of IRF1 from transcriptionally engaged target promoters.
... Crosstalk between phosphorylation and the ubiquitin machinery is important for regulating protein abundance, activity and interactions (18,19). In some contexts phosphorylation generates PTM motifs (phospho-degrons) that are recognised by receptor proteins associated with the ubiquitinproteasome degradation machinery. ...
IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3β (Glycogen Synthase Kinase 3β) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7α (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilised protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear spent molecules of IRF1 from transcriptionally engaged target promoters.
... Compared with low-LET radiation, high-LET radiation causes greater protein modifications via posttranslational and oxidative processes [51][52][53][54][55]. The ubiquitin/proteasome system might modulate the cellular radiation response by affecting protein turnover [56] and acts together with phosphorylation, methylation, and acetylation of, for example, H2AX [57] and p53 [58], ADP-ribosylation, and other ubiquitin-like modifiers [59]. ...
Energetic, charged particles elicit an orchestrated DNA damage response (DDR) during their traversal through healthy tissues and tumors. Complex DNA damage formation, after exposure to high linear energy transfer (LET) charged particles, results in DNA repair foci formation, which begins within seconds. More protein modifications occur after high-LET, compared with low-LET, irradiation. Charged-particle exposure activates several transcription factors that are cytoprotective or cytodestructive, or that upregulate cytokine and chemokine expression, and are involved in bystander signaling. Molecular signaling for a survival or death decision in different tumor types and healthy tissues should be studied as prerequisite for shaping sensitizing and protective strategies. Long-term signaling and gene expression changes were found in various tissues of animals exposed to charged particles, and elucidation of their role in chronic and late effects of charged-particle therapy will help to develop effective preventive measures.
... This rapid and widespread response is made possible by coordinated posttranslational modifications of DNA repair factors and histones at the break site, including phosphorylation, ubiquitination, SUMOylation, PARylation and others [15,16,29,85,126,127,182,192]. In the context of phosphorylation, the DDR is regulated by three highly related PI3 kinase-like kinases (PI3KKs): Ataxia Telangiectasia Mutated (ATM), Ataxiatelangiectasia and RAD3 Related protein (ATR) and the catalytic subunit of the DNA protein kinase (DNA-PKcs) [108]. ...
In response to DNA double strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that coordinate their detection, signaling and repair. Central to this network is the ATM kinase and its substrates at chromatin surrounding DSBs H2AX, MDC1 and 53BP1. In humans, germline inactivation of ATM causes Ataxia Telangiectasia (A-T), an autosomal recessive syndrome of increased proneness to hematological malignancies driven by clonal chromosomal translocations. Studies of cancers arising in A-T patients and in genetically engineered mouse models (GEMM) deficient for ATM and its substrates have revealed complex, multilayered roles for ATM in translocation suppression and identified functional redundancies between ATM and its substrates in this context. "Programmed" DSBs at antigen receptor loci in developing lymphocytes employ ubiquitous DDR factors for signaling and repair and have been particularly useful for mechanistic studies because they are region-specific and can be monitored in vitro and in vivo. In this context, murine thymocytes deficient for ATM recapitulate the molecular events that lead to transformation in T cells from A-T patients and provide a widely used model to study the mechanisms that suppress RAG recombinase-dependent translocations. Similarly, analyses of the fate of Activation induced Cytidine Deaminase (AID)-dependent DSBs during mature B cell Class Switch Recombination (CSR) have defined the genetic requirements for end-joining and translocation suppression in this setting. Moreover, a unique role for 53BP1 in the promotion of synapsis of distant DSBs has emerged from these studies.
... Importantly, a protein sequence can have many different modifiable amino acids (Figure 2), but not necessarily all will be modified at the same time point or within the same copy of that particular protein. Even for a specific residue, there can be a competition between different modifications, as shown, for example, for Lys acetylation and ubiquitination [5], for different lipid modifications [6], and also for O-phosphorylation and O-GlcNac [7,8]. For each modifiable amino acid, there is an equilibrium between the free and different modified versions within a biological system, which can be shifted to precisely drive cellular response in a specific direction. ...
Introduction:
Numerous diseases are caused by changes in post-translational modifications (PTMs). Therefore, the number of clinical proteomics studies that include the analysis of PTMs is increasing. Combining complementary information - e.g., changes in protein abundance, PTM levels, with the genome and transcriptome (proteogenomics) - holds great promise for discovering important drivers and markers of disease, as variations in copy number, expression levels, or mutations without spatial/functional/isoform information is often insufficient or even misleading. Areas covered: We discuss general considerations, requirements, pitfalls, and future perspectives in applying PTM-centric proteomics to clinical samples. This includes samples obtained from a human subject, for instance (i) bodily fluids such as plasma, urine, or cerebrospinal fluid, (ii) primary cells such as reproductive cells, blood cells, and (iii) tissue samples/biopsies. Expert commentary: PTM-centric discovery proteomics can substantially contribute to the understanding of disease mechanisms by identifying signatures with potential diagnostic or even therapeutic relevance but may require coordinated efforts of interdisciplinary and eventually multi-national consortia, such as initiated in the cancer moonshot program. Additionally, robust and standardized mass spectrometry (MS) assays - particularly targeted MS, MALDI imaging, and immuno-MALDI - may be transferred to the clinic to improve patient stratification for precision medicine, and guide therapies.
... K63-linked polyubiquitylation is essential for DNA damage repair (44), inflammation, and the immune response (45,46) by either serving as a protein posttranslational modification or interacting with distinct subsets of protein factors. Here, we identify that, independent of its canonical protein-related roles, K63-linked ubiquitin chains interacted with DNA in a ubiquitin chain length-dependent manner and through a DIP region in ubiquitin that critically includes Thr 9 , Lys 11 , and Glu 34 . ...
Polyubiquitylation is canonically viewed as a posttranslational modification that governs protein stability or protein-protein interactions, in which distinct polyubiquitin linkages ultimately determine the fate of modified protein(s). We explored whether polyubiquitin chains have any nonprotein-related function. Using in vitro pull-down assays with synthetic materials, we found that polyubiquitin chains with the Lys⁶³ (K63) linkage bound to DNA through a motif we called the “DNA-interacting patch” (DIP), which is composed of the adjacent residues Thr⁹, Lys¹¹, and Glu³⁴. Upon DNA damage, the binding of K63-linked polyubiquitin chains to DNA enhanced the recruitment of repair factors through their interaction with an Ile⁴⁴ patch in ubiquitin to facilitate DNA repair. Furthermore, experimental or cancer patient–derived mutations within the DIP impaired the DNA binding capacity of ubiquitin and subsequently attenuated K63-linked polyubiquitin chain accumulation at sites of DNA damage, thereby resulting in defective DNA repair and increased cellular sensitivity to DNA-damaging agents. Our results therefore highlight a critical physiological role for K63-linked polyubiquitin chains in binding to DNA to facilitate DNA damage repair.
