UBE2W interacts with FANCL and regulates the monoubiquitination of fanconi anemia protein FANCD2

Fanconi anemia (FA) is a rare cancer-predisposing genetic disease mostly caused by improper regulation of the monoubiquitination of Fanconi anemia complementation group D2 (FANCD2). Genetic studies have indicated that ubiquitin conjugating enzyme UBE2T and HHR6 could regulate FANCD2 monoubiquitination through distinct mechanisms. However, the exact regulation mechanisms of FANCD2 monoubiquitination in response to different DNA damages remain unclear. Here we report that UBE2W, a new ubiquitin conjugating enzyme, could regulate FANCD2 monoubiquitination by mechanisms different from UBE2T or HHR6. Indeed, UBE2W exhibits ubiquitin conjugating enzyme activity and catalyzes the monoubiquitination of PHD domain of Fanconi anemia complementation group L (FANCL) in vitro. UBE2W binds to FANCL, and the PHD domain is both necessary and sufficient for this interaction in mammalian cells. In addition, over-expression of UBE2W in cells promotes the monoubiquitination of FANCD2 and down-regulated UBE2W markedly reduces the UV irradiation-induced but not MMC-induced FANCD2 monoubiquitination. These results indicate that UBE2W regulates FANCD2 monoubiquitination by mechanisms different from UBE2T and HRR6. It may provide an additional regulatory step in the activation of the FA pathway.     

Histone H2B monoubiquitination in the chromatin of FLOWERING LOCUS C regulates flowering time in Arabidopsis

Ubiquitination is one of many known histone modifications that regulate gene expression. Here, we examine the Arabidopsis thaliana homologs of the yeast E2 and E3 enzymes responsible for H2B monoubiquitination (H2Bub1). Arabidopsis has two E3 homologs (HISTONE MONOUBIQUITINATION1 [HUB1] and HUB2) and three E2 homologs (UBIQUITIN CARRIER PROTEIN [UBC1] to UBC3). hub1 and hub2 mutants show the loss of H2Bub1 and early flowering. By contrast, single ubc1, ubc2, or ubc3 mutants show no flowering defect; only ubc1 ubc2 double mutants, and not double mutants with ubc3, show early flowering and H2Bub1 defects. This suggests that ubc1 and ubc2 are redundant, but ubc3 is not involved in flowering time regulation. Protein interaction analysis showed that HUB1 and HUB2 interact with each other and with UBC1 and UBC2, as well as self-associating. The expression of FLOWERING LOCUS C (FLC) and its homologs was repressed in hub1, hub2, and ubc1 ubc2 mutant plants. Association of H2Bub1 with the chromatin of FLC clade genes depended on UBC1,2 and HUB1,2, as did the dynamics of methylated histones H3K4me3 and H3K36me2. The monoubiquitination of H2B via UBC1,2 and HUB1,2 represents a novel form of histone modification that is involved in flowering time regulation.     

A conserved RING finger protein required for histone H2B monoubiquitination and cell size control

Monoubiquitination of histone H2B is required for methylation of histone H3 on lysine 4 (K4), a modification associated with active chromatin. The identity of the cognate ubiquitin ligase is unknown. We identify Bre1 as an evolutionarily conserved RING finger protein required in vivo for both H2B ubiquitination and H3 K4 methylation. The RING domain of Bre1 is essential for both of these modifications as is Lge1 (Large 1), a protein required for cell size control that copurifies with Bre1. In cells lacking the euchromatin-associated histone variant H2A.Z, BRE1, RAD6, and LGE1 are each essential for cell viability, supporting redundant functions for H2B ubiquitination and H2A substitution in the formation of active chromatin. Notably, analysis of mutants demonstrates a function for Bre1/Lge1-dependent H2B monoubiquitination in the control of cell size.     

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.     

UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination

The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.     

