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 USP6 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 USP6 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
We previously showed that ubiquitin specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in ES cells.
Here we demonstrate that TRE17 activates canonical p65-containing NF-κB through an atypical mechanism that is independent of IκB phosphorylation/degradation.
In sum, these data indicate that TRE17(long) selectively activates classical NF-κB, consisting of either homo- or heterodimeric p65 complexes, in three different cell types.
Our finding that TRE17 activates classical NF-κB in the absence of IκB degradation, and in a manner requiring both IKKα and IKKβ, adds to the growing list of stimuli that activate NF-κB through mechanisms distinct from simplified depiction of the classical pathway ( xref ; xref ).
Optimal activation of NF-κB by TRE17 requires both catalytic subunits of IKK, distinguishing its mechanism from the classical and non-canonical pathways, which require either IKKβ or IKKα, respectively.
Jak1 in this clone was reduced to levels approximating those in control NIH3T3 cells (XREF_SUPPLEMENTARY), demonstrating that upregulation of Jak1 by USP6 is required for full activation of STAT3 by USP6.
Jak1 in this clone was reduced to levels approximating those in control NIH3T3 cells ( xref ), demonstrating that upregulation of Jak1 by USP6 is required for full activation of STAT3 by USP6.
Given that NF-kappaB and STAT3 are often activated coordinately during tumorigenesis, we examined whether STAT3 was activated by USP6 in our established cellular models of ABC and NF.
Given that NF-κB and STAT3 are often activated coordinately during tumorigenesis, we examined whether STAT3 was activated by USP6 in our established cellular models of ABC and NF.
Interestingly, the Wnt antagonist Dickkopf-1 (DKK1) or LRP5/6 siRNA blocked USP6 induced Wnt signalling, suggesting that a Wnt-ligand receptor complex is required for USP6 function.
A genomewide siRNA screen revealed that deubiquitinase USP6 overexpression could activate Wnt signaling by inhibition of Fzd7 endocytosis and its subsequent targeting for degradation [XREF_BIBR].
In a separate whole-genome siRNA screen, USP6 was found to activate Wnt signalling by deubiquitylating Frizzled receptors and increasing their plasma membrane expression 28.
USP6 was a target of miR-130a and the overexpression of miR-130a or inhibition of USP6 in UM MUM-2B and MUM-2C cell lines inhibited the expression of Wnt, β-catenin, and EGFR, and activated SMAD4 expression, while reducing UM cell migration and invasion abilities in vitro.
Lu et al. [6] reported a novel CTNNB1-USP6 fusion in IVF, showing that IVF is a USP6-induced neoplasm and should be included in USP6-rearranged lesions.The most common clinical presentation is a painless, slowly growing mass.
Intra-articular nodular fasciitis and aneurysmal bone cyst seem to belong to the same biological spectrum defined as USP6-induced tumors according to the report [8].
Although the molecular diagnosis was not performed, the findings of present case suggest that these two tumors reside in the same biologic spectrum defined as USP6-induced tumors.
Recently, Oliveira and Chou suggested that aneurismal bone cysts and nodular fasciitis reside in the same biologic spectrum as USP6-induced tumors [9].
Jak1 in this clone was reduced to levels approximating those in control NIH3T3 cells (XREF_SUPPLEMENTARY), demonstrating that upregulation of Jak1 by USP6 is required for full activation of STAT3 by USP6.
Together, this group of experiments indicates that upregulation of Jak1 by USP6 is required not only for the production of autocrine and paracrine factors, but also for heightened STAT3 activation in response to them.
Our data support a model in which Jak1 levels are increased through de-ubiquitination by USP6, rendering cells hyper-sensitive to low levels of Jak1 agonists present in the microenvironment.
XREF_BIBR, XREF_BIBR USP6 was shown to induce transcription of MMP9 through the activation of nuclear factor-kappaB (NF-kappaB) in a USP dependent manner.