... These include the E2 ubiquitin-conjugating enzyme Ubc13, which functions in complex with RNF8 and RNF168. Ubc13 and its γH2AX independent recruitment through the Kat5 complex (130)(131)(132)(133) are potential important factors in CSR. Indeed, H2AX-deficient mice experience reduced CSR (39), while a link with SHM fails to be seen (38). ...
Epigenetic modifications, such as histone modifications, DNA methylation status, and non-coding RNAs (ncRNA), all contribute to antibody maturation during somatic hypermutation (SHM) and class-switch recombination (CSR). Histone modifications alter the chromatin landscape and, together with DNA primary and tertiary structures, they help recruit Activation-Induced Cytidine Deaminase (AID) to the immunoglobulin (Ig) locus. AID is a potent DNA mutator, which catalyzes cytosine-to-uracil deamination on single-stranded DNA to create U:G mismatches. It has been shown that alternate chromatin modifications, in concert with ncRNAs and potentially DNA methylation, regulate AID recruitment and stabilize DNA repair factors. We, hereby, assess the combination of these distinct modifications and discuss how they contribute to initiating differential DNA repair pathways at the Ig locus, which ultimately leads to enhanced antibody–antigen binding affinity (SHM) or antibody isotype switching (CSR). We will also highlight how misregulation of epigenomic regulation during DNA repair can compromise antibody development and lead to a number of immunological syndromes and cancer.
... Post-translational modifications, including phosphorylation, ubiquitination, ADP-ribosylation and methylation, play critical roles in regulating DNA repair factors at ionizing radiation (IR)-induced foci (IRIF) and are essential for the repair of DNA DSBs (2,42,43). Phosphorylation of 53BP1 by ATM is required for the recruitment of the downstream factors PTIP and RIF1 (11)(12)(13)16,17). It also has been shown in the previous study that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, also located in its UDR motif (44,45). ...
P53-binding protein 1 (53BP1) plays critical roles in DNA double strand break (DSB) repair by promoting non-homologous end joining (NHEJ), and loss of 53BP1 abolishes PARPi sensitivity in BRCA1-deficient cells by restoring homologous recombination (HR). 53BP1 is one of the proteins initially recruited to sites of DSBs via recognition of H4K20me2 through the Tudor-UDR domain and H2AK15ub through the UDR motif. Although extensive studies have been conducted, it remains unclear how the post-translational modification of 53BP1 affects DSB repair pathway choice. Here, we identified 53BP1 as an acetylated protein and determined that acetylation of 53BP1 inhibit NHEJ and promote HR by negatively regulating 53BP1 recruitment to DSBs. Mechanistically, CBP-mediated acetylation of K1626/1628 in the UDR motif disrupted the interaction between 53BP1 and nucleosomes, subsequently blocking the recruitment of 53BP1 and its downstream factors PTIP and RIF1 to DSBs. Hyperacetylation of 53BP1, similar to depletion of 53BP1, restored PARPi resistance in BRCA1-deficient cells. Interestingly, 53BP1 acetylation was tightly regulated by HDAC2 to maintain balance between the HR and NHEJ pathways. Together, our results demonstrate that the acetylation status of 53BP1 plays a key role in its recruitment to DSBs and reveal how specific 53BP1 modification modulates the choice of DNA repair pathway.
... Conservation of ubiquitination as a critical regulator of protein function from yeast to humans underscores the importance of this post-translational modification for eukaryotic life. Over the past two decades, ubiquitin has been linked to apoptosis, facilitating protein-protein interactions, promoting DNA transcription and cell repair (Grabbe et al., 2011;Hammond-Martel et al., 2012;Husnjak and Dikic, 2012;Zhao et al., 2014). ...
E3 ligases are essential scaffold proteins, facilitating the transfer of ubiquitin from E2 enzymes to lysine residues of client proteins via isopeptide bonds. The specificity of substrate binding and the expression and localization of E3 ligases can, however, endow these proteins with unique features with variable effects on mitochondrial, metabolic and CNS function. By comparing and contrasting two E3 ligases, Parkin and C-terminus of HSC70-Interacting protein (CHIP) we seek to highlight the biophysical prop- erties that may promote mitochondrial dysfunction, acute stress signaling and critical developmental periods to cease in response to mutations in these genes. Encoded by over 600 human genes, RING-finger proteins are the largest class of E3 ligases. Parkin contains three RING finger domains, with R1 and R2 separated by an in-between region (IBR) domain. Loss-of-function mutations in Parkin were identified in patients with early onset Parkinson's disease. CHIP is a member of the Ubox family of E3 ligases. It contains an N-terminal TPR domain and forms unique asymmetric homodimers. While CHIP can substitute for mutated Parkin and enhance survival, CHIP also has unique functions. The differences between these proteins are underscored by the observation that unlike Parkin-deficient animals, CHIP-null ani- mals age prematurely and have significantly impaired motor function. These properties make these E3 ligases appealing targets for clinical intervention. In this work, we discuss how biophysical and metabolic properties of these E3 ligases have driven rapid progress in identifying roles for E3 ligases in development, proteostasis, mitochondrial biology, and cell health, as well as new data about how these proteins alter the CNS proteome.
... Dynamic ubiquitination and de-ubiquitination is known to be important in transmitting DNA damage signals and in regulating various steps in repair 23 . Upon DNA DSB, ATM is activated and initiates a series of phosphorylation events that ultimately result in the recruitment of two E3 ubiquitin liagases, RNF8 first and then RNF168 (ref. ...
Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APC Cdh1 plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1. We show that the removal of USP1 depends on APC Cdh1 and requires Chk1 activation known to be catalysed by ssDNA-RPA-ATR signalling at the ends designated for HDR, linking the status of end processing to RIF1 or BRCA1 recruitment.
... Abundant evidence has demonstrated that the protein levels of many critical transcription factors are regulated by protein phosphorylation and proteasomal degradation (38). Several histone methyltransferases and demethylases, such as Set2, Jhd2 and Gis1, are degraded by the 26S proteasome system ( Figure 2) (24,25,39). ...
Histone modifiers regulate proper cellular activities in response to various environmental stress by modulating gene expression. In budding yeast, Rph1 transcriptionally represses many DNA damage or autophagy-related gene expression. However, little is known how Rph1 is regulated during these stress conditions. Here, we report that Rph1 is degraded upon DNA damage stress conditions. Notably, this degradation occurs via the autophagy pathway rather than through 26S proteasome proteolysis. Deletion of ATG genes or inhibition of vacuole protease activity compromises Rph1 turnover. We also determine that Rph1 and nuclear export protein Crm1 interact, which is required for Rph1 translocation from the nucleus to the cytoplasm. More importantly, Gcn5 directly acetylates Rph1 in vitro and in vivo, and Gcn5-containing complex, SAGA, is required for autophagic degradation of Rph1. Gcn5-mediated Rph1 acetylation is essential for the association of Rph1 with the nuclear pore protein Nup1. Finally, we show that sustaining high levels of Rph1 during DNA damage stress results in cell growth defects. Thus, we propose that Gcn5-mediated acetylation finely regulates Rph1 protein level and that autophagic degradation of Rph1 is important for cell homeostasis. Our findings may provide a general connection between DNA damage, protein acetylation and autophagy.
