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1.
Nat Rev Chem ; 7(8): 534-535, 2023 08.
Article in English | MEDLINE | ID: mdl-37434011
2.
Org Biomol Chem ; 21(28): 5873-5879, 2023 07 19.
Article in English | MEDLINE | ID: mdl-37417819

ABSTRACT

Nucleoside analogs show useful bioactive properties. A versatile solid-phase synthesis that readily enables the diversification of thymine-containing nucleoside analogs is presented. The utility of the approach is demonstrated with the preparation of a library of compounds for analysis with SNM1A, a DNA damage repair enzyme that contributes to cytotoxicity. This exploration provided the most promising nucleoside-derived inhibitor of SNM1A to date with an IC50 of 12.3 µM.


Subject(s)
Nucleosides , Thymine , Nucleosides/pharmacology , Thymine/pharmacology , Solid-Phase Synthesis Techniques , Exodeoxyribonucleases/metabolism , DNA Repair
3.
Chembiochem ; 24(13): e202200756, 2023 07 03.
Article in English | MEDLINE | ID: mdl-36917742

ABSTRACT

We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide-alkyne cycloaddition reaction. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes.


Subject(s)
Cell Cycle Proteins , Exodeoxyribonucleases , Exodeoxyribonucleases/metabolism , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/metabolism , DNA Repair , Oligonucleotides/chemistry
4.
ChemMedChem ; 17(5): e202100603, 2022 03 04.
Article in English | MEDLINE | ID: mdl-34905656

ABSTRACT

Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.


Subject(s)
Exodeoxyribonucleases , Nucleosides , DNA Repair , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Nucleosides/pharmacology , Phosphates , Phosphorylation , Zinc/pharmacology
5.
ACS Chem Biol ; 16(12): 2719-2730, 2021 12 17.
Article in English | MEDLINE | ID: mdl-34779621

ABSTRACT

Over the past two decades, activity-based probes have enabled a range of discoveries, including the characterization of new enzymes and drug targets. However, their suitability in some labeling experiments can be limited by nonspecific reactivity, poor membrane permeability, or high toxicity. One method for overcoming these issues is through the development of "inducible" activity-based probes. These probes are added to samples in an unreactive state and require in situ transformation to their active form before labeling can occur. In this Review, we discuss a variety of approaches to inducible activity-based probe design, different means of probe activation, and the advancements that have resulted from these applications. Additionally, we highlight recent developments which may provide opportunities for future inducible activity-based probe innovations.


Subject(s)
Molecular Probes/chemistry , Proteins/chemistry , Fluorescent Dyes/chemistry , Photochemical Processes , Protein Conformation , Structure-Activity Relationship
6.
Bioorg Med Chem ; 46: 116369, 2021 09 15.
Article in English | MEDLINE | ID: mdl-34482229

ABSTRACT

SNM1A is a zinc-dependent nuclease involved in the removal of interstrand crosslink lesions from DNA. Inhibition of interstrand crosslink repair enzymes such as SNM1A is a promising strategy for improving the efficacy of crosslinking chemotherapy drugs. Initial studies have demonstrated the feasibility of developing SNM1A inhibitors, but the full potential of this enzyme as a drug target has yet to be explored. Herein, the synthesis of a family of squaramide- and thiosquaramide-bearing nucleoside derivatives and their evaluation as SNM1A inhibitors is reported. A gel electrophoresis assay was used to identify nucleoside derivatives bearing an N-hydroxysquaramide or squaric acid moiety at the 3'-position, and a thymidine derivative bearing a 5'-thiosquaramide, as candidate SNM1A inhibitors. Quantitative IC50 determination showed that a thymidine derivative bearing a 5'-thiosquaramide was the most potent inhibitor, followed by a thymidine derivative bearing a 3'-squaric acid. UV-Vis titrations were carried out to evaluate the binding of the (thio)squaramides to zinc ions, allowing the order of inhibitory potency to be rationalised. The membrane permeability of the active inhibitors was investigated, with several compounds showing promise for future in vivo applications.


Subject(s)
DNA Repair Enzymes/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Quinine/analogs & derivatives , DNA Repair Enzymes/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , Quinine/chemical synthesis , Quinine/chemistry , Quinine/pharmacology , Structure-Activity Relationship
7.
Org Biomol Chem ; 19(10): 2177-2181, 2021 03 18.
Article in English | MEDLINE | ID: mdl-33630007

ABSTRACT

Eosin Y was assessed for its ability to induce a thiol-ene dependent protein-protein reaction in a metal-free, oxygen-tolerant, visible light mediated system. Protein-protein coupling efficiency under these mild conditions was comparable to previously reported UV-dependent conditions. The desired thiol-ene reaction was however limited within more complex biological systems.