In this study we show that tre17 is sufficient to induce expression of mmp-9 and mmp-10, in a manner requiring its usp activity, but not its ability to bind arf6. Tre17 induces transcription of mmp-9 through activation of nuclear factor-kappab (nf-kappab), mediated in part by the gtpase rhoa and its effector kinase, rock.
Loss of the NFκB-binding site abrogated activation of the MMP-9 promoter by TRE17(long) in hFOB1.19 and HeLa ( xref ), indicating that NFκB is the key mediator of TRE17-induced MMP-9 gene transcription.
Loss of either Hrp1 or Hrp3 leads to increased cryptic transcription, similar to that observed upon loss of Isw1 and Chd1 in budding yeast [XREF_BIBR - XREF_BIBR].
Hrp1 forms the B component of the CFI polyadenylation factor and, when overexpressed, increases recognition of a weakened polyadenylation site and suppresses the defective transcription termination.
Not only does USP6 independently induce activation of the IFN signaling mediators JAK1 and STAT1, but it also renders ES cells exquisitely responsive to exogenous IFNs, potentiating activation of STAT1 and STAT3.
Single gene complementation of two Cluster V mutants, Tn : : Rv0712 and Tn : : hrp1 (hypoxic response protein 1, Rv2626c), restored the type I IFN response (XREF_FIG), suggesting that each of these genes is required for induction of type I IFNs.
Thus, Hrp1p and Nab4p specifically interacts with the DSE from PGK1, and this interaction requires the presence of the sequence motifs.We asked whether the inability of hrp1 strains to promote NMD was[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
We show that, in addition to centromeric regions, a low level of CENP-A (Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1 (Chd1) chromatin remodeling factor.
It is perhaps less surprising that the strong increase in H3 occupancy does not restrict chromatin accessibility since previous studies in fission yeast revealed that hrp1 and hrp3 deletions increase promoter H3 occupancy but do not affect MNase sensitivity of promoter regions [XREF_BIBR, XREF_BIBR].
To determine whether TRE1 and TRE2 can mediate the transcriptional regulation of the Dot1L gene by T3 in vivo, we used a reconstituted Xenopus laevis oocyte transcription system, which allows the analysis of the promoter in the context of chromatin in vivo [XREF_BIBR].
Here we show that tre17 (also called tre-2 and usp6), a founding member of the tbc family, targets the arf family gtpase arf6, which regulates plasma membrane-endosome trafficking. Surprisingly, tre17 does not function as a gap for arf6 but rather promotes its activation in vivo. Forced expression of tre17 promotes the localization of arf6 to the plasma membrane, leading to arf6 activation, presumably due to facilitated access to membrane-associated guanine nucleotide exchange factors (gefs).
USP6 was a target of miR-130a and the overexpression of miR-130a or inhibition of USP6 in UM MUM-2B and MUM-2C cell lines inhibited the expression of Wnt, β-catenin, and EGFR, and activated SMAD4 expression, while reducing UM cell migration and invasion abilities in vitro.
USP6 was a target of miR-130a and the overexpression of miR-130a or inhibition of USP6 in UM MUM-2B and MUM-2C cell lines inhibited the expression of Wnt, β-catenin, and EGFR, and activated SMAD4 expression, while reducing UM cell migration and invasion abilities in vitro.
In this study we show that tre17 is sufficient to induce expression of mmp-9 and mmp-10, in a manner requiring its usp activity, but not its ability to bind arf6. Tre17 induces transcription of mmp-9 through activation of nuclear factor-kappab (nf-kappab), mediated in part by the gtpase rhoa and its effector kinase, rock.
Contrary to RNF43, ubiquitin specific protease 6 (USP6) is a deubiquitylase; ectopic expression of USP6 increases cell surface abundance of Fzds and Lrp6 and activates beta-catenin pathway in tumor cells XREF_BIBR.
These experiments reveal that these interactions do not depend on the presence of RNA, as interactions between Caf1 and Hrp1 or Caf1 and Nab2 were not reduced following RNase treatment (XREF_FIG and data not shown).