... Ubiquitination and its crosstalk with other post-translational modifications have recently emerged as key regulatory mechanisms in the cellular response to DNA damage 48,49 . As a consequence, defects in the ubiquitination machinery have been shown to be associated with reduced cellular survival and increased genomic instability in response to DSBs, mainly due to impaired protein-protein interactions causing mislocalization of DSB repair factors [50][51][52] . ...
Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.
... 6 7 Intensive studies have demonstrated that ubiquitination does not work alone, but act synergistically with other post-translational modifications such as phosphorylation, acetylation, and sumoylation. 84 One of the components of SCF ligases, the F-box protein, has been the focus of extensive investigations, as it functions by recruiting substrate proteins and to determine the targeting specificity of the SCF complex. Studies in the past 15 years have identified ∼69 F-box genes in the human genome and F-box proteins were shown to be evolutionarily conserved among eukaryotes. ...
Normal cellular processes such as cell survival, cell division and cell death depend on both temporally and spatially controlled synthesis and degradation of a set of proteins. In higher eukaryotic organisms, regulated protein degradation is mainly controlled by the ubiquitin-proteasome system (UPS) involving two sequential processes: ubiquitination and subsequent degradation by the 26S proteasomes. The Skp1–Cul1–F-box-protein (SCF) E3 ligases are the largest family in ubiquitin ligases that catalyze substrate ubiquitination and play regulatory roles in a wide range of fundamental biological processes. Within the SCF complex, the F-box protein determines the substrate specificity. In this review, we first provide a brief introduction to the UPS system and the SCF E3 ligases, and then discuss the functions of several F-box proteins wellstudied for their roles in several biological processes, including cell cycle progression, developmental patterning, circadian rhythm and cell survival. We will end our review with perspectives of future directions in order to better understand the function of the F-box proteins.
... Accordingly, deregulation of key PTMs such as phosphorylation, SUMOylation and ubiquitylation causes cell cycle defects, genome instability, and malignant transformation or cell death [1]. Crosstalk between PTMs in signal transduction is widespread [2], and has recently come to the fore in the SUMO and ubiquitin field. SUMO and ubiquitin are small protein PTMs that are covalently attached to target proteins via similar enzymatic cascades of E1 activating, E2 conjugating enzymes, and E3 ligases [3]. ...
Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors.
... Cross-talk between PTMs is employed to facilitate the spatiotemporal regulation of DDR signaling [182]. Cross-talk between histone modifications during DDR has been summarized [183,184]. Histone modifications can be activated or suppressed by each other sequentially, or they can function in specific DDR factor recruitment collaboratively. For example, H4K16ac adjacent to H4K20me2 affects 53BP1 binding through disruption of a salt bridge between H4K16 and Glu1551 in the 53BP1 Tudor domain [185,186] (Fig. 2E), while H2A/H2AX ubiquitination at K15 and H4K20me2 are both required for 53BP1 recruitment [111,112] (Fig. 2E). ...
DNA damage response (DDR) signaling network is initiated to protect cells from various exogenous and endogenous damage resources.
Timely and accurate regulation of DDR proteins is required for distinct DNA damage repair pathways. Post-translational modifications of
histone and non-histone proteins play a vital role in the DDR factor foci formation and signaling pathway. Phosphorylation,
ubiquitylation, SUMOylation, neddylation, poly(ADP-ribosyl)ation, acetylation, and methylation are all involved in the spatial–temporal
regulation of DDR, among which phosphorylation and ubiquitylation are well studied. Studies in the past decade also revealed
extensive roles of lysine methylation in response to DNA damage. Lysine methylation is finely regulated by plenty of lysine
methyltransferases, lysine demethylases, and can be recognized by proteins with chromodomain, plant homeodomain, Tudor domain,
malignant brain tumor domain, or proline–tryptophan–tryptophan–proline domain. In this review, we outline the dynamics and
regulation of histone lysine methylation at canonical (H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20) and non-canonical sites
after DNA damage, and discuss their context-specific functions in DDR protein recruitment or extraction, chromatin environment
establishment, and transcriptional regulation. We also present the emerging advances of lysine methylation in non-histone
proteins during DDR.
... Crosstalk between ubiquitin, UBLs and other lysinetargeted modifications. In addition to the widespread crosstalk between ubiquitin and UBLs, a further level of complexity in DSB-induced signalling arises from the interplay between these PTMs and other lysine modifi cations such as acetylation, methylation and ADP-ribosylation 147 . The bivalent recruitment of 53BP1 to DSB sites via its H4K20me2-binding Tudor domains and its UDR motif recognizing H2A ubiquitylated on K15 (REF. ...
DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate lesion repair and protection of genome integrity in mammalian cells. These advances offer new therapeutic opportunities for diseases linked to genetic instability.
Lysine post-translational modifications (PTMs) are widespread and versatile protein PTMs that are involved in diverse biological processes by regulating the fundamental functions of histone and non-histone proteins. Dysregulation of lysine PTMs is implicated in many diseases, and targeting lysine PTM regulatory factors, including writers, erasers, and readers, has become an effective strategy for disease therapy. The continuing development of mass spectrometry technologies coupled with antibody-based affinity enrichment technologies greatly promotes the discovery and decoding of PTMs. The global characterization of lysine PTMs is crucial for deciphering the regulatory networks, molecular functions, and mechanisms of action of lysine PTMs. In this review, we focus on lysine PTMs, and provide a summary of the corresponding regulatory enzymes of lysine PTMs and the proteomics advances in lysine PTMs by mass spectrometry technologies. We also discuss the types and biological functions of lysine PTM crosstalk on histone and non-histone proteins and current druggable targets of lysine PTM regulatory factors for disease therapy.
Cells protect the integrity of the genome against DNA double-strand breaks through several well-characterized mechanisms including nonhomologous end-joining repair, homologous recombination repair, microhomology-mediated end-joining and single-strand annealing. However, aberrant DNA damage responses (DDRs) lead to genome instability and tumorigenesis. Clarification of the mechanisms underlying the DDR following lethal damage will facilitate the identification of therapeutic targets for cancer. Histones are small proteins that play a major role in condensing DNA into chromatin and regulating gene function. Histone modifications commonly occur in several residues including lysine, arginine, serine, threonine and tyrosine, which can be acetylated, methylated, ubiquitinated and phosphorylated. Of these, lysine modifications have been extensively explored during DDRs. Here, we focus on discussing the roles of lysine modifying enzymes involved in acetylation, methylation, and ubiquitination during the DDR. We provide a comprehensive understanding of the basis of potential epigenetic therapies driven by histone lysine modifications.
