USP1 Data Analysis

HGNC Gene Name
ubiquitin specific peptidase 1
HGNC Gene Symbol
USP1
Identifiers
hgnc:12607 NCBIGene:7398 uniprot:O94782
Orthologs
mgi:2385198 rgd:1306461
INDRA Statements
deubiquitinations all statements
Pathway Commons
Search for USP1
Number of Papers
203 retrieved on 2022-05-22

DepMap Analysis

The Dependency Map (DepMap) is a genome-wide pooled CRISPR-Cas9 knockout proliferation screen conducted in more than 700 cancer cell lines spanning many different tumor lineages. Each cell line in the DepMap contains a unique barcode, and each gene knockout is assigned a “dependency score” on a per cell-line basis which quantifies the rate of CRISPR-Cas9 guide drop. It has been found that proteins with similar DepMap scores across cell lines, a phenomenon known as co-dependent genes, have closely related biological functions. This can include activity in the same or parallel pathways or membership in the same protein complex or the same pathway.

We identified the strongest seven co-dependent genes (“Symbol”) for DUBs and ran GO enrichment analysis. We used Biogrid, IntAct, and Pathway Commons PPIDs, and the NURSA protein-protein interaction databases (PPIDs) to determine whether co-dependent genes interact with one another. The “Evidence” column contains the PPIDs in which the interaction appears as well as whether there is support for the association by an INDRA statement. As another approach to identify potential interactors, we looked at proteomics data from the Broad Institute's Cancer Cell Line Encyclopedia (CCLE) for proteins whose expression across ~375 cell lines strongly correlated with the abundance of each DUB; it has previously been observed that proteins in the same complex are frequently significantly co-expressed. The correlations and associated p-values in the CCLE proteomics dataset are provided. And, we determined whether co-dependent genes yield similar transcriptomic signatures in the Broad Institute's Connectivity Map (CMap). A CMap score greater than 90 is considered significantly similar.

DepMap Correlations

Symbol Name DepMap Correlation Evidence CCLE Correlation CCLE Z-score CCLE p-value (adj) CCLE Significant CMAP Score CMAP Type
WDR48 WD repeat domain 48 0.547 BioGRID IntAct Pathway Commons INDRA (26) Reactome (4) 0.26 1.34 1.51e-05
TP53 tumor protein p53 -0.275 INDRA (1) Reactome (1) 0.12 0.58 6.44e-02
FANCM FA complementation group M 0.272 Reactome (2)
MDM4 MDM4 regulator of p53 0.261
LIG4 DNA ligase 4 0.26 Reactome (1) 0.01 -0.01 8.60e-01
APEX2 apurinic/apyrimidinic endodeoxyribonuclease 2 0.25 0.31 1.63 1.96e-05
WDR89 WD repeat domain 89 0.249 0.14 0.66 3.75e-02

Dependency GO Term Enrichment

Gene set enrichment analysis was done on the genes correlated with USP1using the terms from Gene Ontology and gene sets derived from the Gene Ontology Annotations database via MSigDB.

Using the biological processes and other Gene Ontology terms from well characterized DUBs as a positive control, several gene set enrichment analyses were considered. Threshold-less methods like GSEA had relatively poor results. Over-representation analysis with a threshold of of the top 7 highest absolute value Dependency Map correlations yielded the best results and is reported below.

GO Identifier GO Name GO Type p-value p-value (adj.) q-value
GO:0006310 DNA recombination Biological Process 6.80e-07 2.48e-04 7.30e-05
GO:1902525 regulation of protein monoubiquitination Biological Process 1.25e-06 4.55e-04 7.30e-05
GO:0140097 catalytic activity, acting on DNA Molecular Function 1.67e-05 6.09e-03 6.51e-04
GO:0006284 base-excision repair Biological Process 7.13e-05 2.60e-02 1.75e-03
GO:0070646 protein modification by small protein removal Biological Process 7.48e-05 2.72e-02 1.75e-03
GO:0036297 interstrand cross-link repair Biological Process 1.14e-04 4.15e-02 2.05e-03
GO:0010332 response to gamma radiation Biological Process 1.23e-04 4.47e-02 2.05e-03

Literature Mining

INDRA was used to automatically assemble known mechanisms related to USP1 from literature and knowledge bases. The first section shows only DUB activity and the second shows all other results.

Deubiquitinase Activity

psp cbn pc bel_lc signor biogrid lincs_drug tas hprd trrust ctd vhn pe drugbank omnipath conib crog dgi | rlimsp isi tees geneways eidos trips medscan sparser reach
USP1 deubiquitinates FANCD2. 10 / 44
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USP1 combined with USP1-associated factor 1 (UAF1) deubiquitinates PCNA or FANCD2 during DNA repair process such as interstrand cross-link (ICL) repair, homologous recombination (HR) repair, and translesion DNA synthesis (TLS) [87].
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USP1 deubiquitylates FancD2-Ub, and USP3 is thought to act on uH2A.

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USP1 deubiquitinates both ub-FANCD2 [XREF_BIBR] and ub-FANCI [XREF_BIBR], thus reverting the critical event in the activation of the FA pathway.

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Upon DNA damage, USP1 is downregulated by transcriptional shut-off [86], perhaps to facilitate FANCD2 mono-ubiquitination.

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Surprisingly, depletion of UBE2M did not reduce the FANCD2 monoubiquitination that is up-regulated by USP1 depletion (XREF_SUPPLEMENTARY), suggesting that the Nedd8 inhibition specifically abrogates FANCD2 monoubiquitination that is induced by exogenous DNA damage.

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How USP1 eventually deubiquitinates the FANCD2 and FANCI complex following the completion of the DNA repair is still a question.

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Monoubiquitinated FANCD2 interacts with the downstream repair proteins BRCA2/FANCD1 and possibly FANCJ and FANCN to repair the DNA crosslinks and other lesions by homologous recombination.In addition to the monoubiquitination of FANCD2, deubiquitination of FANCD2 by deubiquitination enzyme USP1 is known to be required for ICL repair.

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We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2.

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The exact role of deubiquitination of PCNA and FANCD2 by USP1 and UAF1 in human DNA damage response remains to be elucidated.

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Interestingly, the recently identified deubiquitinating enzyme, USP1, negatively regulates both FANCD2 and PCNA monoubiquitination, suggesting an interaction between these seemingly parallel DNA damag[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 deubiquitinates PCNA. 10 / 42
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The mono-ubiquitination of PCNA is usually reversed by USP1, however, USP1 does not cleave UbL73P chains (XREF_FIG).

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Recently, PCNA deubiquitylation by the USP1 and UAF1 complex in conjunction with ELG1, which directs the USP1-UAF1 to ubiquitin-PCNA, has emerged as an important regulatory mechanism of damage bypass.

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Review

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Later studies showed that USP1 also deubiquitinates proliferating cell nuclear antigen (PCNA) and Fanconi anemia complementation group I (FANCI).

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For example, USP1 deubiquitylates mono-ubiquitylated PCNA, which inhibits recruitment of DNA polymerases in the absence of DNA damage, and thereby leads to regulated DNA repair.

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USP1 deubiquitinates PCNA, thus contributing to prevent unscheduled recruitment of error-prone TLS DNA polymerases [XREF_BIBR].

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Interestingly, the recently identified deubiquitinating enzyme, USP1, negatively regulates both FANCD2 and PCNA monoubiquitination, suggesting an interaction between these seemingly parallel DNA damag[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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The USP1 and UAF1 complex also deubiquitinates PCNA-Ub, and deubiquitination requires the PCNA binding protein hELG1.

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Significantly, we report that PCNA polyubiquitination is negatively regulated by USP1.

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USP1 deubiquitinates PCNA and FANCD2 in cells.
USP1 deubiquitinates FANCI. 10 / 18
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Monoubiquitination or ATR dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was.

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Usp1 deubiquitinates FANCD2 and FANCI in the Falconi anemia pathway, promoting DNA repair.

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We have previously shown that USP1, in complex with its stimulatory binding partner, UAF1, deubiquitinates the Fanconi Anemia (FA) proteins, FANCD2 and FANCI.

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Deubiquitination of FANCD2 and FANCI proteins by the multisubunit protein complex USP1 and UAF1 is required for the completion of the FA pathway.