Different mechanisms of CDK5 and CDK2 activation as revealed by CDK5/p25 and CDK2/cyclin A dynamics

A detailed analysis is presented of the dynamics of human CDK5 in complexes with the protein activator p25 and the purine-like inhibitor roscovitine. These and other findings related to the activation of CDK5 are critically reviewed from a molecular perspective. In addition, the results obtained on the behavior of CDK5 are compared with data on CDK2 to assess the differences and similarities between the two kinases in terms of (i) roscovitine binding, (ii) regulatory subunit association, (iii) conformational changes in the T-loop following CDK/regulatory subunit complex formation, and (iv) specificity in CDK/regulatory subunit recognition. An energy decomposition analysis, used for these purposes, revealed why the binding of p25 alone is sufficient to stabilize the extended active T-loop conformation of CDK5, whereas the equivalent conformational change in CDK2 requires both the binding of cyclin A and phosphorylation of the Thr(160) residue. The interaction energy of the CDK5 T-loop with p25 is about 26 kcal.mol(-1) greater than that of the CDK2 T-loop with cyclin A. The binding pattern between CDK5 and p25 was compared with that of CDK2/cyclin A to find specific regions involved in CDK/regulatory subunit recognition. The analyses performed revealed that the alphaNT-helix of cyclin A interacts with the alpha6-alpha7 loop and the alpha7 helix of CDK2, but these regions do not interact in the CDK5/p25 complex. Further differences between the CDK5/p25 and CDK2/cyclin A systems studied are discussed with respect to their specific functionality.     

Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases,and dramatically changes SUV39H2 functions

Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study.     

BAP1 regulates cell cycle progression through E2F1 target genes and mediates transcriptional silencing via H2A monoubiquitination in uveal melanoma cells

Uveal melanoma (UM) is the most common form of primary intraocular malignancy in adult and has the tendency to metastasize. BAP1 mutations are frequently found in UM and are associated with a poor prognosis. The role of BAP1 in cell cycle regulation is currently a research highlight, but its underlying mechanism is not well understood. Here, we report that BAP1 knockdown can lead to G1 arrest and is accompanied by a decrease in the expression of S phase genes in OCM1 cells. Furthermore, in chromatin immunoprecipitation experiments, BAP1 could bind to E2F1 responsive promoters and the localization of BAP1 to E2F1-responsive promoters is host cell factor-1 dependent. Moreover, BAP1 knockdown leads to increased H2AK119ub1 levels on E2F responsive promoters. Together, these results provide new insight into the mechanisms of BAP1 in cell cycle regulation.     

Exchange of histones H1, H2A, and H2B in vivo

We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.     

Restraining CDK1–cyclin B activation: PP2A on the cUSP(7)

USP7 inhibitors are gaining momentum as a therapeutic strategy to stabilize p53 through their ability to induce MDM2 degradation. However, these inhibitors come with an unexpected p53‐independent toxicity, via an unknown mechanism. In this issue of The EMBO Journal, Galarreta et al report how inhibition of USP7 leads to re‐distribution of PP2A from cytoplasm to nucleus and an increase of deleterious CDK1‐dependent phosphorylation throughout the cell cycle, revealing a new regulatory mechanism for the progression of S‐phase cells toward mitosis to maintain genomic integrity.Subject Categories: Cell Cycle, Post-translational Modifications, Proteolysis & Proteomics

Recent work reveals untimely activation of mitotic cyclin‐dependent kinase as a molecular basis for p53‐independent cell toxicity of USP7 deubiquitinase inhibitors.