Subject(s)
Cysteine Endopeptidases/chemistry , Deubiquitinating Enzymes/chemistry , Eosine Yellowish-(YS)/chemistry , Molecular Probes/chemistry , Alkenes/chemistry , Catalysis/radiation effects , Cysteine/chemistry , Eosine Yellowish-(YS)/radiation effects , HEK293 Cells , Humans , Light , Molecular Probes/radiation effects
8.
Molecules ; 26(2)2021 Jan 09.
Article in English | MEDLINE | ID: mdl-33435514

ABSTRACT

SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5'-position or 3'-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5'-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Exodeoxyribonucleases/antagonists & inhibitors , Nucleosides/pharmacology , Organometallic Compounds/pharmacology , Binding Sites/drug effects , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Esters/chemistry , Esters/pharmacology , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Malonates/chemistry , Malonates/pharmacology , Molecular Structure , Nucleosides/chemistry , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry
9.
Chem Sci ; 11(11): 2967-2972, 2020 Feb 06.
Article in English | MEDLINE | ID: mdl-34122797

ABSTRACT

Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme-probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe's reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.

10.
Chem Commun (Camb) ; 55(78): 11671-11674, 2019 Sep 26.
Article in English | MEDLINE | ID: mdl-31497827

ABSTRACT

We report the design and optimisation of novel oligonucleotide substrates for a sensitive fluorescence assay for high-throughput screening and functional studies of the DNA repair enzyme, XPF-ERCC1, with a view to accelerating inhibitor and drug discovery.


Subject(s)
DNA Repair , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Dimerization , Endonucleases/chemistry , Endonucleases/genetics , Humans , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Substrate Specificity , Temperature
11.
Org Biomol Chem ; 17(35): 8094-8105, 2019 09 21.
Article in English | MEDLINE | ID: mdl-31380542

ABSTRACT

Nine modified nucleosides, incorporating zinc-binding pharmacophores, have been synthesised and evaluated as inhibitors of the DNA repair nuclease SNM1A. The series included oxyamides, hydroxamic acids, hydroxamates, a hydrazide, a squarate ester and a squaramide. A hydroxamic acid-derived nucleoside inhibited the enzyme, offering a novel approach for potential therapeutic development through the use of rationally designed nucleoside derived inhibitors.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Exodeoxyribonucleases/antagonists & inhibitors , Hydroxamic Acids/pharmacology , Cell Cycle Proteins/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Exodeoxyribonucleases/metabolism , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Molecular Structure , Structure-Activity Relationship
12.
Thromb Haemost ; 119(1): 104-116, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30597505

ABSTRACT

Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.


Subject(s)
Blood Platelets/metabolism , Platelet Membrane Glycoproteins/chemistry , Ubiquitin/chemistry , Ubiquitination , Blood Platelets/drug effects , Flow Cytometry , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lysine/chemistry , Mass Spectrometry , P-Selectin/metabolism , Platelet Activation , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational , Protein-Tyrosine Kinases/metabolism , Signal Transduction
13.
Front Chem ; 7: 914, 2019.
Article in English | MEDLINE | ID: mdl-31998698

ABSTRACT

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery.

14.
Nature ; 556(7699): 113-117, 2018 04 05.
Article in English | MEDLINE | ID: mdl-29590092

ABSTRACT

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.


Subject(s)
Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Kelch-Like ECH-Associated Protein 1/chemistry , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/agonists , NF-E2-Related Factor 2/metabolism , Succinates/metabolism , Alkylation , Animals , Carboxy-Lyases , Cattle , Cysteine/chemistry , Cysteine/metabolism , Cytokines/biosynthesis , Cytokines/immunology , Feedback, Physiological , Female , HEK293 Cells , Humans , Hydro-Lyases/biosynthesis , Interferon-beta/immunology , Interferon-beta/pharmacology , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Proteins/metabolism , Rats , Rats, Wistar , Succinates/chemistry
15.
ChemistrySelect ; 3(45): 12824-12829, 2018 Dec 06.
Article in English | MEDLINE | ID: mdl-31414040

ABSTRACT

Phosphate groups are often crucial to biological activity and interactions of oligonucleotides, but confer poor membrane permeability. In addition, the group's lability to enzymatic hydrolysis is an obstacle to its use in therapeutics and in biological tools. We present the synthesis of N-oxyamide and squaramide modifications at the 5'-end of oligonucleotides as phosphate replacements and their biological evaluation using the 5'-exonuclease SNM1A. The squaryl diamide modification showed minimal recognition as a 5'-phosphate mimic; however, modest inhibition of SNM1A, postulated to occur through metal coordination at the active site, was observed. Their facile incorporation after solid-phase synthesis and recognition by the exonuclease makes squaryl diamides attractive neutral 5'-phosphate replacements for oligonucleotides. This work is the first example of squaryl diamide modifications at the 5'-terminal position of oligonucleotides and of the potential use of modified oligonucleotides to bind to the metal center of SNM1A.