Reversible lysine acetylation of histones is a key epigenetic regulatory process controlling gene expression. Reversible histone acetylation is mediated by two opposing enzyme families: histone acetyltransferases (HATs) and histone deacetylases (HDACs). Moreover, many non-histone targets of HATs and HDACs are known, suggesting a crucial role for lysine acetylation as a posttranslational modification on the cellular proteome and protein function far beyond chromatin-mediated gene regulation. The HDAC family consists of 18 members and pan-HDAC inhibitors (HDACi) are clinically used for the treatment of certain types of cancer. HDACi or individual HDAC member-deficient (cell lineage-specific) mice have also been tested in a large number of preclinical mouse models for several autoimmune and autoinflammatory diseases and in most cases HDACi treatment results in an attenuation of clinical disease severity. A reduction of disease severity has also been observed in mice lacking certain HDAC members. This indicates a high therapeutic potential of isoform-selective HDACi for immune-mediated diseases. Isoform-selective HDACi and thus targeted inactivation of HDAC isoforms might also overcome the adverse effects of current clinically approved pan-HDACi. This review provides a brief overview about the fundamental function of HDACs as epigenetic regulators, highlights the roles of HDACs beyond chromatin-mediated control of gene expression and summarizes the studies showing the impact of HDAC inhibitors and genetic deficiencies of HDAC members for the outcome of autoimmune and autoinflammatory diseases with a focus on rheumatoid arthritis, inflammatory bowel disease and experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis.
Background:
Triple Negative Breast cancer (TNBC) is incurable cancer with higher rates of relapse and shorter overall survival compared with other subtypes of breast cancer. Cellular retinoic acid binding protein 2 (CRABP2) belongs to fatty acid binding protein (FABP) family which binds with all-trans retinoic acid (RA). Previous studies from the database have reported the patients with high expression of CRABP2 showed different prognosis in ER+ and ER- breast cancer. However, its biological role and exact mechanism in breast cancer remain unknown. This aim of this study was to explore how CRABP2 regulated invasion and metastasis based on the estrogen receptor-α (herein called ER) status in breast cancer.
Methods:
Immunohistochemical staining method was used to analyze the expression of CRABP2 in human breast cancer tissues. Lentivirus vector-based shRNA technique was used to test the functional relevance of CRABP2 knockdown in breast tumors. Tail vein injection model was used to examine the lung metastasis. Co-immunoprecipitation, Western blotting, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were conducted to investigate the underlying mechanism that influenced the ER to the regulation of CRABP2 to Lats1.
Results:
We observed that knockdown of CRABP2 promotes EMT, invasion and metastasis of ER+ breast cancer cells in vitro and in vivo, whereas overexpression of CRABP2 yields the reverse results. In ER+ mammary cancer cells, the interaction of CRABP2 and Lats1 suppress the ubiquitination of Lats1 to activate Hippo pathway to inhibit the invasion and metastasis of ER+ mammary cancer. However, in ER- mammary cancer cells, the interaction of CRABP2 and Lats1 promote the ubiquitination of Lats1 to inactivate Hippo pathway to promote the invasion and metastasis of ER- mammary cancer.
Conclusions:
Our findings indicate that CRABP2 can suppress invasion and metastasis of ER+ breast cancer and promote invasion and metastasis of ER- breast cancer by regulating the stability of Lats1 in vitro and in vivo, and it provides new ideas for breast cancer therapy.
Cadmium (Cd) is a dispensable element for the human body and is usually considered a carcinogen. Occupational and environmental Cd exposure leads to sustained cellular proliferation in some tissues and tumorigenesis via an unclear mechanism. Here, we evaluated the role of Cd in the DNA damage response (DDR). We found that Cd exposure causes extensive DNA double-strand breaks (DSBs) and prevents accumulation of ubiquitination signals at these sites of DNA damage. Cd treatment compromises 53BP1 and BRCA1 recruitment to DSBs induced by itself or DNA damaging agents and partially inactivates the G2/M checkpoint. Mechanistically, Cd directly binds to the E3 ubiquitin ligase RNF168, induces the ubiquitin-proteasome pathway that mediates RNF168 degradation and suppresses RNF168 ubiquitin-ligase activity in vitro. Our study raises the possibility that Cd may target RNF168 to disrupt proper DSB signaling in cultured cells. This pathway may represent a novel mechanism for carcinogenesis induced by Cd.
Purpose:
Protein sumoylation is a highly dynamic and reversible post-translational modification, involving covalently conjugation of the small ubiquitin-like modifier (SUMO) to the lysine residue of the target protein. Similar to ubiquitination, sumoylation is catalyzed by E1, E2 and several E3 ligases. However, sumoylation usually does not cause protein degradation but alter the target function through diverse mechanisms. Increasing evidences have shown that sumoylation plays pivotal roles in the pathogenesis of human diseases, including neuron degeneration, cancer and heart disease, etc. We and others have shown that sumoylation is critically implicated in mouse eye development. However, the expression of sumoylation machinery has not been characterized in normal and pathogenic retina. Worldwide, age-related macular degeneration (AMD) is the leading cause of irreversible blindness in aged person. In the present study, we investigated the expression of the major sumoylation enzymes in normal mice and sodium iodateinduced AMD mouse model.
Methods:
Four-week-old C57BL/6J mice were used in our experiment. A sterile 1% NaIO3 solution was freshly prepared in PBS from solid NaIO3. Experimental mice were injected with 70 mg/kg NaIO3, and similar volumes of PBS as control. Eyes were enucleated and immersion in FAA fixation overnight and processed for eye cross-sections. After fixation, cross sections eyes were dehydrated, embedded in paraffin, and 6 mm transverse sections were cut using the rotary microtome. Then paraffin sections were stained with hematoxylin and eosin (H&E), and mouse retinal thickness was observed to assess the histopathologic changes.
Results:
Significantly declined RNA levels of E1, E2 and E3 ligase PIAS1 in NaIO3-injected mouse RPE one day-post treatment. Consistently, the protein level of PIAS1 was also decreased at this time point. At the late stage of treatment (three days post-injection), significantly reduced expression of E1 enzyme SAE1/UBA2 was detected in NaIO3-injected mouse retinas. In the contrary, dramatically increased E3 ligase RanBP2 was found in the injected-retinas.
Conclusion:
Together, our results demonstrated for the first time the dynamic expression of sumoylation pathway enzymes during the progression of retina degeneration induced by oxidative stress. Dynamic expression of E1, E2 and E3 enzymes were found during the time course of RPE and retina degeneration, which revealed the potential regulatory roles of sumoylation in AMD pathogenesis.
The differentiation of T helper cell subsets and their acquisition of effector functions are accompanied by changes in gene expression programmes, which in part are regulated and maintained by epigenetic processes. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are key epigenetic regulators that function by mediating dynamic changes in the acetylation of histones at lysine residues. In addition, many non-histone proteins are also acetylated, and reversible acetylation affects their functional properties, demonstrating that HDACs mediate effects beyond the epigenetic regulation of gene expression. In this Review, we discuss studies revealing that HDACs are key regulators of CD4+ T cell-mediated immunity in mice and humans and that HDACs are promising targets in T cell-mediated immune diseases. Finally, we discuss unanswered questions and future research directions to promote the concept that isoform-selective HDAC inhibitors might broaden the clinical application of HDAC inhibitors beyond their current use in certain types of cancer.