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Deubiquitination of FANCD2 and FANCI by USP1 in complex with UAF1 is also important for FA pathway function.

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Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was.

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However, in addition to this opposition to activate and monoubiquitinate FANCD2/I, USP1 deubiquitination of FANCI has also been associated with promoting core complex recruitment to the site of DNA da[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Deficiency of USP1 or FANCD2 promotes haematopoietic stem cells defects [50] , and siRNA knockdown experiments have shown that USP1 also deubiquitinates FANCI [48] , but whether this step is indispensable for ICL repair needs to be ascertained.The sliding clamp of DNA replication, also known as the proliferative cell nuclear antigen (PCNA), plays a leading role in the regulation of replicative lesion bypass (reviewed in [51] ).

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The activated FA pathway must be inactivated for completion and recycling of the functional pathway, and this event is regulated by the USP1 and UAF1 deubiquitinating enzyme complex, which deubiquitinates FANCD2 and FANCI.

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How USP1 eventually deubiquitinates the FANCD2 and FANCI complex following the completion of the DNA repair is still a question.
USP1 deubiquitinates ID1. 10 / 12
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Once the lesion has been repaired, the ID complex is deubiquitinated by the USP1 and UAF1 complex, which also acts on monoubiquitinated PCNA when associated to the RFC subunit ELG1.

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Williams et al. also recently reported that USP1 and UAF1 deubiquitinates and prevents proteasomal degradation of ID (inhibitor of DNA binding) proteins, 13 which have been shown to activate multiple pathways involved in tumor progression, including preservation of the cancer stem cell phenotype.

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USP1 also deubiquitinates and stabilizes the transcriptional regulators ID1, ID2 and ID3 [XREF_BIBR - XREF_BIBR].

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The FA ID complex is deubiquitinated by the UAF1 and USP1 complex.

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Therefore, we sought a deubiquitinating enzyme that counters ID ubiquitination.We show that USP1 deubiquitinates and stabilizes ID1, ID2, and ID3, resulting in their increased abundance.

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One of the responsible mechanisms for OS pathogenesis is the deubiquitination of ID proteins by enzyme USP1, which helps maintain the OS cell stemness.

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By deubiquitinating ID proteins, USP1 contributes to prevent bHLH mediated differentiation, and thus maintain stem-cell characteristics in osteosarcoma cells [XREF_BIBR].

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To identify a biological context in which USP1 deubiquitinates ID proteins, we examined USP1 expression patterns.

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Another piece of evidence for a tight coordination between the two pathways is the notion that the isopeptidase USP1 mediates deubiquitylation of both PCNA and the ID complex.
USP1 deubiquitinates ID2. 5 / 5
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Mechanically, we demonstrated that USP1 promoted GC metastasis via upregulating ID2 expression and further confirmed that USP1 stabilized ID2 expression through deubiquitinating ID2 in GC.

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To address whether USP1 deubiquitinated ID2 directly, ubiquitinated ID2 purified from 293T cells was incubated in vitro with either wild-type USP1 or USP1 C90S purified separately from 293T cells.

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Therefore, we sought a deubiquitinating enzyme that counters ID ubiquitination.We show that USP1 deubiquitinates and stabilizes ID1, ID2, and ID3, resulting in their increased abundance.

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USP1 also deubiquitinates and stabilizes the transcriptional regulators ID1, ID2 and ID3 [XREF_BIBR - XREF_BIBR].
USP1 deubiquitinates ubiquitinated PCNA. 4 / 4
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In vitro, the USP1 and UAF1 complex can de-ubiquitinate mono-ubiquitinated PCNA more efficiently than USP1 alone [59].

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USP1 (ubiquitin specific peptidase 1), which promotes de-ubiquitination of mono-ubiquitinated PCNA [57], forms a stable complex with UAF1 (USP1 associated factor 1), which stabilizes and activates USP[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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They observed an increased mutation frequency in cells in which USP1 was inactivated, a result expected if steady-state levels of monoubiquitinated PCNA are upregulated because of reduced deubiquitina[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Following DNA synthesis on damaged DNA templates by TLS polymerase (Mailand et al., 2013), the ubiquitinated PCNA is deubiquitinated by USP1, causing the release of TLS polymerases and re-association [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 deubiquitinates KDM4A. 3 / 3
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3.4 USP1 deubiquitinates KDM4A.

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9, 27, 28 As KDM4A plays a key role in human cancer development, it is possible that in human cancers USP1 promotes the deubiquitination and stabilization of KDM4A.

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A significant decrease in polyubiquitylated KDM4A protein was observed in cells transfected with GST fusion USP1, whereas GST fusion USP1 (CS) was not able to reduce KDM4A ubiquitination.
USP1 deubiquitinates AKT. 3 / 3
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USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signaling in muscle during prolonged starvation.

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USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signaling in muscle during prolonged starvation.

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USP1, similarly to USP12 and USP46, deubiquitinates AKT phosphatases PHLPP and PHLPPL [XREF_BIBR] and relies on UAF1 for its enzymatic activity, while it does not bind to WDR20 or any other additional proteins [XREF_BIBR].
USP1 deubiquitinates ID3. 3 / 3
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Review

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Therefore, we sought a deubiquitinating enzyme that counters ID ubiquitination.We show that USP1 deubiquitinates and stabilizes ID1, ID2, and ID3, resulting in their increased abundance.

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USP1 also deubiquitinates and stabilizes the transcriptional regulators ID1, ID2 and ID3 [XREF_BIBR - XREF_BIBR].
USP1 deubiquitinates PHLPP1. 2 / 2
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USP1 [137] and USP46 [138] are other DUBs known to deubiquitinate PHLPP1, with a similar outcome in other cancers as well.

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USP1, similarly to USP12 and USP46, deubiquitinates AKT phosphatases PHLPP and PHLPPL [XREF_BIBR] and relies on UAF1 for its enzymatic activity, while it does not bind to WDR20 or any other additional proteins [XREF_BIBR].
USP1 deubiquitinates ubiquitinated FANCD2. 2 / 2
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Furthermore, the USP1 and UAF1 complex can readily, and rapidly, de-ubiquitinate mono-ubiquitinated FANCD2 in an in vitro de-ubiquitination reaction [XREF_BIBR].

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Furthermore, the USP1 and UAF1 complex can readily, and rapidly, de-ubiquitinate mono-ubiquitinated FANCD2 in an in vitro de-ubiquitination reaction [165].
USP1 deubiquitinates ubiquitinated ID2. 1 / 1
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To address whether USP1 deubiquitinated ID2 directly, ubiquitinated ID2 purified from 293T cells was incubated in vitro with either wild-type USP1 or USP1 C90S purified separately from 293T cells.
USP1 deubiquitinates EZH2. 1 / 1
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beta-catenin and TCF4 activated transcription of the deubiquitinase USP1, which then interacted with and deubiquitinated EZH2 directly.
Modified USP1 leads to the deubiquitination of ID2. 1 / 1
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Both basal and USP1 induced ID2 deubiquitination was enhanced by coexpression of USP1 cofactor WDR48 (Cohn et al., 2007).
Unubiquitinated USP1 leads to the deubiquitination of PCNA. 1 / 1
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These include the deubiquitylation of PCNA and FANCD2 catalysed by USP1, deubiquitylation and stabilisation of 53BP1 or claspin by USP28 and more recently is has been reported that USP3 catalyses the deubiquitylation of H2AX and gamma-H2AX.
Modified USP1 leads to the deubiquitination of USP1. 1 / 1
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To further validate this point, experimental evidence has suggested that higher than normal levels of USP1 will inhibit the DNA damage induced monoubiquitination of USP1 targets, such as FANCD2, FANCI, and PCNA, whereas the loss of USP1 has been shown to cause chromosomal instability and elevated perinatal lethality in mice.
Modified mutated USP1 leads to the deubiquitination of PCNA. 1 / 1
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Importantly, expression of a USP1 mutant that can not be degraded via APC/C (Cdh1) inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites.
Unubiquitinated USP1 leads to the deubiquitination of FANCD2. 1 / 1
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These include the deubiquitylation of PCNA and FANCD2 catalysed by USP1, deubiquitylation and stabilisation of 53BP1 or claspin by USP28 and more recently is has been reported that USP3 catalyses the deubiquitylation of H2AX and gamma-H2AX.
USP1 leads to the deubiquitination of TBK1. 1 / 1
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USP1 greatly inhibited TBK1 ubiquitination, whereas USP12 and USP46 had no effects on TBK1 ubiquitination.
USP1 leads to the deubiquitination of CCP110. 1 / 1
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In addition, over-expression of two unrelated DUBs, USP1 and USP37, did not promote CP110 de-ubiquitylation (XREF_FIG and data not shown).
USP1 deubiquitinates PHLPP2. 1 / 1
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USP1, similarly to USP12 and USP46, deubiquitinates AKT phosphatases PHLPP and PHLPPL [XREF_BIBR] and relies on UAF1 for its enzymatic activity, while it does not bind to WDR20 or any other additional proteins [XREF_BIBR].
USP1 deubiquitinates IDS. 1 / 1
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Deubiquitination of IDs by USP1 promotes ID protein stability and prevents stem cell differentiation.
Modified USP1 leads to the deubiquitination of PCNA. 1 / 1
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Conversely, overexpression of USP1 when cells are treated with hydroxyurea inhibits monoubiquitination of PCNA.