The G2‐M transition in the eukaryotic cell cycle is a critical point to ensure that cells with damaged DNA are unable to enter the mitotic phase. This checkpoint is highly regulated by a number of kinases, including ATR, ATM and WEE1, and ends upon activation of the CDK1–cyclin B1 kinase complex (Visconti et al, 2016). Since premature activation of CDK1–cyclin B1 causes replication fork collapse, DNA damage, apoptosis, and mitotic catastrophe (Szmyd et al, 2019 and references therein), restricting CDK1–cyclin B1 activity prior to mitosis is key to maintaining genomic integrity.A body of recent work has suggested that the deubiquitinase USP7 is a master regulator of genomic integrity; it is required for DNA replication in numerous ways, including indirect regulation of cyclin A2 during the S‐phase, origin firing, and replication fork progression. USP7 also regulates mitotic entry by stabilizing PLK1, another kinase which is highly active in the M phase and ensures proper alignment of chromatids prior to segregation. Notably, USP7 inhibitors have become an attractive cancer therapeutic strategy based on their ability to trigger degradation of MDM2, and thereby stabilize p53 (Valles et al, 2020). However, there is growing evidence of USP7 inhibitor‐related toxicity that is not mediated through p53 (Lecona et al, 2016; Agathanggelou et al, 2017), indicating that USP7 inhibitors impact other cellular processes. Therefore, Galarreta et al (2021) investigated the potential functional relationship between USP7 and CDK1, given the role of both factors in regulating the cell cycle.Through a series of in vitro experiments, the authors confirmed that five USP7 inhibitors induce premature mitotic kinase activity, including increased MPM2 signal (indicative of mitosis‐specific phosphorylation events) and phosphorylation of histone H3 Ser10 (H3S10P) in all cells, regardless of where they are in the cell cycle. To determine whether USP7 affects CDK1 during the cell cycle, Galarreta et al (2021) demonstrate that cell lines treated with USP7 inhibitors exhibit reduced levels of inhibitory Tyr‐15 phosphorylation on CDK1 and increased cyclin B1 presence in the nucleus, suggesting activation of the CDK1–cyclin B1 complex. Furthermore, treatment with the CDK1 inhibitor RO3306 rescues the USP7 inhibitor‐dependent increase of mitotic activity.These observations suggest that CDK1 has the potential to catalyze mitosis‐specific phosphorylation irrespective of cell cycle phase and that cells rely on USP7‐specific deubiquitination to suppress or reverse premature CDK1 activity. Surprisingly, despite the nuclear localization of cyclin B and decrease in inhibitory CDK1 Tyr‐15 phosphorylation, USP7 inhibitors failed to drive cells into mitosis. How might this be? Nuclear localization of cyclin B normally occurs just minutes before the onset of mitosis and nuclear envelope breakdown (Santos et al, 2012), yet the nucleus remains intact following USP7 inhibition. Moreover, the decrease in Tyr‐15 phosphorylation suggests the ATR‐ and WEE1‐dependent G2/M checkpoint is inactivated by USP7 inhibition. Do these data hint at the presence of an additional, unknown regulatory mechanism controlling mitotic entry independent of the G2/M checkpoint and nuclear localization of the CDK1–cyclin B complex?To determine whether CDK1 is the driver of USP7 inhibitor toxicity, Galarreta et al exposed cells to CDK1 inhibitors and observed a reduction in apoptosis. Furthermore, CDK1 inhibitors promote cell survival in cells treated with three structurally unrelated USP7 inhibitors. Finally, CDC25A‐deficient mouse embryonic stem cells, which constitutively express low levels of CDK1, resist USP7 inhibition. Together, these data suggest that the USP7 inhibitor‐dependent toxicity is the result of CDK1‐mediated cell death. The authors note that the phosphatase PP2A is an antagonist for CDK1 in addition to being a candidate USP7 substrate (Lecona et al, 2016; Wlodarchak & Xing, 2016), and thus, they turned their attention to elucidating the connection between USP7 and PP2A. Combining biochemical and immunofluorescence studies, Galarreta et al (2021) demonstrate that USP7 interacts with two subunits of PP2A, and this interaction increases in response to USP7 inhibition. Inhibiting USP7 furthermore triggers PP2A re‐localization from the cytoplasm to the nucleus and increases the phosphorylation levels of PP2A substrates, such as AKT and PRC1. DT‐061, a chemical activator of PP2A, reduces CDK1 phosphorylation events, suggesting that PP2A deregulation is a key mediator of USP7 inhibitor‐related toxicity. Using phosphoproteomics to analyze cells treated with a USP7 inhibitor or PP2A‐inhibiting okadaic acid, the authors reveal that both treatments share