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signaling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks1–3. Understanding the upstream signaling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation4–6. Here, we demonstrate that human cells have a previously unrecognized signaling mechanism for sensing damage induced by alkylation. We find that the ASCC alkylation repair complex⁷ relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE domain of ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RNF113A as the E3 ligase responsible for upstream ubiquitin signaling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy (TTD), which harbor a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a heretofore unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked TTD.
Bloom syndrome is an autosomal recessive disease with phenotypes of cancer predisposition and premature aging caused by mutations of the blm gene. BLM belongs to the RecQ DNA helicase family and functions in maintaining genomic stability. In this study, we found that several lysine residues of BLM were acetylated in cells. The dynamic acetylation levels of BLM were regulated by CBP/p300 and SIRT1. We further identified that five lysines, K476, K863, K1010, K1329, and K1411, are the major acetylation sites. Treating cells with different DNA damage agents found that acetylation of BLM was different in response to etoposide and hydroxyurea, suggesting that BLM acetylation may have multiple functions in DNA repair.
Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.Oncogene advance online publication, 14 November 2016; doi:10.1038/onc.2016.392.
Histone demethylation is known to regulate transcription, but its role in other processes is largely unknown. We report a role for the histone demethylase LSD1/KDM1A in the DNA damage response (DDR). We show that LSD1 is recruited directly to sites of DNA damage. H3K4 dimethylation, a major substrate for LSD1, is reduced at sites of DNA damage in an LSD1-dependent manner. The E3 ubiquitin ligase RNF168 physically interacts with LSD1 and we find this interaction to be important for LSD1 recruitment to DNA damage sites. Although loss of LSD1 did not affect the initial formation of pH2A.X foci, 53BP1 and BRCA1 complex recruitment were reduced upon LSD1 knockdown. Mechanistically, this was likely a result of compromised histone ubiquitylation preferentially in late S/G2. Consistent with a role in the DDR, knockdown of LSD1 resulted in moderate hypersensitivity to γ-irradiation and increased homologous recombination. Our findings uncover a direct role for LSD1 in the DDR and place LSD1 downstream of RNF168 in the DDR pathway.
Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or StUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled:Ubiquitin-Proteasome System.
Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response.
53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.
Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13-Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response.
The BAL1 macrodomain-containing protein and its partner E3 ligase, BBAP, are overexpressed in chemotherapy-resistant lymphomas.
BBAP selectively ubiquitylates histone H4 and indirectly promotes early 53BP1 recruitment to DNA damage sites. However, neither
BBAP nor BAL1 has been directly associated with a DNA damage response (DDR), and the function of BAL1 remains undefined. Herein,
we describe a direct link between rapid and short-lived poly(ADP-ribose) (PAR) polymerase 1 (PARP1) activation and PARylation
at DNA damage sites, PAR-dependent recruitment of the BAL1 macrodomain-containing protein and its partner E3 ligase, local
BBAP-mediated ubiquitylation, and subsequent recruitment of the checkpoint mediators 53BP1 and BRCA1. The PARP1-dependent
localization of BAL1-BBAP functionally limits both early and delayed DNA damage and enhances cellular viability independent
of ATM, MDC1, and RNF8. These data establish that BAL1 and BBAP are bona fide members of a DNA damage response pathway and
are directly associated with PARP1 activation, BRCA1 recruitment, and double-strand break repair.
Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.
The histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand
breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment
of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in
H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated.
Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were
identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects,
including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system
for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions
for H2AX in human cells.
Chromatin-remodeling enzymes play essential roles in many biological processes, including gene expression, DNA replication and repair, and cell division. Although one such complex, SWI/SNF, has been extensively studied, new discoveries are still being made. Here, we review SWI/SNF biochemistry; highlight recent genomic and proteomic advances; and address the role of SWI/SNF in human diseases, including cancer and viral infections. These studies have greatly increased our understanding of complex nuclear processes.
Ubiquitination of histones plays a critical role in the regulation of several processes within the nucleus, including maintenance of genome stability and transcriptional regulation. The only known ubiquitination site on histones is represented by a conserved Lys residue located at the C terminus of the protein. Here, we describe a novel ubiquitin mark at the N-terminal tail of histone H2As consisting of two Lys residues at positions 13 and 15 (K13/K15). This "bidentate" site is a target of the DNA damage response (DDR) ubiquitin ligases RNF8 and RNF168. Histone mutants lacking the K13/K15 site impair RNF168- and DNA damage-dependent ubiquitination. Conversely, inactivation of the canonical C-terminal site prevents the constitutive monoubiquitination of histone H2As but does not abolish the ubiquitination induced by RNF168. A ubiquitination-defective mutant is obtained by inactivating both the N- and the C-terminal sites, suggesting that these are unique, non-redundant acceptors of ubiquitination on histone H2As. This unprecedented result implies that RNF168 generates a qualitatively different Ub mark on chromatin.
Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.
Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylation (γH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and γH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems.
In response to DNA damage, many DNA damage factors, such as MDC1 and 53BP1, redistribute to sites of DNA damage. The mechanism governing the turnover of these factors at DNA damage sites, however, remains enigmatic. Here, we show that MDC1 is sumoylated following DNA damage, and the sumoylation of MDC1 at Lys1840 is required for MDC1 degradation and removal of MDC1 and 53BP1 from sites of DNA damage. Sumoylated MDC1 is recognized and ubiquitinated by the SUMO-targeted E3 ubiquitin ligase RNF4. Mutation of the MDC1 Lys 1840 (K1840R) results in impaired CtIP, replication protein A, and Rad51 accumulation at sites of DNA damage and defective homologous recombination (HR). The HR defect caused by MDC1K1840R mutation could be rescued by 53BP1 downregulation. These results reveal the intricate dynamics governing the assembly and disassembly of DNA damage factors at sites of DNA damage for prompt response to DNA damage.
The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.
Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel, SUMOylation of so-far elusive upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response. We show that HERC2 and RNF168 are novel DNA damage-dependent SUMOylation targets in human cells. In response to DSBs, both HERC2 and RNF168 were specifically modified with SUMO1 at DSB sites in a manner dependent on the SUMO E3 ligase PIAS4. SUMOylation of HERC2 was required for its DSB-induced association with RNF8 and for stabilizing the RNF8-Ubc13 complex. We also demonstrate that the ZZ Zinc finger in HERC2 defined a novel SUMO-specific binding module, which together with its concomitant SUMOylation and T4827 phosphorylation promoted binding to RNF8. Our findings provide novel insight into the regulatory complexity of how ubiquitylation and SUMOylation cooperate to orchestrate protein interactions with DSB repair foci.
The chromatin organization modifier domain (chromodomain) was first identified as a motif associated with chromatin silencing in Drosophila. There is growing evidence that chromodomains are evolutionary conserved across different eukaryotic species to control diverse aspects of epigenetic regulation. Although originally reported as histone H3 methyllysine readers, the chromodomain functions have now expanded to recognition of other histone and non-histone partners as well as interaction with nucleic acids. Chromodomain binding to a diverse group of targets is mediated by a conserved substructure called the chromobox homology region. This motif can be used to predict methyllysine binding and distinguish chromodomains from related Tudor "Royal" family members. In this review, we discuss and classify various chromodomains according to their context, structure and the mechanism of target recognition.
