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1.
Nucleic Acids Res ; 51(16): 8413-8433, 2023 09 08.
Article in English | MEDLINE | ID: mdl-37462077

ABSTRACT

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.


Subject(s)
Daunorubicin , Leukemia, Myeloid, Acute , Humans , Daunorubicin/pharmacology , Daunorubicin/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Esters/therapeutic use , Chromatin/genetics
2.
Am J Hum Genet ; 102(2): 249-265, 2018 02 01.
Article in English | MEDLINE | ID: mdl-29395072

ABSTRACT

Townes-Brocks syndrome (TBS) is characterized by a spectrum of malformations in the digits, ears, and kidneys. These anomalies overlap those seen in a growing number of ciliopathies, which are genetic syndromes linked to defects in the formation or function of the primary cilia. TBS is caused by mutations in the gene encoding the transcriptional repressor SALL1 and is associated with the presence of a truncated protein that localizes to the cytoplasm. Here, we provide evidence that SALL1 mutations might cause TBS by means beyond its transcriptional capacity. By using proximity proteomics, we show that truncated SALL1 interacts with factors related to cilia function, including the negative regulators of ciliogenesis CCP110 and CEP97. This most likely contributes to more frequent cilia formation in TBS-derived fibroblasts, as well as in a CRISPR/Cas9-generated model cell line and in TBS-modeled mouse embryonic fibroblasts, than in wild-type controls. Furthermore, TBS-like cells show changes in cilia length and disassembly rates in combination with aberrant SHH signaling transduction. These findings support the hypothesis that aberrations in primary cilia and SHH signaling are contributing factors in TBS phenotypes, representing a paradigm shift in understanding TBS etiology. These results open possibilities for the treatment of TBS.


Subject(s)
Abnormalities, Multiple/genetics , Anus, Imperforate/genetics , Cilia/metabolism , Hearing Loss, Sensorineural/genetics , Mutation/genetics , Thumb/abnormalities , Transcription Factors/genetics , Animals , Cytoplasm/metabolism , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , HEK293 Cells , Hedgehog Proteins/metabolism , Humans , Infant, Newborn , Mice , Phenotype , Protein Binding , Proteomics , Signal Transduction
3.
Cell Rep ; 21(2): 546-558, 2017 Oct 10.
Article in English | MEDLINE | ID: mdl-29020638

ABSTRACT

The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.


Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Replication , Proteome/metabolism , Sumoylation , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , DNA Damage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , Stress, Physiological
4.
Sci Rep ; 7: 40756, 2017 01 18.
Article in English | MEDLINE | ID: mdl-28098257

ABSTRACT

Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.


Subject(s)
Ubiquitins/metabolism , Animals , Animals, Genetically Modified , Biotinylation , Cell Line , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Humans , Protein Processing, Post-Translational , Repressor Proteins/genetics , Repressor Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins , Sumoylation , Ubiquitins/genetics
5.
Mol Cell ; 63(4): 686-695, 2016 08 18.
Article in English | MEDLINE | ID: mdl-27453045

ABSTRACT

Dynamic protein phosphorylation is a fundamental mechanism regulating biological processes in all organisms. Protein phosphatase 2A (PP2A) is the main source of phosphatase activity in the cell, but the molecular details of substrate recognition are unknown. Here, we report that a conserved surface-exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues within the motif increases B56 binding, allowing integration of kinase and phosphatase activity. We identify conserved LxxIxE motifs in essential proteins throughout the eukaryotic domain of life and in human viruses, suggesting that the motifs are required for basic cellular function. Our study provides a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes.


Subject(s)
Protein Phosphatase 2/metabolism , Amino Acid Sequence , Animals , Binding Sites , Computational Biology , Conserved Sequence , Databases, Protein , Forkhead Box Protein O3/metabolism , GTPase-Activating Proteins/metabolism , HeLa Cells , Humans , Molecular Docking Simulation , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Phosphatase 2/chemistry , Protein Phosphatase 2/genetics , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Transfection
6.
Mol Cell Proteomics ; 14(5): 1419-34, 2015 May.
Article in English | MEDLINE | ID: mdl-25755297

ABSTRACT

Genotoxic agents can cause replication fork stalling in dividing cells because of DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 h of hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and two proteins were down-regulated for SUMOylation, whereas after 24 h, 35 proteins were up-regulated for SUMOylation, and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1000 SUMO-2 acceptor lysines in target proteins. The methodology is generic and is widely applicable in the ubiquitin field. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1, and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the aim of dealing with the induced DNA damage.


Subject(s)
Hydroxyurea/pharmacology , Lysine/metabolism , Nucleic Acid Synthesis Inhibitors/pharmacology , Osteoblasts/drug effects , Recombinant Fusion Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , BRCA1 Protein/chemistry , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line, Tumor , Chromatin Assembly Factor-1/chemistry , Chromatin Assembly Factor-1/genetics , Chromatin Assembly Factor-1/metabolism , DNA Damage , DNA Replication , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Gene Expression , Genomic Instability , Humans , Lysine/chemistry , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Protein Interaction Mapping , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Signal Transduction , Small Ubiquitin-Related Modifier Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins/genetics , Sumoylation , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors
7.
Mol Cell ; 53(6): 1053-66, 2014 Mar 20.
Article in English | MEDLINE | ID: mdl-24582501

ABSTRACT

Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell-cycle progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation. SUMOylation of FoxM1 peaks during G2 and M phase, when FoxM1 transcriptional activity is required. We found that a SUMOylation-deficient FoxM1 mutant was less active compared to wild-type FoxM1, implying that SUMOylation of the protein enhances its transcriptional activity. Mechanistically, SUMOylation blocks the dimerization of FoxM1, thereby relieving FoxM1 autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. Our findings contribute to understanding the role of SUMOylation during cell-cycle progression.


Subject(s)
Cell Cycle/genetics , Chromosome Segregation , Forkhead Transcription Factors/genetics , Small Ubiquitin-Related Modifier Proteins/genetics , Transcription, Genetic , Amino Acid Sequence , Forkhead Box Protein M1 , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Genomic Instability , HeLa Cells , Humans , Molecular Sequence Data , Protein Multimerization , Signal Transduction , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation
8.
EMBO Rep ; 15(3): 282-90, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24477933

ABSTRACT

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying entry into anaphase until all sister chromatids have become bi-oriented. A key component of the SAC is the Mad2 protein, which can adopt either an inactive open (O-Mad2) or active closed (C-Mad2) conformation. The conversion of O-Mad2 into C-Mad2 at unattached kinetochores is thought to be a key step in activating the SAC. The "template model" proposes that this is achieved by the recruitment of soluble O-Mad2 to C-Mad2 bound at kinetochores through its interaction with Mad1. Whether Mad1 has additional roles in the SAC beyond recruitment of C-Mad2 to kinetochores has not yet been addressed. Here, we show that Mad1 is required for mitotic arrest even when C-Mad2 is artificially recruited to kinetochores, indicating that it has indeed an additional function in promoting the checkpoint. The C-terminal globular domain of Mad1 and conserved residues in this region are required for this unexpected function of Mad1.


Subject(s)
Cell Cycle Proteins/metabolism , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints , Mad2 Proteins/metabolism , Nuclear Proteins/metabolism , Cell Cycle Proteins/chemistry , HeLa Cells , Humans , Nuclear Proteins/chemistry , Protein Binding , Protein Structure, Tertiary
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