a significant number of altered phosphorylated targets—especially those related to mitosis, the cell cycle, and epitopes with a CDK‐dependent motif. Thus, the effects of USP7 inhibitors on CDK1 appear to be mediated through PP2A localization to the nucleus.These unexpected findings raise several questions that potentially impact the current view of cell cycle regulation. For example, how does USP7 regulate PP2A localization and is this important for reversing CDK1‐dependent phosphorylation of mitotic substrates prior to mitosis? Does PP2A accumulation in the nucleus explain the failure of USP7‐inhibited cells to enter mitosis despite cyclin B1 nuclear localization? A role for ubiquitin signaling as a regulator of CDK1 in interphase cells has not been reported, and accordingly, new investigations will be needed to unravel the mechanisms by which USP7 controls PP2A localization.Another important question that arises is whether or not CDK1 has sufficient basal activity to phosphorylate numerous mitotic proteins independent of cell cycle phase. The observation that USP7 and PP2A act to prevent the improper accumulation of CDK1‐dependent phosphorylation even in G1 phase cells suggests this to be the case. Alternatively, USP7 activity may be required to prevent abnormal pairing of CDK1 with a cyclin that is ubiquitously expressed across the cell cycle. If so, more research will be needed to uncover how ubiquitin signaling ensures CDK1 only pairs with cyclin A and cyclin B once they accumulate later in the cell cycle.Interestingly, USP7 inhibition also causes a rapid loss in DNA synthesis of S‐phase cells, prompting the authors to perform a time course experiment to decipher the order of events following treatment (i.e., does CDK1 activation precede or follow termination of DNA replication?). High‐throughput microscopy and flow cytometry analysis reveal an immediate reduction of DNA replication, an increase of CDK1 activity, and elevated DNA damage before a detectable increase in H3S10P. Long‐term exposure of USP7 inhibitors leads to DNA damage restricted only to cells with corresponding high levels of H3S10P and MPM2. Overall, these results illustrate how inhibition of USP7 activates CDK1, disrupting DNA replication and inducing DNA damage (Fig 1).Open in a separate windowFigure 1USP7 regulates CDK1In untreated cells, CDK1 is suppressed by USP7 and PP2A, and CDK1‐cyclin B is only active during the G2/M transition. In response to treatment, USP7 facilitates PP2A localization to the nucleus. This allows CDK1 to initiate premature mitotic activity throughout the cell cycle, resulting in increased DNA damage and cellular toxicity.The finding that USP7 inhibitors caused a rapid shutdown of DNA replication brings to mind the recent findings by several groups, that CDK1 activation occurs concomitantly with the S/G2 transition and that premature CDK1 activation in S‐phase terminates replication (Akopyan et al, 2014; Lemmens et al, 2018; Saldivar et al, 2018; Deng et al, 2019; Branigan et al, 2021). According to these studies, coupling of CDK1 activation to the S/G2 transition is regulated by ATR‐CHK1 signaling, a pathway activated by DNA replication to restrain CDK1 through Tyr‐15 phosphorylation. Galarreta et al''s observation that USP7 inhibition overrides ATR‐CHK1 (i.e., Tyr‐15 phosphorylation) highlights the fundamental importance of ubiquitin signaling, and potentially PP2A localization, for ensuring proper S‐to‐M progression and genome maintenance. Ultimately, the mechanistic details of Galarreta et al''s observations remain to be elucidated, and undoubtedly, their findings will inspire future investigations. Moreover, their discovery may lead to a new strategy targeting CDK1 to mitigate unwanted toxicities in the clinic.     

Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.

Despite the ubiquity of histones in eukaryotes and their important role in determining the structure and function of chromatin, no detailed studies of the evolution of the histones have been reported. We have constructed phylogenetic trees for the core histones H2A, H2B, H3, and H4. Histones which form dimers (H2A/H2B and H3/H4) have very similar trees and appear to have co-evolved, with the exception of the divergent sea urchin testis H2Bs, for which no corresponding divergent H2As have been identified. The trees for H2A and H2B also support the theory that animals and fungi have a common ancestor. H3 and H4 are 10-fold less divergent than H2A and H2B. Three evolutionary histories are observed for histone variants. H2A.F/Z-type variants arose once early in evolution, while H2A.X variants arose separately, during the evolution of multicellular animals. H3.3-type variants have arisen in multiple independent events.