Mutations in the tumour suppressor gene BRCA1 lead to breast and/or ovarian cancer. Here we show that loss of Brca1 in mice results in transcriptional de-repression of the tandemly repeated satellite DNA. Brca1 deficiency is accompanied by a reduction of condensed DNA regions in the genome and loss of ubiquitylation of histone H2A at satellite repeats. BRCA1 binds to satellite DNA regions and ubiquitylates H2A in vivo. Ectopic expression of H2A fused to ubiquitin reverses the effects of BRCA1 loss, indicating that BRCA1 maintains heterochromatin structure via ubiquitylation of histone H2A. Satellite DNA de-repression was also observed in mouse and human BRCA1-deficient breast cancers. Ectopic expression of satellite DNA can phenocopy BRCA1 loss in centrosome amplification, cell-cycle checkpoint defects, DNA damage and genomic instability. We propose that the role of BRCA1 in maintaining global heterochromatin integrity accounts for many of its tumour suppressor functions.
Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.
Protein ubiquitination is a crucial component of the DNA damage response. To study the mechanism of the DNA damage-induced ubiquitination pathway, we analyzed the impact of the loss of two E3 ubiquitin ligases, RNF8 and Chfr. Notably, DNA damage-induced activation of ATM kinase is suppressed in cells deficient in both RNF8 and Chfr (double-knockout, or DKO), and DKO mice develop thymic lymphomas that are nearly diploid but harbor clonal chromosome translocations. Moreover, DKO mice and cells are hypersensitive to ionizing radiation. We present evidence that RNF8 and Chfr synergistically regulate histone ubiquitination to control histone H4 Lys16 acetylation through MRG15-dependent acetyltransferase complexes. Through these complexes, RNF8 and Chfr affect chromatin relaxation and modulate ATM activation and DNA damage response pathways. Collectively, our findings demonstrate that two chromatin-remodeling factors, RNF8 and Chfr, function together to activate ATM and maintain genomic stability in vivo.
p53-binding protein 1 (53BP1) is known to be an important mediator of the DNA damage response, with dimethylation of histone H4 lysine 20 (H4K20me2) critical to the recruitment of 53BP1 to double-strand breaks (DSBs). However, it is not clear how 53BP1 is specifically targeted to the sites of DNA damage, as the overall level of H4K20me2 does not seem to increase following DNA damage. It has been proposed that DNA breaks may cause exposure of methylated H4K20 previously buried within the chromosome; however, experimental evidence for such a model is lacking. Here we found that H4K20 methylation actually increases locally upon the induction of DSBs and that methylation of H4K20 at DSBs is mediated by the histone methyltransferase MMSET (also known as NSD2 or WHSC1) in mammals. Downregulation of MMSET significantly decreases H4K20 methylation at DSBs and the subsequent accumulation of 53BP1. Furthermore, we found that the recruitment of MMSET to DSBs requires the γH2AX-MDC1 pathway; specifically, the interaction between the MDC1 BRCT domain and phosphorylated Ser 102 of MMSET. Thus, we propose that a pathway involving γH2AX-MDC1-MMSET regulates the induction of H4K20 methylation on histones around DSBs, which, in turn, facilitates 53BP1 recruitment.
Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair.
The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.
Cells respond to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin-remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs.
The chromatin remodelling factor chromodomain helicase DNA-binding protein 4 (CHD4) is a catalytic subunit of the NuRD transcriptional repressor complex. Here, we reveal novel functions for CHD4 in the DNA-damage response (DDR) and cell-cycle control. We show that CHD4 mediates rapid poly(ADP-ribose)-dependent recruitment of the NuRD complex to DNA-damage sites, and we identify CHD4 as a phosphorylation target for the apical DDR kinase ataxia-telangiectasia mutated. Functionally, we show that CHD4 promotes repair of DNA double-strand breaks and cell survival after DNA damage. In addition, we show that CHD4 acts as an important regulator of the G1/S cell-cycle transition by controlling p53 deacetylation. These results provide new insights into how the chromatin remodelling complex NuRD contributes to maintaining genome stability.
Regulatory ubiquitylation is emerging as an important mechanism to protect genome integrity in cells exposed to DNA damage1, 2, 3, 4, 5, 6, 7, 8, 9. However, the spectrum of known ubiquitin regulators of the DNA damage response (DDR) is limited and their functional interplay is poorly understood. Here, we identify HERC2 as a factor that regulates ubiquitin-dependent retention of repair proteins on damaged chromosomes. In response to ionising radiation (IR), HERC2 forms a complex with RNF8, a ubiquitin ligase involved in the DDR3, 4, 5, 6. The HERC2–RNF8 interaction requires IR-inducible phosphorylation of HERC2 at Thr 4827, which in turn binds to the forkhead-associated (FHA) domain of RNF8. Mechanistically, we provide evidence that HERC2 facilitates assembly of the ubiquitin-conjugating enzyme Ubc13 with RNF8, thereby promoting DNA damage-induced formation of Lys 63-linked ubiquitin chains. We also show that HERC2 interacts with, and maintains the levels of, RNF168, another ubiquitin ligase operating downstream of RNF8 (Refs 7, 8). Consequently, knockdown of HERC2 abrogates ubiquitin-dependent retention of repair factors such as 53BP1, RAP80 and BRCA1. Together with the increased radiosensitivity of HERC2-depleted cells, these results uncover a regulatory layer in the orchestration of protein interactions on damaged chromosomes and they underscore the role of ubiquitin-mediated signalling in genome maintenance.
Cells are constantly exposed to genotoxic events that can damage DNA. To counter this, cells have evolved a series of highly conserved DNA repair pathways to maintain genomic integrity. The ATM protein kinase is a master regulator of the DNA double-strand break (DSB) repair pathway. DSBs activate ATM's kinase activity, promoting the phosphorylation of proteins involved in both checkpoint activation and DNA repair. Recent work has revealed that two DNA damage response proteins, the Tip60 acetyltransferase and the mre11- rad50-nbs1 (MRN) complex, co-operate in the activation of ATM in response to DSBs. MRN functions to target ATM and the Tip60 acetyltransferase to DSBs. Tip60's chromodomain then interacts with histone H3 trimethylated on lysine 9, activating Tip60's acetyltransferase activity and stimulating the subsequent acetylation and activation of ATM's kinase activity. These results underscore the importance of chromatin structure in regulating DNA damage signaling and emphasize how histone modifications co-ordinate DNA repair. In addition, human tumors frequently exhibit altered patterns of histone methylation. This rewriting of the histone methylation code in tumor cells may impact the efficiency of DSB repair, increasing genomic instability and contributing to the initiation and progression of cancer.
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.