These experiments reveal that these interactions do not depend on the presence of RNA, as interactions between Caf1 and Hrp1 or Caf1 and Nab2 were not reduced following RNase treatment (XREF_FIG and data not shown).
USP6 positively modulates GluN1 expression in transfected cells, and USP6 down-regulation impedes focal GluN1 distribution at postsynaptic densities and impairs synaptic function in neurons derived from human embryonic stem cells.
USP6 positively modulates GluN1 expression in transfected cells, and USP6 down-regulation impedes focal GluN1 distribution at postsynaptic densities and impairs synaptic function in neurons derived from human embryonic stem cells.
USP6 was a target of miR-130a and the overexpression of miR-130a or inhibition of USP6 in UM MUM-2B and MUM-2C cell lines inhibited the expression of Wnt, β-catenin, and EGFR, and activated SMAD4 expression, while reducing UM cell migration and invasion abilities in vitro.
Contrary to RNF43, ubiquitin specific protease 6 (USP6) is a deubiquitylase; ectopic expression of USP6 increases cell surface abundance of Fzds and Lrp6 and activates beta-catenin pathway in tumor cells XREF_BIBR.
MTT proliferation assays revealed that each TGFB isoform decreased HRP-1 cell growth in a dose dependent manner, whereas RCHO-1 cells were resistant to the growth-suppressive effect of TGFB1 and TGFB3.
Activation of ERK, MAPK14 (p38 MAPK), or SMAD pathways is known to play a role in cell proliferation, and we found that TGFB activates these pathways in both HRP-1 and RCHO-1 cells in an isoform specific manner.
Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively.
In quiescent cells, tre17 is localized to intracellular filamentous and punctate structures in the cytoplasm, folded in an inactive conformation. Upon growth factor addition, cdc42 and rac1 become activated and recruit tre17 to the plasma membrane. Stable membrane localization of tre17 also requires polymerized actin. This recruitment process leads to a conformational change in tre17, such that the n-terminal portion of the molecule further stimulates the accumulation of cortical actin.
Strikingly, we found that Jak1 levels were significantly increased in NIH3T3 and MC3T3 cell lines expressing USP6, in a dox dependent manner (XREF_FIG).
Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively.
In quiescent cells, tre17 is localized to intracellular filamentous and punctate structures in the cytoplasm, folded in an inactive conformation. Upon growth factor addition, cdc42 and rac1 become activated and recruit tre17 to the plasma membrane. Stable membrane localization of tre17 also requires polymerized actin. This recruitment process leads to a conformational change in tre17, such that the n-terminal portion of the molecule further stimulates the accumulation of cortical actin.
We then examined if histamine may enhance NGF promoter activity in keratinocytes.It is reported that human NGF gene contains two TREs, TRE1 (-62/-56 bp) and TRE2 (+35/+41 bp), and these may act as enh[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Conversely, glucose depletion enhanced only the formation of clear foci of Pab1-GFP, Pbp1-GFP, and Hrp1-GFP, but not Rpg1-GFP, Prt1-GFP, and Nip1-GFP in the cytoplasm.
Inhibition of Wnt signaling using Dickkopf-1 (DKK1) or a Porcupine (PORCN) inhibitor significantly decreased the growth of USP6 driven xenograft tumors, indicating that Wnt signaling is a key target of USP6 during tumorigenesis.
Two members of the tre-2 and USP6, BUB2, cdc16 (TBC1) domain family of proteins, Akt substrate of 160 kDa (also known as AS160 or TBC1D4) and TBC1D1, have been proposed as potential sites for the convergence of insulin and exercise signaling to stimulate glucose transport in skeletal muscle.
In ABC, both neoplastic cells and other cells in the microenvironment exhibited P-STAT3 activation (XREF_FIG, left), suggesting that USP6 might induce paracrine signaling (as was confirmed below).
For example, HRP1 and CLP1, both induced in response to MMS, are subunits of cleavage factor I, required for the cleavage and polyadenylation of 3 ' mRNA ends [XREF_BIBR].