Other Statements

psp cbn pc bel_lc signor biogrid lincs_drug tas hprd trrust ctd vhn pe drugbank omnipath conib crog dgi | rlimsp isi tees geneways eidos trips medscan sparser reach
WDR48 affects USP1
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WDR48 activates USP1.
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WDR48 activates USP1. 10 / 54
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UAF1 stimulates not only USP1, but also two other DUB enzymes, USP12 and USP46.

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WDR48 stimulates the activity of three DUBs, USP12, USP46 and USP1, a DUB which regulates the Fanconi anemia DNA damage pathway (Cohn et al., 2007, 2009; Faesen et al., 2011).

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For example, USP1, USP12, and USP46 are activated by the WD40-repeat containing UAF1, and USP7 is activated by GMPS (Cohn et al., 2007, 2009; Faesen et al., 2011; van der Knaap et al., 2005).

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WDR48 and UAF1 is an activator of USP1 and more recently has been shown to activate USP12 and USP46.

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UAF1 increases the basicity of the USP1 catalytic histidine, increasing its potency as a general base.

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The activation of USP1 by UAF1 takes place through an allosteric mechanism where the catalytic turnover (k cat ) of the enzyme is increased several fold (Cohn et al , xref ).

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Mutation of Ser313 to Ala on USP1 abolished the complex formation between USP1 and UAF1, thus prevented the activation of USP1 by UAF1.

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Although ATAD5 depletion does not alter protein levels of USP1 [XREF_BIBR], since UAF1 increases USP1 activity [XREF_BIBR], it is still possible that ATAD5 regulates deubiquitination activity of USP1 through UAF1.

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Our results support a regulatory mechanism in which UAF1 activates USP1 by modulating its active site conformation.

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For example, in contrast to the USP46 BL1 residue F258, which supports the tail of Ub, the corresponding USP12 residue F262 is pushed into the catalytic cleft and would collide with the substrate.Usin[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
WDR48 bound to USP1 activates USP1. 1 / 1
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The formation of USP1 and UAF1 complex greatly stimulates the DUB activity of USP1.
WDR48 inhibits USP1.
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WDR48 inhibits USP1. 1 / 1
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In vitro, USP1 activity is greatly stimulated by USP1-associated factor 1 (UAF1; also known as WDR48), enhancing USP1 catalytic turnover (k cat ) but not affinity (K m ) for monoubiquitylated substrates 74 .
WDR48 deubiquitinates USP1.
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WDR48 deubiquitinates USP1. 1 / 1
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The E1 protein from anogenital HPV types interacts with the UAF1 associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome.
USP1 affects ID1
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USP1 activates ID1.
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USP1 activates ID1. 10 / 16
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These data suggest that ATAD5 regulates interactions between UAF1 and ID1 and this regulatory mechanism for ID1 is different from USP1 mediated ID1 regulation.

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USP1 enhanced protein stability of the ID1 and CHEK1, important regulators of DNA damage response and stem cell maintenance (Lee et al., 2016).

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Collectively, these results indicate that the novel small molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells.

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In contrast to USP1 knockdown, USP1 overexpression in primary human mesenchymal stem cells (hMSCs) caused ID protein accumulation and interfered with normal differentiation.

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The small molecule inhibitor of USP1 promotes ID1 degradation and has cytotoxicity to leukemic cells [XREF_BIBR].

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More importantly , inhibition of USP1 leads to the downregulated expression of ID1 , further causing the inactivation of PI3K / AKT pathway .

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Besides DNA repair proteins, USP1 also promotes stabilization of ID (ID1-ID4) proteins 31.

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Here, we demonstrate that targeting USP1 with a small molecule, can promote ID1 degradation and cell death in multiple leukemic cell lines.

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Another USP1 selective inhibitor (C527) promotes the degradation of ID1 and induces cytotoxicity in leukemic cells [XREF_BIBR].

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A doxycycline induced USP1 shRNA suppressed USP1 expression and reduced ID1 and ID2 in the xenografts.
USP1 inhibits ID1.
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USP1 inhibits ID1. 6 / 6
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Moreover, depletion of USP1 by siRNA promoted ID1 degradation in K562 cells (XREF_SUPPLEMENTARY).

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A doxycycline induced USP1 shRNA suppressed USP1 expression and reduced ID1 and ID2 in the xenografts.

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USP1 knockdown in osteosarcoma cells caused ID protein destabilization, p53 independent induction of CDKN1A encoding cyclin dependent kinase inhibitor (CDKI) p21, and cell-cycle arrest.

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Small molecule inhibitors of USP1 target ID1 degradation in leukemic cells.

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small-molecule inhibitors of USP1 promote the degradation of inhibitor of DNA binding 1 (ID1), and are cytotoxic to leukemic cells [XREF_BIBR].

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shRNA resistant wild-type USP1, but not USP1 C90S, suppressed E box driven reporter activity caused by USP1 knockdown, confirming that USP1 catalytic activity is required for bHLH dependent transcript[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1-C90S inhibits ID1. 1 / 1
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shRNA resistant wild-type USP1, but not USP1 C90S, suppressed E box driven reporter activity caused by USP1 knockdown, confirming that USP1 catalytic activity is required for bHLH dependent transcript[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 increases the amount of ID1.
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USP1 increases the amount of ID1. 1 / 1
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Finally, we found that inhibition of USP1 downregulated the expression of ID1 and p-AKT, and upregulated ID1 expression could reverse the suppressive effects of USP1 inhibitor in B-ALL cells.
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Thus, USP1 overexpression perturbs normal osteoblast differentiation, which is characterized by p53 independent upregulation of multiple CDKIs (Funato et al., 2001; Kenner et al., 2004; Matsumoto et a[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Considering the action of the proteins ID, USP1 contributes to prevent differentiation mediated by bHLH and, consequently, keep CSC original characteristics.

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ID2 or USP1 overexpression in mesenchymal stem cells inhibited osteogenic differentiation and promoted retention of mesenchymal stem cell features.

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Finally, USP1 knockdown in osteosarcoma cells causes cell‐cycle arrest and osteogenic differentiation.

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These data, together with a study showing that misregulated ID expression inhibits osteogenic differentiation (Peng et al., 2004), prompted us to investigate whether USP1 overexpression disrupts hMSC [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation.

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Importantly, USP1 knockdown results in decreased mesenchymal cell proliferation, and enhanced differentiation of osteosarcoma cells which overexpress USP1 and ID1, providing a rationale for differentiation therapy of many cancer types including leukemia (e.g. acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML)).

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Collectively, our observations suggest that overexpression of USP1 or ID2 is sufficient to block osteoblastic differentiation, promote retention of stem like features, and render cells resistant to di[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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USP1 overexpression not only was necessary for the proliferation of several osteosarcoma cell lines, it also was sufficient to prevent normal mesenchymal cell differentiation, capturing the cells in a[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Finally, USP1 inhibits cell differentiation by stabilizing tumor promoting inhibitor of DNA binding (ID) proteins XREF_BIBR, XREF_BIBR.
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Recently, the deubiquitinase USP1 was shown to stabilize Id2 and modulate cellular differentiation in osteosarcomas.

