Yael David

The E2 ubiquitin conjugating enzymes direct polyubiquitination to preferred lysines

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.

Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries

Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone post-translational modification (PTM) signatures remains a daunting task in the epigenetics field. We introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semisynthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries, once they have been treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome, is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how preexisting PTMs, alone or synergistically, affect further PTM deposition via cross-talk mechanisms. We anticipate that the high throughput and sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.

Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins

Post-translational modification of the histone proteins in chromatin plays a central role in epigenetic control of DNA-templated processes in eukaryotic cells. Developing methods that enable the structure of histones to be manipulated is therefore essential to understand the biochemical mechanisms underlying genomic regulation. Here we present a synthetic biology method to engineer histones bearing site-specific modifications on cellular chromatin using protein trans-splicing. We genetically fused the N-terminal fragment of ultrafast split-intein to the C-terminus of histone H2B, which upon reaction with a complementary synthetic C-intein, generated labeled histone. Using this approach, we incorporated various non-native chemical modifications to chromatin in vivo with temporal control. Furthermore, the time and concentration dependence of protein trans-splicing performed in nucleo enabled us to examine differences in the accessibility of the euchromatin and heterochromatin regions of the epigenome. Finally, we used protein trans-splicing to semi-synthesize a native histone modification, H2BK120 ubiquitination, in isolated nuclei, and show that this can trigger downstream epigenetic cross-talk of H3K79 methylation.

E3 Ligases Determine Ubiquitination Site and Conjugate Type by Enforcing Specificity on E2 Enzymes

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono- and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Significance This article applies a range of protein ligation methods at the level of chromatin to understand the cross-talk mechanism between the well-established biomedical target the human Dot1 (hDot1L) methyltransferase and the ubiquitylation of H2B at lysine 120. Through a systematic structure–activity relationship study of the ubiquitin surface in regard to hDot1L-mediated H3K79 methylation and further investigation with precisely engineered chromatin substrates, a functional hotspot within ubiquitin was identified that is essential to the stimulation of hDot1L activity. More broadly, this work shows how chemical synthesis approaches can be used to precisely tailor protein posttranslational modifications to afford mechanistic insights that would be impossible by other methods. Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure–activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub–induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.

Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13

Background: The double-capped 26S proteasome complex is viewed as a symmetrical particle. Results: The ubiquitin receptor subunit Rpn13 is found in an asymmetrical ratio. Conclusion: Rpn13 ubiquitin receptor may dictate preference in substrate recruitment and processing. Significance: The ubiquitin receptor Rpn13 defines asymmetry and thus imposes directionality in the 26S proteasome. The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps.

SUMOylation of Blimp-1 promotes its proteasomal degradation

SUMO1 physically interacts with Blimp1 by anti tag coimmunoprecipitation (View interaction).

From ubiquitin-proteasomal degradation to CDK1 inactivation: requirements for the first polar body extrusion in mouse oocytes

Completion of the first meiotic division, manifested by extrusion of the first polar body (PBI), depends on proteasomal degradation of cyclin B1 and securin and the subsequent respective CDK1 inactivation and chromosome segregation. We aimed at identifying the polyubiquitin signal that mediates proteasomal action and at a better characterization of the role of CDK1 inactivation at this stage of meiosis. Microinjections of mutated ubiquitin proteins into mouse oocytes revealed that interference with lysine‐11 polyubiquitin chains abrogated chromosome segregation and reduced PBI extrusion by 63% as compared to WT ubiquitin‐injected controls. Inactivation of CDK1 in oocytes arrested at first metaphase by a proteasome inhibitor fully rescued PBI extrusion. However, removal of CDK1 inhibition failed to allow progression to the second metaphase, rather, inducing PBI reengulfment in 62% of the oocytes. Inhibition of either PLK1 or MEK1/2 during the first anaphase changed spindle dimensions. The PLK1 inhibitor also blocked PBI emission and prevented RhoA translocation. Our results identified lysine‐11 rather than the canonic lysine‐48 ubiquitin chains as the degradation signal in oocytes resuming meiosis, further disclosing that CDK1 inactivation is necessary and sufficient for PBI emission. This information significantly contributes to our understanding of faulty chromosome segregation that may lead to aneuploidy.—Pomerantz, Y., Elbaz, J., Ben‐Eliezer, I., Reizel, Y., David, Y., Galiani, D., Nevo, N., Navon, A., Dekel, N. From ubiquitin‐proteasomal degradation to CDK1 inactivation: requirements for the first polar body extrusion in mouse oocytes. FASEB J. 26, 4495–4505 (2012). www.fasebj.org

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Significance Programmable DNA-binding proteins such as nuclease-deficient Cas9 (dCas9) offer a simple system for genomic localization of genetically encodable biomolecules. These tools have enabled characterization of numerous different chromatin effectors at specific genetic elements. The delivery of fully synthetic cargos to specific loci is currently limited by a lack of adequate technologies. Here we used protein trans-splicing to ligate chemical moieties to dCas9 in vitro and subsequently deliver these molecules to live cells. We demonstrate the effectiveness and versatility of this method by delivering both small-molecule and proteinaceous epigenetic probes to endogenous genomic sites where they interact with their target proteins. This facile and modular strategy will find broad use in locus-specific targeting of chemical cargos to study nuclear processes. Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.