Mutations in BRCA1 are associated with a high risk of breast and ovarian cancer. BRCA1 participates in the DNA damage response and acts as a ubiquitin ligase. However, its regulation remains poorly understood. Here we report that BRCA1 is modified by small ubiquitin-like modifier (SUMO) in response to genotoxic stress, and co-localizes at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO-conjugating enzyme Ubc9. PIAS SUMO E3 ligases co-localize with and modulate SUMO modification of BRCA1, and are required for BRCA1 ubiquitin ligase activity in cells. In vitro SUMO modification of the BRCA1/BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO-regulated ubiquitin ligase (SRUbL). Further, PIAS SUMO ligases are required for complete accumulation of double-stranded DNA (dsDNA) damage-repair proteins subsequent to RNF8 accrual, and for proficient double-strand break repair. These data demonstrate that the SUMOylation pathway plays a significant role in mammalian DNA damage response.
Although the BBAP E3 ligase and its binding partner BAL are overexpressed in chemotherapy-resistant lymphomas, the role of these proteins in DNA damage responses remains undefined. Because BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. Herein, we demonstrate that BBAP selectively monoubiquitylates histone H4 lysine 91 and protects cells exposed to DNA-damaging agents. Disruption of BBAP-mediated monoubiquitylation of histone H4K91 is associated with the loss of chromatin-associated H4K20 methylase, mono- and dimethyl H4K20, and a delay in the kinetics of 53BP1 foci formation at sites of DNA damage. Because 53BP1 localizes to DNA damage sites, in part, via an interaction with dimethyl H4K20, these data directly implicate BBAP in the monoubiquitylation and additional posttranslational modification of histone H4 and an associated DNA damage response.
DNA double-strand break (DSB) repair involves complex interactions between chromatin and repair proteins, including Tip60, a tumour suppressor. Tip60 is an acetyltransferase that acetylates both histones and ATM (ataxia telangiectasia mutated) kinase. Inactivation of Tip60 leads to defective DNA repair and increased cancer risk. However, how DNA damage activates the acetyltransferase activity of Tip60 is not known. Here, we show that direct interaction between the chromodomain of Tip60 and histone H3 trimethylated on lysine 9 (H3K9me3) at DSBs activates the acetyltransferase activity of Tip60. Depletion of intracellular H3K9me3 blocks activation of the acetyltransferase activity of Tip60, resulting in defective ATM activation and widespread defects in DSB repair. In addition, the ability of Tip60 to access H3K9me3 is dependent on the DNA damage-induced displacement of HP1beta (heterochromatin protein 1beta) from H3K9me3. Finally, we demonstrate that the Mre11-Rad50-Nbs1 (MRN) complex targets Tip60 to H3K9me3, and is required to activate the acetyltransferase activity of Tip60. These results reveal a new function for H3K9me3 in coordinating activation of Tip60-dependent DNA repair pathways, and imply that aberrant patterns of histone methylation may contribute to cancer by altering the efficiency of DSB repair.
Cancer is a heterogeneous disease which arises primarily from the accumulation of mutations and deletions in the cells DNA. However, recent work has implicated both DNA methylation and histone modifications in the etiology and progression of cancer. For example, abnormal patterns of histone methylation have been identified in breast cancer, raising the possibility that altered patterns of histone methylation can be exploited both as a biomarker to monitor disease status and as a novel therapeutic target.
Here, we report on a novel role for histone methylation in regulating the ability of the Tip60 acetyltransferase to repair DNA double strand breaks (DSBs). Tip60 is a haplo‐insufficient tumor suppressor, and loss of heterozygosity at the Tip60 locus and reduced levels of nuclear Tip60 have been detected in both breast and prostate cancer. Tip60 is an acetyltransferase, and can acetylate several DNA repair proteins implicated in breast cancer, including myc, p53 and ATM. Previously, we demonstrated that Tip60 acetylates and activates the ATM protein, a key component of the cells DSB repair mechanism. Here, we demonstrate that methylation of histones creates docking sites for the recruitment of the Tip60 DNA repair complex to sites of DNA damage. Further, interaction between Tip60 and methylated histones serves to specifically activate Tip60'fs acetyltransferase activity at DSBs. Reduction in histone methylation levels compromises Tip60 activation, leading to reduced DNA repair and increased genomic instability. These results indicate that the level and distribution of specific histone methylation marks across the chromatin is a critical determinant of cells ability to repair and survive DNA damage. Thus histone methylation may regulate the genomic stability of cells and their intrinsic sensitivity to agents, including radio‐ and chemo‐therapy, which directly damage DNA. Altered histone methylation associated with breast cancer may therefore contribute to both genomic instability and to resistance to therapy. Finally, since histone methylation is reversible, the enzymes controlling histone methylation represent an exciting new target for therapy in breast cancer.
Citation Information: Cancer Res 2009;69(23 Suppl):B40.
The DNA damage response factor 53BP1 functions at the intersection of two major double strand break (DSB) repair pathways - promoting nonhomologous end-joining (NHEJ) and inhibiting homology-directed repair (HDR) - and integrates cellular inputs to ensure their timely execution in the proper cellular contexts. Recent work has revealed that 53BP1 controls 5' end resection at DNA ends, mediates synapsis of DNA ends, promotes the mobility of damaged chromatin, improves DSB repair in heterochromatic regions, and contributes to lethal mis-repair of DSBs in BRCA1-deficient cells. Here we review these aspects of 53BP1 and discuss new data revealing how 53BP1 is loaded onto chromatin and uses its interacting factors Rif1 and PTIP to promote NHEJ and inhibit HDR.
Reversible modification of proteins by SUMO (small ubiquitin-like modifier) affects a large number of cellular processes. In striking contrast to the related ubiquitin pathway, only a few enzymes participate in the SUMO system, although this pathway has numerous substrates as well. Emerging evidence suggests that SUMOylation frequently targets entire groups of physically interacting proteins rather than individual proteins. Protein-group SUMOylation appears to be triggered by recruitment of SUMO ligases to preassembled protein complexes. Because SUMOylation typically affects groups of proteins that bear SUMO-interaction motifs (SIMs), protein-group SUMOylation may foster physical interactions between proteins through multiple SUMO-SIM interactions. Individual SUMO modifications may act redundantly or additively, yet they may mediate dedicated functions as well. In this review, we focus on the unorthodox principles of this pathway and give examples for SUMO-controlled nuclear activities. We propose that collective SUMOylation is typical for nuclear assemblies and argue that SUMO serves as a distinguishing mark for functionally engaged protein fractions. Expected final online publication date for the Annual Review of Genetics Volume 47 is November 23, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.
Genome integrity is challenged by DNA damage from both endogenous and environmental sources. This damage must be repaired to allow both RNA and DNA polymerases to accurately read and duplicate the information in the genome. Multiple repair enzymes scan the DNA for problems, remove the offending damage, and restore the DNA duplex. These repair mechanisms are regulated by DNA damage response kinases including DNA-PKcs, ATM, and ATR that are activated at DNA lesions. These kinases improve the efficiency of DNA repair by phosphorylating repair proteins to modify their activities, by initiating a complex series of changes in the local chromatin structure near the damage site, and by altering the overall cellular environment to make it more conducive to repair. In this review, we focus on these three levels of regulation to illustrate how the DNA damage kinases promote efficient repair to maintain genome integrity and prevent disease.