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In light of the role that IDs play in stem cell maintenance and regulation of differentiation, we investigated whether either USP1 or ID knockdown in osteosarcoma would trigger osteoblastic differenti[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Considering the action of the proteins ID, USP1 contributes to prevent differentiation mediated by bHLH and, consequently, keep CSC original characteristics.

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Suppression of IDs 1-3 blocked colony formation in soft agar, indicating that the IDs are essential for USP1 transformation of NIH 3T3 cells.Because USP1 overexpression impaired osteoblastic different[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 affects FANCD2
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USP1 activates FANCD2.
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USP1 activates FANCD2. 7 / 8
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The uncoupling of monoubiquitination and chromatin enrichment from nuclear foci formation and effective ICL repair has previously been observed : Usp1 -/- murine embryonic fibroblasts display elevated levels of monoubiquitinated Fancd2 and Fancd2 chromatin localization, but fail to support Fancd2 nuclear foci formation and exhibit ICL-hypersensitivity XREF_BIBR.

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This observation indicates that UAF1 activates the DUB enzyme activity of USP1 and that it functions as a scaffolding protein for USP1 targeting FANCD2 [XREF_BIBR, XREF_BIBR].

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USP1 targets FANCD2, FANC1, and PCNA for deubiquitylation.

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USP1 depletion, which also causes an increase in FANCD2-Ub, even in the absence of DNA damage, increased promoter binding of FANCD2-Ub (XREF_FIG).

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Because USP1 is reported to target PCNA and FANCD2 during DNA repair (Nijman et al., 2005; Huang et al., 2006), we also checked if USP1 knockdown produced DNA damage.

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In USP1 depleted cells this fraction is also enriched in SQSTM1 (sequestosome 1), the aggresome marker HDAC6 (histone deacetylase 6), and the prototype of USP1 targets FANCD2 (FA complementation group D2).

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In addition, DUBs involved in DNA damage signaling are USP1 that targets PCNA (proliferating cell nuclear antigen) [76], FANCD2 and FANCI (the Fanconi anemia proteins) [93,94], and USP3 and USP16 that[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 inhibits FANCD2.
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USP1 inhibits FANCD2. 3 / 5
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Depletion of Usp1 decreases epidermal proliferation in Fancd2 (+/-) mice.

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However, in FA pathway, disruption of USP1 gene causes accumulation of the monoubiquitinated forms of FANCD2 and FANCI but increases ICL hypersensitivity and genomic instability [XREF_BIBR, XREF_BIBR].

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However, in FA pathway, disruption of USP1 gene causes accumulation of the monoubiquitinated forms of FANCD2/FANCI but increases ICL hypersensitivity and genomic instability [49, 77] .
USP1 decreases the amount of FANCD2.
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USP1 decreases the amount of FANCD2. 2 / 2
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Small molecule inhibitors of Usp1 may enhance FANCD2-Ub levels and TAp63 levels and perhaps promote cellular senescence.

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We found that USP1 inhibition by SJB increased the levels of both Ub-FANCD2 and Ub-FANCI in MM cells.
USP1 bound to WDR48 decreases the amount of FANCD2. 1 / 1
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Disruption of USP1/UAF1 complex promote level of FANCD2/FANCI ubiquitination and DNA repair defects, suggesting a failure in the completion of FA pathway (Nijman et al., 2005b, Smogorzewska et al., 2007).
Modified USP1 decreases the amount of FANCD2. 1 / 1
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These data further indicate that a decrease in Usp1 expression causes an increase in Fancd2-Ub levels, and that this increase may account for the decreased epidermal cell proliferation in the double heterozygote animals.
USP1 increases the amount of FANCD2.
| 1
USP1 increases the amount of FANCD2. 1 / 1
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In wild-type MEFs, Usp1 depletion increased the levels of Fancd2-Ub, TAp63, and p21 (XREF_FIG, lanes 1, 2) and enhanced OIS (XREF_FIG).

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Gene silencing of USP1 by lentivirus effectively inhibits proliferation and invasion of human osteosarcoma cells.

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Notably , downregulation of USP1 could inhibit cell proliferation and promote apoptosis in a variety of solid tumors 9,11,12 .

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Consistent with increased p21, USP1 knockdown reduced U2-OS cell proliferation.

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Similarly , pharmacological inhibition of USP1 by SJB3-019A significantly repressed cell proliferation and triggered B-ALL cell apoptosis .

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Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation.

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Recent studies have demonstrated that USP1 inhibition induced apoptosis and suppressed cell proliferation in acute myeloid leukemia ( AML ) and MM cells 12 , 13 .

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USP1 knockdown similarly reduced proliferation in HOS, SAOS, and SJSA, but not MG-63 osteosarcoma cells.

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Similarly, pharmacological inhibition of USP1 by SJB3-019A significantly repressed cell proliferation and triggered B-ALL cell apoptosis.

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In addition, inhibition of USP1 decreased PC proliferation and promoted response to therapeutic agent enzalutamide in a KDM4A dependent manner.

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USP1 knockdown inhibited cell proliferation by more than 2-fold within 72h of seeding and showed a simultaneous increase in caspase activity.

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shRNA resistant wild-type USP1, but neither USP1 C90S nor USP1Delta260-300, restored cell proliferation, indicating that both USP1 catalytic activity and ID substrate recognition are required to maint[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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USP1 downregulation decreased expressions of fibrosis related genes, suppressed proliferation, and promoted apoptosis in TGF-beta1-induced LX-2 cells, which were reversed by SNAIL overexpression.

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Depletion of Usp1 decreases epidermal proliferation in Fancd2 (+/-) mice.

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Collectively, our results suggest that silencing USP1 inhibits cell proliferation and invasion in U2OS cells.
USP1 affects PCNA
| 15
USP1 activates PCNA.
| 8
USP1 activates PCNA. 6 / 7
| 6

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USP1 also targets PCNA during replication stress.

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Whereas UAF1 displays no detectable activity, and isolated Usp1 shows only low levels of activity toward PCNA-Ub, stoichiometric mixtures of Usp1 and UAF1 are highly active, confirming that the PCNA-Ub can be targeted by Usp1 and UAF1 (XREF_FIG).

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In addition, DUBs involved in DNA damage signaling are USP1 that targets PCNA (proliferating cell nuclear antigen) [76], FANCD2 and FANCI (the Fanconi anemia proteins) [93,94], and USP3 and USP16 that[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Because USP1 is reported to target PCNA and FANCD2 during DNA repair (Nijman et al., 2005; Huang et al., 2006), we also checked if USP1 knockdown produced DNA damage.

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It has been reported that USP1 depletion from Rad18 deficient cells induces PCNA monoubiqutinatiton, indicating that other E3 ubiquitin ligase (s) carry out this function independent of Rad18.

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Similar results have been seen in the case of Usp1 and Uaf -1 where knockdown of either protein alone altered the ubiquitination status of Usp1 targets PCNA and FANCD2, in this example an epistatic relationship between Usp1 and Uaf-1 was observed [XREF_BIBR].
USP1 activates ubiquitinated PCNA. 1 / 1
| 1

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Oxidative stress not only induces DNA damages, but also inactivates USP1, allowing ubiquitinated PCNA to rapidly accumulate in cells to launch the TLS programme.
USP1 bound to WDR48 activates PCNA. 1 / 1
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The USP1 and UAF1 complex promotes homologous recombination and DNA cross-link repair via deubiquitylation of two critical DNA repair substrates : Fanconi anaemia protein (FANCD2) [XREF_BIBR] and proliferating cell nuclear antigen (PCNA) [XREF_BIBR, XREF_BIBR].
USP1 decreases the amount of PCNA.
| 3
USP1 decreases the amount of PCNA. 3 / 4
| 3

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Notably, exogenous expression of HA tagged C-SDE2 was able to antagonize the elevated PCNA-Ub levels caused by USP1 knockdown and UVC damage, suggesting that activated SDE2 regulates PCNA modification process independently of USP1 (XREF_FIG).

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This decrease in USP1 activity allows PCNA ubiquitylation levels to increase and promote TLS [XREF_BIBR].