Two members of the tre-2 and USP6, BUB2, cdc16 (TBC1) domain family of proteins, Akt substrate of 160 kDa (also known as AS160 or TBC1D4) and TBC1D1, have been proposed as potential sites for the convergence of insulin and exercise signaling to stimulate glucose transport in skeletal muscle.
In contrast, cohesin mutants rad21 and psc3 were rescued by loss of the RNA elimination pathway (Erh1, Mmi1, and Red1), and loader mutant mis4 was rescued by loss of Hrp1 mediated chromatin remodeling.
USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly (ADP-ribose) polymerase-1 (PARP-1) protein.
Studies have used the TRE3G (Tet-On-3G) and TRE2 Tet-On promoters to activate Cas9 proteins in an animal model and human immortalised cell lines respectively under the control of administered doxycycline [ xref , xref ].
It also illustrates the added value of genomic analyses in the setting of an ABC lesion : complex clonal aberrations argues for a lesion secondary to a malignant proliferation whereas USP6 rearrangement allows the diagnosis of primary ABC.
To determine whether RhoA was activated by TRE17, we performed pulldown assays using the Rho-binding domain (RBD) of Rhotekin (which binds to RhoA in a GTP-dependent manner) as an affinity reagent.
USP6 was competent to induce Erk activation and prevent PARP cleavage upon serum starvation, and this effect was abrogated by depletion of Jak1 or STAT3 (XREF_FIG).
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.
XREF_BIBR At the hrp1 + locus loss of HIRA function resulted in marked changes to the MNase profile which extended throughout the entire gene and into the neighboring genes (atg12 + and pap1 +) (XREF_FIG; Fig.
USP6 inhibited ES xenograft growth in nude but not NSG mice and was accompanied by increased intra-tumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the anti-tumorigenic effects of USP6.
In particular, USP6 overexpression inhibits the expression of bone morphogenetic protein BMP4, a key regulator of osteogenesis, and simultaneously augments expression the BMP antagonist Gremlin-1 [53][MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP6 was competent to induce Erk activation and prevent PARP cleavage upon serum starvation, and this effect was abrogated by depletion of Jak1 or STAT3 (XREF_FIG).
USP6 was a target of miR-130a and the overexpression of miR-130a or inhibition of USP6 in UM MUM-2B and MUM-2C cell lines inhibited the expression of Wnt, β-catenin, and EGFR, and activated SMAD4 expression, while reducing UM cell migration and invasion abilities in vitro.
In contrast, cohesin mutants rad21 and psc3 were rescued by loss of the RNA elimination pathway (Erh1, Mmi1, and Red1), and loader mutant mis4 was rescued by loss of Hrp1 mediated chromatin remodeling.
In particular, USP6 overexpression inhibits the expression of bone morphogenetic protein BMP4, a key regulator of osteogenesis, and simultaneously augments expression the BMP antagonist Gremlin-1 [53][MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
In particular, USP6 overexpression inhibits the expression of bone morphogenetic protein BMP4, a key regulator of osteogenesis, and simultaneously augments expression the BMP antagonist Gremlin-1 [53][MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Similarly, the inactivation of the evolutionarily related DUB in yeast, Ubp6, increases the cellular fitness of aneuploid cells, which have been shown to incur increased proteotoxic stresses due to chromosomal imbalance [66].
As many as 69% of primary ABCs demonstrate a characteristic clonal t(16;17) genetic translocation which can lead to an upregulation of the TRE17/USP6 oncogene [4].
To test this, we examined STAT3 activation in USP6 and NIH3T3 cells in which NF-kappaB was inhibited through expression of IkappaB-alpha super repressor.
Previously, we demonstrated that silencing of cyclic AMP (cAMP) response element binding protein 3 like 1 (CREB3L1), a cellular transcription factor that inhibits HCV replication, allows HCV to replicate in HRP1 cells, a subclone of Huh-7 cells permissive for HCV replication.
Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner.