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.
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
Transcriptomics
The following table shows the significantly differentially expressed genes after knocking
out USP27X using CRISPR-Cas9.
There were too few differentially expressed genes to run a meaningful GSEA.
Literature Mining
INDRA was used to automatically assemble known mechanisms
related to USP27X from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
The requirement of ATXN7L3 for H2B deubiquitination by USP22, USP27x, and USP51 suggests that the ATXN7L3 zinc finger plays a role analogous to that of the Sgf11 zinc finger in docking human SAGA DUB [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The requirement of ATXN7L3 for H2B deubiquitination by USP22, USP27x, and USP51 suggests that all three use the ATXN7L3 zinc finger to dock the H2A and H2B acidic patch in a manner similar to that shown in the structure of the yeast DUB module bound to ubiquitinated nucleosomes.
Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK-dependent phosphorylation and can upregulate its expression levels.
Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti-apoptotic effects of ERK activity, and therefore acts as a tumour suppressor.
The observation that USP27 interacts with and deubiquitinates Cyclin E led us to think that USP27 might involve in cell cycle progression by regulating Cyclin E stability.
Knockdown of Usp22 shortened the half-life of Hes1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis-expression of Usp27x reduced neuronal differentiation.
In contrast, depletion of non-enzymatic components, ATXN7L3 or ENY2, results in increased H2Bub1. These observations led us to discover two H2Bub1 DUBs, USP27X and USP51, which function independently of SAGA and compete with USP22 for ATXN7L3 and ENY2 for activity.
Overexpression of Usp27x reduces ERK dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA stimulated cells, as well as in tumour cells with a constitutively active Raf and ERK pathway.
We found that Hes1 was deubiquitinated and stabilized by Usp27x and its homologs ubiquitin specific protease 22 (Usp22) and ubiquitin specific protease 51 (Usp51).
Expression of WT-USP27X on its own led to slightly increased proliferation, whereas expression of the C285S mutant led to slightly reduced proliferation.
The observation that USP27 promotes cell cycle progression and cell proliferation prompted us to imagine that depletion of USP27 expression might suppress tumor progression.
Herein, we demonstrate that USP27 regulates Cyclin E abundance to accelerate cell cycle progression, cell proliferation, and tumor cell growth as well.
Furthermore, cell proliferation was also remarkably reduced in the combination of USP27 knockdown and 5-FU treatment, whereas overexpression of USP27 or addition of Cyclin E in USP27 knockdown cells could raise cell proliferation.
XREF_FIG, catalytically inactive USP27/CA mutant failed to protect Cyclin E from degradation, implying that the DUB activity is required for USP27 mediated Cyclin E stabilization.
USP27 mediated Cyclin E stabilization is involved in tumorigenesis, suggesting that targeting USP27 may represent a new therapeutic strategy to treat cancers with aberrant overexpression of Cyclin E protein.
Our discovery that USP27 mediated Cyclin E stabilization implied that USP27 might modulate cell cycle and cell proliferation through Cyclin E regulation.
We found that USP27 knockdown suppresses cell cycle progression by increasing the percentage of cells in G0/G1 phase, which can be reversed by the addition of Cyclin E expression.
Herein, we demonstrate that USP27 regulates Cyclin E abundance to accelerate cell cycle progression, cell proliferation, and tumor cell growth as well.
The observation that USP27 promotes cell cycle progression and cell proliferation prompted us to imagine that depletion of USP27 expression might suppress tumor progression.
To further support our notion that USP27 promotes cell cycle progression, we detected a statistically significant reduction in the growth of Hep3B and MHCC97H cells compared with that of control cells with single stable knockdown of USP27.
XREF_FIG, USP27 knockdown suppresses cell cycle progression by increasing the percentage of cells in G1/S phase but decreasing the percentage of cells in G2/M phase, similar to 5-FU treatment.
Although Cyclin E has been reported to involve in metastasis [XREF_BIBR], how USP27 promotes migration and metastasis also need further detailed exploration.
Overexpression of Usp27x induces low levels of apoptosis in melanoma and non small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling.
Overexpression of Usp27x reduces ERK dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA stimulated cells, as well as in tumour cells with a constitutively active Raf and ERK pathway.
USP27X was upregulated by TGFbeta during EMT and was required for TGFbeta induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF.
To determine whether USP22 blocks association of USP27X and USP51 with SAGA, we isolated GCN5 associated proteins after shRNA mediated depletion of USP22 (XREF_FIG, lanes 2 and 3 and 5 and 6).
To determine whether USP22 blocks association of USP27X and USP51 with SAGA, we isolated GCN5 associated proteins after shRNA mediated depletion of USP22.
Equal numbers of cells expressing shRNAs that specifically target USP27X or USP51, but not USP22, or expressing control shRNA, were seeded and monitored for proliferation by cell counts 72 hr later.
Equal numbers of cells expressing shRNAs that specifically target USP27X or USP51, but not USP22 (XREF_FIG), or expressing control shRNA, were seeded and monitored for proliferation by cell counts 72 hours later.
Cathepsin K inhibition induced mitochondrial ROS enhances sensitivity of cancer cells to anti-cancer drugs through USP27x mediated Bim protein stabilization.
Down-regulation of Raptor expression increased mitochondrial ROS production, and mitochondria specific superoxide scavengers prevented USP27x mediated stabilization of Bim by inhibition of Cat K. Moreover, combined treatment with Cat K inhibitor (odanacatib) and tumor necrosis factor related apoptosis inducing ligand (TRAIL) reduced tumor growth and induced cell death in a xenograft model.
As shown in Figure S5A-D, depletion of USP27 expression significantly decreased cell migration compared with the control in Hep3B and MHCC97H cells using wound healing assay.
Herein, we demonstrate that USP27 regulates Cyclin E abundance to accelerate cell cycle progression, cell proliferation, and tumor cell growth as well.
Knockdown of Usp22 shortened the half-life of Hes1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis expression of Usp27x reduced neuronal differentiation.
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.
Interestingly, the DUBs USP26 and USP27 were found to modulate RNF168 mediated protein ubiquitylation at DSB sites, preventing excessive spreading of RAP80-BRCA1, promoting association of BRCA1 with P[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Overexpression of the Ubiquitin Specific Proteases USP43, USP41, USP27x and USP6 in Osteosarcoma Cell Lines: Inhibition of Osteosarcoma Tumor Growth and Lung Metastasis Development by the USP Antagonist PR619.
Among the DUBs that interact with cGAS or MDA5, USP27X (98) and USP29 (99) stabilize cGAS and thus positively regulate IFN production and antiviral activities.
Our results now reveal that in addition to USP22, ATXN7L3 and ENY2 activate two previously uncharacterized deubiquitinating enzymes, USP27X and USP51, which are not part of SAGA.
Interestingly, USP27 expression might also be regulated by Fbxw7, a well-known E3 ubiquitin ligase of Cyclin E, which interacts with and degrades USP27.
Our results now reveal that in addition to USP22, ATXN7L3 and ENY2 activate two previously uncharacterized deubiquitinating enzymes, USP27X and USP51, which are not part of SAGA.