The detection of DNA lesions within chromatin represents a critical step in cellular responses to DNA damage. However, the regulatory mechanisms that couple chromatin sensing to DNA-damage signalling in mammalian cells are not well understood. Here we show that tyrosine phosphorylation of the protein acetyltransferase KAT5 (also known as TIP60) increases after DNA damage in a manner that promotes KAT5 binding to the histone mark H3K9me3. This triggers KAT5-mediated acetylation of the ATM kinase, promoting DNA-damage-checkpoint activation and cell survival. We also establish that chromatin alterations can themselves enhance KAT5 tyrosine phosphorylation and ATM-dependent signalling, and identify the proto-oncogene c-Abl as a mediator of this modification. These findings define KAT5 tyrosine phosphorylation as a key event in the sensing of genomic and chromatin perturbations, and highlight a key role for c-Abl in such processes.
Carriers of BRCA1 germline mutations are predisposed to breast and ovarian cancers. Accumulated evidence shows that BRCA1 is quickly recruited to DNA lesions and plays an important role in the DNA damage response. However, the mechanism by which BRCA1 is recruited to DNA damage sites remains elusive. BRCA1 forms a Ring-domain heterodimer with BARD1, a major partner of BRCA1 that contains tandem BRCA1 C-terminus (BRCT) motifs. Here, we identify the BRCTs of BARD1 as a poly(ADP-ribose) (PAR)-binding module. The binding of the BARD1 BRCTs to PAR targets the BRCA1/BARD1 heterodimer to DNA damage sites. Thus, our study uncovers a PAR-dependent mechanism of rapid recruitment of BRCA1/BARD1 to DNA damage sites.
Ubiquitin-like proteins have been shown to be covalently conjugated to targets. However, the functions of these ubiquitin-like proteins are largely unknown. Here, we have screened most known ubiquitin-like proteins after DNA damage and found that NEDD8 is involved in the DNA damage response. Following various DNA damage stimuli, NEDD8 accumulated at DNA damage sites; this accumulation was dependent on an E2 enzyme (UBE2M) and an E3 ubiquitin ligase (RNF111). We further found that histone H4 was polyneddylated in response to DNA damage, and NEDD8 was conjugated to the N-terminal lysine residues of H4. Interestingly, the DNA damage-induced polyneddylation chain could be recognized by the MIU (motif interacting with ubiquitin) domain of RNF168. Loss of DNA damage-induced neddylation negatively regulated DNA damage-induced foci formation of RNF168 and its downstream functional partners, such as 53BP1 and BRCA1, thus affecting the normal DNA damage repair process.
The nucleosome remodeling and deacetylase (NuRD) complex regulates chromatin organization, gene transcription, genomic stability and developmental signaling. NuRD has a unique dual enzymatic activity, containing an ATPase and a histone deacetylase among its six core subunits. Recent studies indicate that NuRD composition and the interplay between subunits may dictate the diverse functions of the complex. In this review, we examine the structures and biological roles of the NuRD subunits and discuss new avenues of research to advance our understanding of the NuRD-mediated signaling network.
The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double-strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. We demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin- and SUMO-dependent DSB recognition, revealing that RNF4-synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.
The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.
Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.
Poly(ADP-ribosyl)ation (PARylation), a protein post-translational modification that was originally connected to the DNA damage response, is now known to engage in a continuously increasing number of biological processes. Despite extensive research and ceaseless, important findings about its role and mode of action, poly(ADP-ribose) remains an enigma regarding its structural complexity and diversity. The recent identification and structural characterization of four different poly(ADP-ribose) binding motifs represents a quantum leap in the comprehension of how this molecule can be decoded. Moreover, the recent discovery of a direct connection between PARylation and poly-ubiquitylation in targeting proteins for degradation by the proteasome has paved the way for a new interpretation of this protein modification. These two novel aspects, poly(ADP-ribose) recognition and readout by the ubiquitylation/proteasome system are developed here.
The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response.
Histones, the fundamental packaging elements of eukaryotic DNA, are highly decorated with a diverse set of post-translational modifications (PTMs) that are recognized to govern the structure and function of chromatin. Ten years ago, we put forward the histone code hypothesis, which provided a model to explain how single and/or combinatorial PTMs on histones regulate the diverse activities associated with chromatin (e.g., gene transcription). At that time, there was a limited understanding of both the number of PTMs that occur on histones and the proteins that place, remove, and interpret them. Since the conception of this hypothesis, the field has witnessed an unprecedented advance in our understanding of the enzymes that contribute to the establishment of histone PTMs, as well as the diverse effector proteins that bind them. While debate continues as to whether histone PTMs truly constitute a strict "code," it is becoming clear that PTMs on histone proteins function in elaborate combinations to regulate the many activities associated with chromatin. In this special issue, we celebrate the 50th anniversary of the landmark publication of the lac operon with a review that provides a current view of the histone code hypothesis, the lessons we have learned over the last decade, and the technologies that will drive our understanding of histone PTMs forward in the future.
Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here, we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.
With the exception of identical twins, individuals have different genetic makeup, which results from two key processes. During
meiosis, maternal and paternal homologous chromosomes assort randomly to form daughter cells (gametes), thus generating different
combinations of maternal and paternal chromosomes. Additional variation is generated by recombinations or crossovers, in which
parts of homologous chromosomes are exchanged, resulting in a new combination of parental alleles. On pages 835, 836, and
876 of this issue, Parvanov et al. (1), Baudat et al. (2), and Myers et al. (3) report the identification of a mammalian gene—PR domain containing 9 (PRDM9)—that controls the extent to which crossovers occur in preferred chromosomal locations, known as “hotspots” (see the figure).
The breast and ovarian cancer type 1 susceptibility protein (BRCA1) has pivotal roles in the maintenance of genome stability. Studies support that BRCA1 exerts its tumour suppression function primarily through its involvement in cell cycle checkpoint control and DNA damage repair. In addition, recent proteomic and genetic studies have revealed the presence of distinct BRCA1 complexes in vivo, each of which governs a specific cellular response to DNA damage. Thus, BRCA1 is emerging as the master regulator of the genome through its ability to execute and coordinate various aspects of the DNA damage response.
The Mre11/Rad50/Nbs1 (MRN) complex has a central function in facilitating activation of the ATM protein kinase at sites of DNA double-strand breaks (DSBs). However, several other factors are also required in human cells for efficient signalling through MRN and ATM, including the tumour suppressor proteins p53-binding protein 1 (53BP1) and BRCA1. In this study, we investigate the functions of these mediator proteins in ATM activation and find that the presence of 53BP1 and BRCA1 can amplify the effects of MRN when interactions between MRN and ATM are compromised. This effect is dependent on a direct interaction between MRN and the tandem breast cancer carboxy-terminal (BRCT) repeats in 53BP1, and is accompanied by hyper-phosphorylation of both Nbs1 and 53BP1. We also find that the BRCT domains of 53BP1 affect the overall structure of 53BP1 multimers and that this structure is important for promoting ATM phosphorylation of substrates as well as for the repair of DNA DSBs in mammalian cells.