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The loss of USP1 activity increases the level of mono-ubiquitinated DNA clamp PCNA, impairing DNA repair.
USP1 inhibits PCNA.
| 2
USP1 bound to WDR48 inhibits ubiquitinated PCNA. 1 / 1
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The USP1 and UAF1 complex reduces the accumulation of mono-ubiquitinated PCNA during normal cell division, which prevents mutagenesis by error-prone TLS polymerases.
USP1 inhibits ubiquitinated PCNA. 1 / 1
| 1

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In addition, in response to H 2 O 2 treatment, we found that USP1 reduced the accumulation of ubiquitinated PCNA but not inhibited NEDDylated PCNA, suggesting that USP1 does not affect PCNA NEDDylation.
USP1 increases the amount of PCNA.
| 2
USP1 increases the amount of ubiquitinated PCNA. 1 / 1
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Depletion of Usp1 also elevated the levels of ubiquitinated PCNA after DNA damage (XREF_FIG), suggesting that Usp1 counteracts Rad18 at stressed forks.
USP1 increases the amount of PCNA. 1 / 1
| 1

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USP1 knockdown or ML-323 treatment reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1.
| 16
| 12

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Despite ectopic expression of ID1 or -2, USP1 depleted cells (shUSP1 and ID1 and shUSP1 and ID2) underwent senescence with the same kinetics as control cells depleted for USP1 alone (shC and pBABE [B0[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Together, these data are consistent with the notion that senescence mediated by USP1 depletion does not produce DNA lesions that cause PCNA mono-ubiquitination.

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Finally, we addressed the question of whether or not USP1 mediated senescence is dependent on ID HLH transcription factors.

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Consequently, ID protein overexpression might rescue USP1 mediated senescence by suppressing CDKN1A expression.

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Usp1 depletion failed to induce senescence in the absence of TAp63, whereas Usp1 depletion in wild-type MEFs enhanced cellular senescence.

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If an aberrant localization of FD2-Ub was indeed a gatekeeper for senescence induced by USP1 depletion, then reduced FD2 levels should mitigate the maintenance of senescence upon USP1 depletion.

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Taken together, the data demonstrate that senescence induced by USP1 depletion is associated with a persistent ATR-CHK1-p53-CDKN1A-dependent DDR, perturbed replisome dynamics, and genomic instability,[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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These results suggest that senescence compromised cancer cells expressing low levels of USP1 or treatment of cancer cells expressing high levels of USP1 with USP1 inhibitors may be more susceptible to[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Together, these results show p53 and its major transcriptional target CDKN1A to be key factors required for the senescence arrest induced by USP1 depletion.

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Taken together, these data further indicate that depletion of USP1 results in increased cellular levels of FANCD2-Ub, causing increased senescence and decreased epithelial tumor growth in vivo.
| 4

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In our human fibroblast senescence model, TAp63 upregulation was absent, indicating that acute loss of USP1 function likely produces qualitatively different senescence responses depending on cell type[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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However, in addition to this opposition to activate and monoubiquitinate FANCD2/I, USP1 deubiquitination of FANCI has also been associated with promoting core complex recruitment to the site of DNA da[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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These results suggest that senescence compromised cancer cells expressing low levels of USP1 or treatment of cancer cells expressing high levels of USP1 with USP1 inhibitors may be more susceptible to[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Usp1 knockdown promotes senescence and reduces tumor growth in vivo.
| 4 9
| 4 4

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Recent studies have demonstrated that USP1 inhibition induced apoptosis and suppressed cell proliferation in acute myeloid leukemia ( AML ) and MM cells 12 , 13 .

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Blockade of Deubiquitylating Enzyme USP1 Inhibits DNA Repair and Triggers Apoptosis in Multiple Myeloma Cells.

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USP1 downregulation decreased expressions of fibrosis-related genes , suppressed proliferation , and promoted apoptosis in TGF-beta1-induced LX-2 cells , which were reversed by SNAIL overexpression .

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Similarly, pharmacological inhibition of USP1 by SJB3-019A significantly repressed cell proliferation and triggered B-ALL cell apoptosis.

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Inhibition of USP1 induces apoptosis via ID1 / AKT pathway in B-cell acute lymphoblastic leukemia cells Deubiquitylating enzyme ubiquitin-specific protease 1 ( USP1 ) has been reported to be aberrantly overexpressed in cancers , and it plays a critical role in regulating various cellular processes , such as cell proliferation , apoptosis , and cell differentiation .

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Inhibition of USP1 induces apoptosis via ID1 and AKT pathway in B-cell acute lymphoblastic leukemia cells.

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Notably , downregulation of USP1 could inhibit cell proliferation and promote apoptosis in a variety of solid tumors 9,11,12 .

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A recent study demonstrated that USP1 inhibition induces apoptosis in leukemic cells 30.
USP1 activates apoptotic process.
| 5

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Overexpression of USP1 promoted cell proliferation, apoptosis, migration and invasion, and decreased cell chemo-sensitivity; however, it could be partially reversed via the overexpression of miR-192-5p in osteosarcoma cell lines.

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Our data show that inhibition of USP1 efficiently blocked human beta-cell apoptosis without compromising function.Complex intracellular networks, namely, the DDR, monitor genome integrity and stabilit[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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USP1 downregulation decreased expressions of fibrosis related genes, suppressed proliferation, and promoted apoptosis in TGF-beta1-induced LX-2 cells, which were reversed by SNAIL overexpression.

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Knockdown of USP1 by siRNA decreased B-ALL cell growth and induced apoptosis.

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Loss of Deubiquitinase USP1 Blocks Pancreatic beta-Cell Apoptosis by Inhibiting DNA Damage Response.
Pimozide affects USP1
| 12
Pimozide inhibits USP1.
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In this study, we showed that pimozide is a potent inhibitor of a human DUB, USP1 and UAF1, and provided insight into the molecular basis for the antineoplastic activity of pimozide.

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Pimozide and GW7647 inhibit USP1 and UAF1 by a noncompetitive mechanism.

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Another study confirmed that pimozide could inhibit USP1 and in turn decrease the clonal growth of glioblastoma cells as well as increase their sensitivity to irradiation, though they are commonly resistant to chemotherapeutic and irradiation treatments.

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Pimozide and GW7647 inactivate USP1 and UAF1 in cells.

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Another study confirmed that pimozide could inhibit USP1 and in turn decrease the clonal growth of glioblastoma cells as well as increase their sensitivity to irradiation, though they are commonly resistant to chemotherapeutic and irradiation treatments (Lee et al., 2016).

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In order to demonstrate that pimozide and GW7647 inhibit the USP1 and UAF1 activity in cells, we developed an assay to determine the levels of Ub-PCNA or Ub-FANCD2 in cells treated with cisplatin, pimozide and GW7647 or a combination of cisplatin and pimozide and GW7647.

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Further studies of the USP1/UAF1 inhibitors suggested that pimozide and GW7647 inhibit USP1/UAF1 by a non‐competitive mechanism and bind at a site other than the active site of USP1/UAF1 [113].
Pimozide activates USP1.
| 3
Pimozide activates USP1. 3 / 3
| 3

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The top inhibitors, pimozide and GW7647, blocked USP1 and UAF1 noncompetitively with a Ki of 0.5 and 0.7 muM, respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase, [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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The top inhibitors, pimozide and GW7647, inhibited USP1 and UAF1 noncompetitively with a K i of 0.5 and 0.7 muM respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase and cysteine proteases.

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The most potent inhibitors, pimozide and GW7647, inhibited USP1 and UAF1 reversibly with a micromolar IC 50.
Pimozide decreases the amount of USP1.
| 2
Pimozide decreases the amount of USP1. 2 / 2
| 2

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Importantly, pimozide lowered USP1 levels, triggered ID1 degradation and inhibited K562 cell growth in vitro and in vivo (Mistry et al., 2013).

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Interestingly, Pimozide promoted ID1 degradation, decreased USP1 levels, and caused a dose dependent inhibition of K562 cell growth in vitro with induction of apoptosis (XREF_FIG), though at a higher drug concentrations as compared to SJB2-043.
USP1 affects Ubiquitin
| 9
USP1 inhibits Ubiquitin.
| 5
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In the past year, two other deubiquitinating enzymes, USP27X and USP1, have been reported to impede ubiquitin mediated degradation of Snail1 in various biological contexts XREF_BIBR, XREF_BIBR.

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We therefore reasoned that pharmacologic inhibition of USP1 would promote ubiquitin mediated degradation of ID1 protein, resulting in differentiation and growth inhibition of immature leukemic cells.

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However, a structural relationship to ubiquitin has been recently proposed for human USP1, which is inactivated by auto‐cleavage after an internal ubiquitin‐like di‐glycine motif.

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Wild-type USP1, but not USP1 C90S, reduced the amount of ID2 modified with HA tagged ubiquitin.
USP1-C90S inhibits Ubiquitin. 1 / 1
| 1

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Wild-type USP1, but not USP1 C90S, reduced the amount of ID2 modified with HA tagged ubiquitin.
USP1 activates Ubiquitin.
| 4
| 4

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The specificity of SJB was confirmed by various experiments : first, we show that SJB potently and selectively block USP1 activity without inhibiting other DUBs (USP2/USP5/USP7/USP14/UCH37); second, SJB inhibited binding of USP1 with HA-Ub-VS probe, but it did not affect labeling of other DUBs with probe; and third, SJB inhibited USP1, but not USP2 or USP7, triggered cleavage of ubiquitin tetramer chains.

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Interestingly, the ubiquitin removal from both PCNA and FANCD2 and FANCI is mediated by the same deubiquitinating enzyme USP1 [86,125] and is required for successful DNA crosslink repair [126].

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Furthermore, when we depleted USP1, which has been shown to increase endogenous ubiquitin conjugated PCNA species, an increased level of ubiquitylated PCNA could be pulled down by Spartan (XREF_FIG C).

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The WD-repeat protein WDR48 (USP1 associated factor UAF-1) stimulates activity of ubiquitin specific proteases USP1, USP12, and USP46.
USP1 affects CHEK1
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USP1 activates CHEK1.
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USP1 activates CHEK1. 5 / 6
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USP1 limits DDB1 dependent degradation of CHK1.

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Finally, to ascertain if elements other than mono-Ub-FANCD2 participate in USP1 mediated CHK1 downregulation, we depleted USP1 in FA-C, FA-G and FA-D2 fibroblasts.

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Two E3 ligase complexes, CUL4A and DDB1 and CUL1 and FBXO6, have shown to be responsible for Chk1 polyubiquitination and degradation; whereas deubiquitinases (DUB), USP1 and USP7, have been reported to promote Chk1 stabilization.

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USP1 enhanced protein stability of the ID1 and CHEK1, important regulators of DNA damage response and stem cell maintenance (Lee et al., 2016).

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It has been previously reported that deubiquitinase USP1 maintains both total and phosphorylated Chk1 level and that USP1 deletion promotes degradation of activated Chk1 in a DDB1 dependent manner.
USP1 inhibits CHEK1.
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USP1 inhibits CHEK1. 1 / 3
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Other USP1 activities include involvement in a feedback loop to limit DDR CHK1 protein kinase activity 72 , and the regulation of cellular differentiation in osteosarcoma cells by deubiquitylating and hence affecting the stability of inhibitors of DNA-binding proteins 73 .
USP1 increases the amount of CHEK1.
| 1
USP1 increases the amount of CHEK1. 1 / 1
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Co-depletion of USP1, together with the FANCcore complex proteins FANCC or FANCD2, rescued the steady-state level of CHK1.
USP1 affects FANCI
| 6
USP1 decreases the amount of FANCI.
| 2
USP1 decreases the amount of FANCI. 1 / 3
| 1

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We found that USP1 inhibition by SJB increased the levels of both Ub-FANCD2 and Ub-FANCI in MM cells.
USP1 bound to WDR48 decreases the amount of FANCI. 1 / 1
| 1

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Disruption of USP1/UAF1 complex promote level of FANCD2/FANCI ubiquitination and DNA repair defects, suggesting a failure in the completion of FA pathway (Nijman et al., 2005b, Smogorzewska et al., 2007).
USP1 inhibits FANCI.
| 2
USP1 inhibits FANCI. 2 / 3
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However, in FA pathway, disruption of USP1 gene causes accumulation of the monoubiquitinated forms of FANCD2 and FANCI but increases ICL hypersensitivity and genomic instability [XREF_BIBR, XREF_BIBR].

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However, in FA pathway, disruption of USP1 gene causes accumulation of the monoubiquitinated forms of FANCD2/FANCI but increases ICL hypersensitivity and genomic instability [49, 77] .
USP1 activates FANCI.
| 2
USP1 activates FANCI. 1 / 2
| 1

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In addition, DUBs involved in DNA damage signaling are USP1 that targets PCNA (proliferating cell nuclear antigen) [76], FANCD2 and FANCI (the Fanconi anemia proteins) [93,94], and USP3 and USP16 that[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 activates ubiquitinated FANCI. 1 / 1
| 1

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Alternatively, USP1 deficiency may cause the accumulation of monoubiquitinated FANCI at genomic sites away from DNA damage, where it can not undergo S559 and S565 phosphorylation by ATR.
USP1 affects WDR48
| 8
USP1 activates WDR48.
| 6
USP1 activates WDR48. 6 / 7
| 6

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The possibility that the NLS inactivating mutations might simultaneously interfere with UAF1 binding can not be formally ruled out, but these results suggest that the inability of USP1 (235-408) to promote UAF1 relocation can not be ascribed to competition with the nuclear import machinery.

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USP1, the major DUB of FANCD2 and FANCI, inactivates these two proteins mediated by the USP1 activating factor UAF1 once DNA damage repair is finished [XREF_BIBR].

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This assay is based on the nuclear accumulation of UAF1 induced by USP1 co-expression and, by using fluorescently tagged versions of these proteins, the results can be observed in live cells, before processing for microscopy analysis.

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Moreover, we verified that (i) UAF1-USP1 is no more effective than UAF1 in the D-loop reaction (XREF_SUPPLEMENTARY), and (ii) USP1 fails to restore functional synergy of the UAF1 EEA mutant with RAD51AP1 (XREF_SUPPLEMENTARY).

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Further studies of the USP1/UAF1 inhibitors suggested that pimozide and GW7647 inhibit USP1/UAF1 by a non‐competitive mechanism and bind at a site other than the active site of USP1/UAF1 [113].

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Moreover, we verified that UAF1-USP1 is no more effective than UAF1 in the D-loop reaction and that USP1 fails to restore functional synergy of the UAF1 EEA mutant with RAD51AP1.
USP1 inhibits WDR48.
| 2
USP1-E444K inhibits WDR48. 1 / 1
| 1

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Likewise, mutation of USP1 E444K, analogous to the impactful USP46 E186K mutation, abrogated WDR48 binding and stimulation of USP1 deubiquitinase activity.
USP1 inhibits WDR48. 1 / 1
| 1

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An analogous mutation in USP1 similarly blocked WDR48 dependent activation.
GW 7647 affects USP1
| 8
GW 7647 inhibits USP1.
| 5
GW 7647 inhibits USP1. 5 / 5
| 5

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Although two inhibitors namely pimozide and GW7647 were reported to selectively inhibit USP1 and UAF1, they also inhibited other DUBs, deSUMOlyase and cysteine proteases [XREF_BIBR].

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Pimozide and GW7647 inactivate USP1 and UAF1 in cells.

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Further studies of the USP1/UAF1 inhibitors suggested that pimozide and GW7647 inhibit USP1/UAF1 by a non‐competitive mechanism and bind at a site other than the active site of USP1/UAF1 [113].

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Pimozide and GW7647 inhibit USP1 and UAF1 by a noncompetitive mechanism.

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In order to demonstrate that pimozide and GW7647 inhibit the USP1 and UAF1 activity in cells, we developed an assay to determine the levels of Ub-PCNA or Ub-FANCD2 in cells treated with cisplatin, pimozide and GW7647 or a combination of cisplatin and pimozide and GW7647.
GW 7647 activates USP1.
| 3
GW 7647 activates USP1. 3 / 3
| 3

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The top inhibitors, pimozide and GW7647, inhibited USP1 and UAF1 noncompetitively with a K i of 0.5 and 0.7 muM respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase and cysteine proteases.

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The most potent inhibitors, pimozide and GW7647, inhibited USP1 and UAF1 reversibly with a micromolar IC 50.

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The top inhibitors, pimozide and GW7647, blocked USP1 and UAF1 noncompetitively with a Ki of 0.5 and 0.7 muM, respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase, [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 affects KDM4A
| 1 7
USP1 increases the amount of KDM4A.
| 5
USP1 increases the amount of KDM4A. 4 / 4
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USP1 knockdown in RV1, LNCAP, and PC3 cells significantly decreased KDM4A protein level, but did not affect KDM4A mRNA level.

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As shown in Figure XREF_FIG, USP1 knockdown dramatically reduced levels of KDM4A and c-Myc, whereas the reconstitution of KDM4A was able to efficiently rescue their expression.

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Similarly, USP1 knockdown in RV1 cells using 2 specific shRNAs significantly decreased KDM4A protein levels and increased cellular sensitivity to enzalutamide treatment, whereas overexpression of KDM4A in depleted cells with USP1 reversed hypersensitivity to enzalutamide treatment.

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We previously found that USP1 knockdown decreased levels of KDM4A in PC cells.
Modified USP1 increases the amount of KDM4A. 1 / 1
| 1

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Furthermore, overexpression of WT USP1 but not the catalytic inactive mutant (CS mutant) in RV1 cells could dramatically increase KDM4A protein level in the cells with depletion of USP1.
USP1 activates KDM4A.
| 1 2
USP1 activates KDM4A. 3 / 3
| 1 2

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We observed that the USP1 inhibitor promoted KDM4A degradation ( Figure7D ) .

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Taken together, these results suggest that USP1 directly regulates KDM4A stability.

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Furthermore, we found that USP1 overexpression decreased the polyubiquitination levels in WT, K6R, K11R, K27R, K29R, K33R, and K63R HA-ubiquitin mutants but not in K48R mutant, suggesting USP1 promotes KDM4A K48 linked deubiquitin; however, which E3 ubiquitin ligase mediated KDM4A ubiquitin and degradation is still unclear.
USP1 affects CDKN1A
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USP1 inhibits CDKN1A.
| 3
USP1 inhibits CDKN1A. 2 / 2
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Taken together, our results demonstrate that USP1 suppresses p21 via ID protein stabilization and inhibition of bHLH transcriptional activity in osteosarcoma.Osteosarcomas are heterogeneous tumors com[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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shRNA resistant wild-type USP1, but not USP1 C90S, reduced p21 to levels observed in control cells, confirming knockdown specificity in this setting.
USP1-C90S inhibits CDKN1A. 1 / 1
| 1

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shRNA resistant wild-type USP1, but not USP1 C90S, reduced p21 to levels observed in control cells, confirming knockdown specificity in this setting.
USP1 increases the amount of CDKN1A.
| 2
USP1 increases the amount of CDKN1A. 2 / 2
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In wild-type MEFs, Usp1 depletion increased the levels of Fancd2-Ub, TAp63, and p21 (XREF_FIG, lanes 1, 2) and enhanced OIS (XREF_FIG).

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In contrast, Usp1 depletion failed to induce TAp63 or p21 expression levels and only slightly induced senescence in Fancd2 (-/-) MEFs.
USP1 activates CDKN1A.
| 2
USP1 activates CDKN1A. 2 / 2
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Increased USP1 levels were also observed upon siRNA mediated p21 depletion (see XREF_FIG, lanes 5 and 6).

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Significantly, CDKN1A siRNAs restored S phase entry in USP1 deficient U2-OS cells, indicating that p21 is essential for the cell-cycle arrest induced by USP1 knockdown.If ID degradation in the absence[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP1 decreases the amount of CDKN1A.
| 1
Modified USP1 decreases the amount of CDKN1A. 1 / 1
| 1

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USP1 was overexpressed frequently in primary osteosarcomas and osteosarcoma cell lines, and by deubiquitinating the ID proteins, inhibited bHLH dependent expression of CDKI p21 resulting in unchecked [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
| 6

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USP1 Promotes GC Metastasis via Stabilizing ID2.

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USP1 dependent RPS16 protein stability drives growth and metastasis of human hepatocellular carcinoma cells.

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USP1 inhibition destabilizes KPNA2 and suppresses breast cancer metastasis.

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In conclusion, our study showed that USP1 promoted GC metastasis via stabilizing ID2 expression, which provides a potential biomarker and therapy target for GC.

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Mechanically, we demonstrated that USP1 promoted GC metastasis via upregulating ID2 expression and further confirmed that USP1 stabilized ID2 expression through deubiquitinating ID2 in GC.

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Then, real-time cellular analysis (RTCA) showed that USP1 knockdown inhibited GC metastasis both in vitro and in vivo.
| 1

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Most importantly, pharmacological intervention of USP1 function by pimozide or ML323 significantly represses breast cancer metastasis in mice, suggesting a rationale for using USP1 inhibitors for treatment of patients with breast cancer.
USP1 affects DNA repair
| 7
USP1 activates DNA repair.
| 4
| 4

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Usp1 modulates DNA replication, polymerase choice and DNA repair by PCNA and as a result Usp1 knock-out mice are genetically unstable and hypersensitive to DNA damage [XREF_BIBR], [XREF_BIBR].

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Because CHK1 is a cell cycle regulated and DNA damage checkpoint protein, we asked whether (a) the downregulation of CHK1 in USP1 depleted cells is an experimental artefact resulting from modifications of the cell cycle and/or altered DNA repair capabilities caused by USP1 depletion and (b) USP1 depletion modifies the G2 checkpoint in response to DNA damage.

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The deubiquitinating enzyme, USP1 is known to promote DNA repair via complexing with UAF1.

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Blockade of Deubiquitylating Enzyme USP1 Inhibits DNA Repair and Triggers Apoptosis in Multiple Myeloma Cells.
USP1 inhibits DNA repair.
| 3
| 2

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Initial evidence suggested that USP1 inhibition leads to constitutive DNA repair and protects cells from MMC induced chromosome aberrations [45].

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Mutation of aspartic acid 199 in USP1 disrupts its deubiquitinating activity and impairs DNA repair.
USP1 bound to WDR48 inhibits DNA repair. 1 / 1
| 1

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Disruption of USP1/UAF1 complex promote level of FANCD2/FANCI ubiquitination and DNA repair defects, suggesting a failure in the completion of FA pathway (Nijman et al., 2005b, Smogorzewska et al., 2007).
PCNA affects USP1
| 7
PCNA activates USP1.
| 3
PCNA activates USP1. 3 / 3
| 3

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Usp1 modulates DNA replication, polymerase choice and DNA repair by PCNA and as a result Usp1 knock-out mice are genetically unstable and hypersensitive to DNA damage [XREF_BIBR], [XREF_BIBR].

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However, pathway (s) responsible for the regulation of USP1 as well as the molecular mechanism by which USP1 regulates DDR in beta-cells, especially in the diabetic environment, remains unknown and wa[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Similar results have been seen in the case of Usp1 and Uaf -1 where knockdown of either protein alone altered the ubiquitination status of Usp1 targets PCNA and FANCD2, in this example an epistatic relationship between Usp1 and Uaf-1 was observed [XREF_BIBR].
PCNA inhibits USP1.
| 2
PCNA inhibits USP1. 2 / 2
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USP1 dependent PCNA deubiquitylation is regulated, on one hand, by UV irradiation dependent inactivation of USP1 through an autocleavage event and, on the other hand, by the PCNA interacting ELG1 protein that is essential for recruiting USP1-UAF1 to the stalled fork.

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(2) PCNA deubiquitination to limit the DNA damage bypass pathway by recruiting the USP1 and USP1 associated factor 1 (UAF1) complex [XREF_BIBR].
PCNA deubiquitinates USP1.
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PCNA deubiquitinates USP1. 2 / 2
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To provide additional confirmation, we aimed to test the effect of decreasing the level of ubiquitin conjugated PCNA, which can be carried out by expressing the PCNA deubiquitylating enzyme USP1 as described.

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XREF_BIBR The levels of PCNA modification are additionally controlled by the clamp loader Elg1, which binds to and unloads sumoylated PCNA from chromatin in yeast XREF_BIBR and targets the PCNA deubiquitinating enzyme USP1 to replicative DNA lesions in humans.
USP1 affects USP1
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USP1 decreases the amount of USP1.
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USP1 decreases the amount of USP1. 2 / 3
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A doxycycline induced USP1 shRNA suppressed USP1 expression and reduced ID1 and ID2 in the xenografts.

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The important regulatory role of USP1 in DNA repair, supported by the finding that USP1 gene knockout in model systems leads to DNA damage hypersensitivity [XREF_BIBR - XREF_BIBR], together with the observation that USP1 is frequently overexpressed in tumors, suggests that USP1 could be a relevant target for cancer therapy, whose inhibition might contribute to overcome chemoresistance.
USP1 activates USP1.
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USP1 activates USP1. 1 / 2
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In addition, USP1 overexpression activated phosphoinositide 3-kinase (PI3K) signaling, whereas a selective PI3K inhibitor (S2739) could reverse the effects of USP1 on NPC cells in vitro.
USP1 bound to WDR48 activates USP1. 1 / 1
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Several studies showed that UAF1 forms a complex with USP1 and stimulates the deubiquitination activity of USP1 both in vitro and in vivo.
USP1 increases the amount of USP1.
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USP1 increases the amount of USP1. 1 / 1
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The important regulatory role of USP1 in DNA repair, supported by the finding that USP1 gene knockout in model systems leads to DNA damage hypersensitivity [XREF_BIBR - XREF_BIBR], together with the observation that USP1 is frequently overexpressed in tumors, suggests that USP1 could be a relevant target for cancer therapy, whose inhibition might contribute to overcome chemoresistance.
SJB2-043 affects USP1
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USP1 inhibitor SJB2-043 inhibits the enzyme activity of native USP1.

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We next determined whether SJB2-043 inhibits the DUB activity of native USP1 complexes isolated from human cells (XREF_FIG).

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SJB2-043 blocked the deubiquitinating enzyme activity of USP1 in vitro with an IC50 in the nanomolar range (Mistry et al., 2013).

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No evidence text available

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These results demonstrate that USP1 inhibitor SJB2-043 inactivates native USP1 in primary AML cells and exhibits cytotoxicity.

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In addition, SJB2-043 inhibited the labeling of USP1 with Ub-VS in a dose dependent manner (XREF_SUPPLEMENTARY).
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We confirmed the inhibition of USP1 by PEITC by monitoring the hydrolysis of di-Ub by purified USP1 in complex with its activator UAF1.

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PEITC treatment decreased the ratio of labeled to unlabeled USP1, demonstrating that PEITC inhibits USP1 in living cells.

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We confirmed the inhibition of USP1 by PEITC by monitoring the hydrolysis of di-Ub by purified USP1 in complex with its activator UAF1 (Figure xref ).

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PEITC treatment decreased the ratio of labeled to unlabeled USP1, demonstrating that PEITC inhibits USP1 in living cells (Figure xref and xref ).
Phenethyl isothiocyanate decreases the amount of USP1.
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Phenethyl isothiocyanate decreases the amount of USP1. 2 / 2
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PEITC decreases USP1 levels.

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Surprisingly, PEITC also decreased total USP1 protein levels in a dose dependent manner.
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Gene silencing of USP1 by lentivirus effectively inhibits proliferation and invasion of human osteosarcoma cells.

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It remains to be investigated if our small molecule inhibitors of USP1 can reduce the invasiveness of leukemic cells.

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USP1 enhances the expression of a number of pro metastatic genes in breast cancer cells, promotes cell migration and invasion in vitro, and facilitates lung metastasis of breast cancer cells.

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Overexpression of USP1 promoted cell proliferation, apoptosis, migration and invasion, and decreased cell chemo-sensitivity; however, it could be partially reversed via the overexpression of miR-192-5p in osteosarcoma cell lines.
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Furthermore, pimozide, a well-known antipsychotic drug, upregulates NgR1 by post-translational targeting of USP1, which sensitizes glioma stem cells to myelin inhibition and suppresses myelin associated infiltration in vivo.

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Collectively, our results suggest that silencing USP1 inhibits cell proliferation and invasion in U2OS cells.
USP1 affects ID2
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USP1 activates ID2.
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USP1 activates ID2. 3 / 3
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USP1 knockdown specificity was confirmed with shRNA resistant USP1, which restored ID1, ID2, and ID3 to basal levels.

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A doxycycline induced USP1 shRNA suppressed USP1 expression and reduced ID1 and ID2 in the xenografts.

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Mechanistically, we found that PDGF signaling regulated the expression of the E2F transcription factors, which directly bound to and activated Usp1 Furthermore, PDGF mediated expression of USP1 led to the stabilization of Inhibitor of DNA binding 2 (ID2), which we found to be required for glioma cell survival.
USP1 decreases the amount of ID2.
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USP1 decreases the amount of ID2. 1 / 1
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Usp1 deficiency resulted in a gradual loss of memory CD8 + T cells over time and reduced Id2 protein levels and proliferation of effector CD8 + T cell following reinfection.
USP1 decreases the amount of ubiquitinated ID2. 1 / 1
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In keeping with this scenario, USP1 knockdown in MG-132-treated U2-OS cells increased the amount of ubiquitinated ID2.
USP1 inhibits ID2.
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USP1 inhibits ID2. 1 / 1
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A doxycycline induced USP1 shRNA suppressed USP1 expression and reduced ID1 and ID2 in the xenografts.
Methoprene affects USP1
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Methoprene increases the amount of USP1.
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Methoprene increases the amount of USP1. 3 / 3
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Simultaneously, RNAi of HaCal suppressed the transcript levels of USP1, PKC, and JHi but not of Met1 and Br-Z2 induced by methoprene in the JH signaling (lane O compared to I).

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Knockdown of Met1 suppressed the transcript levels of USP1, PKC, HaCal, and JHi induced by methoprene (lane L compared to I).

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However, the transcript levels of USP1, PKC, and Br-Z2 induced by methoprene were not suppressed (lane J compared to I).
Methoprene activates USP1.
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USP1, PKC, Br-Z2, and HaCal were induced by both 20E and methoprene.

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In the methoprene treated cells, the non phosphorylated calponin bound with USP1 was necessary for methoprene induced USP1 up-regulation along with that of JHi.
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However, in addition to this opposition to activate and monoubiquitinate FANCD2/I, USP1 deubiquitination of FANCI has also been associated with promoting core complex recruitment to the site of DNA da[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Controlling the ubiquitylation of these two key players appears to be essential for proper replication fork maintenance, as loss of USP1 causes high levels of genome instability and mutagenesis.

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However, in FA pathway, disruption of USP1 gene causes accumulation of the monoubiquitinated forms of FANCD2 and FANCI but increases ICL hypersensitivity and genomic instability [XREF_BIBR, XREF_BIBR].
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Interestingly, siRNA mediated knock-down of FAN1 and USP1, the enzyme responsible for deubiquitylating PCNA, caused similar genomic instability in U2OS cells, but the number of DSBs in the double knock-down was increased approximately two-fold, thus indicating that an increase in ub-PCNA may worsen the genomic instability in FAN1 depleted cells.

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It is tempting to speculate that USP1 deficiency preferably produces replication stress and genomic instability at fragile sites.
USP1 affects BRCA1
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USP1 activates BRCA1.
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USP1 activates BRCA1. 4 / 4
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In contrast to the overexpression, the depletion of USP1 increased the number of BRCA1 foci but decreased the number of RIF1 foci in S/G2 cells (XREF_FIG, XREF_SUPPLEMENTARY).

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As expected, the number of BRCA1 foci reduced dramatically in Cdh1 depleted cells treated with HU, and the simultaneous depletion of USP1 with that of Cdh1 essentially restored BRCA1 focus number (XREF_FIG).

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As shown in XREF_FIG, the depletion of USP1 in control cells (no ectopic USP1 expression) expectedly caused an increase of BRCA1 IRIF but a decrease of RIF1 IRIF (as seen in XREF_FIG).

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Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1 deficient cells, revealing a synthetic lethal relationship.
USP1 inhibits BRCA1.
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USP1 inhibits BRCA1. 1 / 1
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Given that USP1 suppresses BRCA1, we expect that compromising APC function would also lead to an apparent suppression on BRCA1 focus formation.
USP1 affects TBK1
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USP1 increases the amount of TBK1.
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