16.
Nature ; 550(7677): 481-486, 2017 10 26.
Article in English | MEDLINE | ID: mdl-29045389

ABSTRACT

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.


Subject(s)
Piperidines/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Animals , Apoenzymes/antagonists & inhibitors , Apoenzymes/chemistry , Apoenzymes/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Female , Humans , Mice , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/pathology , Piperidines/chemical synthesis , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , Substrate Specificity , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitination/drug effects , Xenograft Model Antitumor Assays
17.
EMBO J ; 36(14): 2047-2060, 2017 07 14.
Article in English | MEDLINE | ID: mdl-28607004

ABSTRACT

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.


Subject(s)
DNA Repair , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Exodeoxyribonucleases/metabolism , Replication Protein A/metabolism , Cell Cycle Proteins , Humans , Models, Biological
18.
J Virol ; 91(13)2017 07 01.
Article in English | MEDLINE | ID: mdl-28424279

ABSTRACT

Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10-kDa auxiliary protein produced in the very late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeleton-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated perinuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate the horizontal transmission of the virus between insect hosts. Here, using mass spectrometric analysis, it is demonstrated that the C terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed aberrant formation of the perinuclear tubules. Thus, the phosphorylation of serine 93 may induce the aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects the structural conformation of P10.IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for the survival of its progeny, and therefore, P10 presents an enigma. As P10 polymerizes to form organized cytoskeletal structures that colocalize with host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus may potentially be responsible for its dissemination and survival.


Subject(s)
Protein Processing, Post-Translational , Viral Proteins/chemistry , Viral Proteins/metabolism , Animals , Cell Line , DNA Mutational Analysis , Insecta , Mass Spectrometry , Phosphorylation , Protein Conformation , Protein Multimerization , Viral Proteins/genetics
19.
Methods Mol Biol ; 1491: 131-144, 2017.
Article in English | MEDLINE | ID: mdl-27778286

ABSTRACT

Posttranslational modification of proteins with the small ubiquitin-like modifier (SUMO) regulates protein function in the context of cell cycle and DNA repair. The occurrence of SUMOylation is less frequent as compared to protein modification with ubiquitin, and appears to be controlled by a smaller pool of conjugating and deconjugating enzymes. Mass spectrometry has been instrumental in defining specific as well as proteome-wide views of SUMO-dependent biological processes, and several methodological approaches have been developed in the recent past. Here, we provide an overview of the latest experimental approaches to the study of SUMOylation, and also describe hands-on protocols using a combination of biochemistry and mass spectrometry-based technologies to profile proteins that are SUMOylated in human cells.


Subject(s)
Mass Spectrometry/methods , Sumoylation , DNA Repair , Humans
20.
Development ; 143(15): 2716-23, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27317809

ABSTRACT

The Polycomb repressive complexes PRC1 and PRC2 are key mediators of heritable gene silencing in multicellular organisms. Here, we characterise AEBP2, a known PRC2 co-factor which, in vitro, has been shown to stimulate PRC2 activity. We show that AEBP2 localises specifically to PRC2 target loci, including the inactive X chromosome. Proteomic analysis confirms that AEBP2 associates exclusively with PRC2 complexes. However, analysis of embryos homozygous for a targeted mutation of Aebp2 unexpectedly revealed a Trithorax phenotype, normally linked to antagonism of Polycomb function. Consistent with this, we observe elevated levels of PRC2-mediated histone H3K27 methylation at target loci in Aebp2 mutant embryonic stem cells (ESCs). We further demonstrate that mutant ESCs assemble atypical hybrid PRC2 subcomplexes, potentially accounting for enhancement of Polycomb activity, and suggesting that AEBP2 normally plays a role in defining the mutually exclusive composition of PRC2 subcomplexes.


Subject(s)
DNA-Binding Proteins/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Nuclear Proteins/metabolism , Polycomb Repressive Complex 2/metabolism , Proteomics/methods , Animals , Cell Line , DNA-Binding Proteins/genetics , Female , Histones/metabolism , Mice , Mutation/genetics , Nuclear Proteins/genetics , Polycomb Repressive Complex 2/genetics , Repressor Proteins
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