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 USP26 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 USP26 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
Taken together, the effects of AR deubiquitinated by USP26 could modulate sperm maturation and spermatogenesis through the androgen receptor signaling pathway.
Elevated cellular ROS levels might then inhibit USP26 activity to increase the ubiquitination of androgen receptor (AR) and AR splice variant 7 (ARv7) and their ubiquitin and proteasome dependent degradation, which contributed to the increase of Enz sensitivity.
USP26 deubiquitinates androgen receptor (AR) in the maintenance of sperm maturation and spermatogenesis through the androgen receptor signaling pathway.
Our data suggest that USP26 assembles with AR and other cofactors in subnuclear foci, and serves to counteract hormone-induced AR ubiquitination, thereby contributing to the regulation of AR transcriptional activity.
USP26 can deubiquitylate AR in the presence of androgen, while USP10 and 2A-DUB both regulate AR transcriptional activity but do not alter protein stability.
USP26 deubiquitinates androgen receptor (AR) in the maintenance of sperm maturation and spermatogenesis through the androgen receptor signaling pathway.
Examples include ubiquitin specific protease 10 (USP10), which can act on SMAD4 to make it deubiquitinated and stable, further promoting TGF-beta signaling [XREF_BIBR], and USP26, which promotes SMAD7 deubiquitination, thereby amplifying the inhibitory effect of SMAD7 and strengthening the inhibition of the TGF-beta signaling pathway [XREF_BIBR].
In contrast, loss of USP26 rapidly degrades SMAD7 stabilizing the TGF-beta receptors leading to enhanced TGF-beta activity as observed by increased levels of phosphorylated SMAD2.
As observed with USP26 knockdown, ectopic expression of the DUB dead mutant recapitulated the augmentation of SMAD7 induction by TGF-beta whereas wild-type USP26 significantly diminished the induction of SMAD7 in the presence of TGF-beta (Fig XREF_FIG F).
Conversely, lesser extent of CBX7 protein binding to the promoters of Cbx4 and Cbx6 is observed during Usp26 mediated ESC differentiation and RA induced ESC differentiation.
Correlative with the Oct4 gene expression data, occupancy at the Oct4 promoter by USP26, CBX4, CBX6, and H2A-ubi1 was low even during USP26 mediated ESC differentiation.
Using screening of shRNA specific for the DUB subfamily USPs, we identified a novel cellular reprogramming repressor gene Usp26, which blocked somatic cell reprogramming into iPSCs and promoted differentiation by stabilizing PRC1 complexes.
Given that USP26 and USP37 are able to reverse RNF8/168 mediated ubiquitylation and promote HR, these enzymes would be ideal candidates to facilitate the repositioning of ubiquitylated substrates away from the DSB.
We reasoned that USP26 and USP37 may antagonize the RAP80 dependent sequestration of BRCA1 by removing RNF8 and RNF168 mediated ubiquitylation and thereby promote HR.
Taken together, these findings confirmed our hypothesis that loss of USP26 enhances TGF-beta signaling and confers poor prognosis in glioblastoma patients.
Taken together, these data indicate that USP26 inhibits TGF-beta pathway activity by limiting Lys48 ubiquitin mediated degradation of SMAD7 consequently resulting in enhanced ubiquitylation and degradation of the TbetaR complex.
As knockdown of USP26 potently activated the TGF-beta pathway in breast cancer, we investigated whether depletion of USP26 in the TGF-beta-responsive metastatic cell line MDA-MB-231 enhanced cellular motility and invasion.
In line with our other models, knockdown of USP26 in this patient derived cell line enhanced TGF-beta activity as evidenced by increased levels of phospho-SMAD2 and increased expression of TGF-beta target genes CTGF, LIF, and SMAD7 (Fig XREF_FIG D and E).
Together, these results suggest that USP26 and USP37 promote the BRCA1 dependent loading of PALB2 and RAD51 by counteracting the repressive impact of RAP80 dependent BRCA1 sequestration and RAP80 dependent inhibition of end-resection during HR.
We reasoned that USP26 and USP37 may antagonize the RAP80 dependent sequestration of BRCA1 by removing RNF8 and RNF168 mediated ubiquitylation and thereby promote HR.
In agreement with this hypothesis, we found that USP26 or USP37 depletion inhibits the efficient association of BRCA1 with PALB2, a unique subunit of the BRCC complex.
Together, these results suggest that USP26 and USP37 promote the BRCA1 dependent loading of PALB2 and RAD51 by counteracting the repressive impact of RAP80 dependent BRCA1 sequestration and RAP80 dependent inhibition of end-resection during HR.
We demonstrate that TGF-beta rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin mediated turnover of SMAD7.
Our data suggest that as part of a negative feedback loop, TGF-beta enhances the expression of not only SMAD7 but also USP26, which acts to deubiquitinate and stabilize SMAD7.
To verify whether USP26 induction by TGF-beta is prevalent in other cell types, we analyzed the induction of USP26 and SMAD7 at early time points in the TGF-beta-responsive breast cancer cell lines MCF7, MDA-MB-231, T47D, and CAL51 and glioma cell lines U373, PCTC, and A172 (Fig XREF_FIG A-G).
This result suggests that the increased Usp26 expression during ESC differentiation may lead to increased CBX4 and CBX6 protein expression not only by CBX4 and CBX6 protein stabilization but also indirectly by reversing the repression of their gene expression.
Taken together, these results provide molecular mechanisms of Usp26 mediated increased CBX4 and CBX6 protein stability for suppressing pluripotent gene expression.
We reasoned that USP26 and USP37 may antagonize the RAP80 dependent sequestration of BRCA1 by removing RNF8 and RNF168 mediated ubiquitylation and thereby promote HR.
Together, these results suggest that USP26 and USP37 promote the BRCA1 dependent loading of PALB2 and RAD51 by counteracting the repressive impact of RAP80 dependent BRCA1 sequestration and RAP80 dependent inhibition of end-resection during HR.
Our results showed that after Usp26 knockdown during Dox induced OSKM mediated MEF reprogramming led to increased mRNA levels of Sox2 and Nanog, but not Oct4, compared with control.
Our results showed that after Usp26 knockdown during Dox induced OSKM mediated MEF reprogramming led to increased mRNA levels of Sox2 and Nanog, but not Oct4, compared with control.
Taken together, these results provide molecular mechanisms of Usp26 mediated increased CBX4 and CBX6 protein stability for suppressing pluripotent gene expression.
In our current study, we screened several AR related USPs, including USP7, USP12, USP14, USP22, and USP26 and found that the USP26 activity was repressed by the induced cellular ROS, which is responsible for AR/ARv7 protein degradation and Enz sensitivity increase by ABT263.
An in vitro DUB assay was applied to further detect USP26 activity, and results revealed that ABT263 could significantly decrease USP26 activity, which could be reversed by inhibition of cellular ROS (XREF_FIG H-J).
In comparison, stable expression of USP26, but not the catalytically inactive mutant USP26 C/S, attenuated invasion of MDA-MB-231 cells following exposure to TGF-beta (Fig XREF_FIG E-G).
As knockdown of USP26 potently activated the TGF-beta pathway in breast cancer, we investigated whether depletion of USP26 in the TGF-beta-responsive metastatic cell line MDA-MB-231 enhanced cellular motility and invasion.
Correlative with the Oct4 gene expression data, occupancy at the Oct4 promoter by USP26, CBX4, CBX6, and H2A-ubi1 was low even during USP26 mediated ESC differentiation.
Our results showed that after Usp26 knockdown during Dox induced OSKM mediated MEF reprogramming led to increased mRNA levels of Sox2 and Nanog, but not Oct4, compared with control.
Together, these results suggest that USP26 and USP37 promote the BRCA1 dependent loading of PALB2 and RAD51 by counteracting the repressive impact of RAP80 dependent BRCA1 sequestration and RAP80 dependent inhibition of end-resection during HR.
Together, these results suggest that USP26 and USP37 promote the BRCA1 dependent loading of PALB2 and RAD51 by counteracting the repressive impact of RAP80 dependent BRCA1 sequestration and RAP80 dependent inhibition of end-resection during HR.
Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26 mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production.
Overexpression of the catalytically inactive USP26 C/S mutant also enhanced the levels of p-SMAD2 whereas ectopic expression of USP26 slightly diminished the levels of phosphorylated SMAD2 (Fig XREF_FIG B).
Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGFbeta receptor stabilization and enhanced levels of p-SMAD2, ultimately enhancing cellular proliferation, invasion, and metastasis [XREF_BIBR].
To further dissect the molecular mechanism (s) how miR-367-3p increases AR expression, according to the screening strategy and bioinformatics analysis, we focused on the 2 miR-367-3p predicted targets, MDM2 and USP26, that were reported to modulate AR protein expression.
In LNCaP cells, Usp26 depletion by siRNA enhances AR dependent transcription, suggesting it acts on a factor that is negatively regulated by ubiquitination (Dirac and Bernards, 2010).
In LNCaP cells, Usp26 depletion by siRNA enhances AR dependent transcription, suggesting it acts on a factor that is negatively regulated by ubiquitination (Dirac and Bernards, 2010).
Conversely, lesser extent of CBX7 protein binding to the promoters of Cbx4 and Cbx6 is observed during Usp26 mediated ESC differentiation and RA induced ESC differentiation.
These data suggest that USP26 and USP37 limit the magnitude of BRCA1-A complex assembly in DSB neighboring chromatin by counteracting RNF168 mediated H2A ubiquitylation.
Moreover , the results from co-immunoprecipitation ( CO-IP ) , immunofluorescence and western blot assays showed that the physiological process due to USP26 interacted with AR and influenced AR deubiquitination , thus upregulating the proteins CCND1 and SPATA46 - which are associated with cell cycle progression and spermatogenesis - as well as decreasing the expression of TP73 .
We isolated the two hairpin vectors from the USP26 DUB pool, which had previously been demonstrated to most effectively inhibit USP26 expression and tested their ability to knockdown USP26 24.
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]
Sox2 and Nanog mRNA levels, but not Oct4, dramatically increased in Cbx4 or Cbx6 knockdown cells, similar to the increased Sox2 and Nanog mRNA levels in Usp26 knockdown cells.
Sox2 and Nanog mRNA levels, but not Oct4, dramatically increased in Cbx4 or Cbx6 knockdown cells, similar to the increased Sox2 and Nanog mRNA levels in Usp26 knockdown cells.
To this end, we co-transfected HEK293T cells with expression plasmids encoding a constitutively active TbetaR (TRICA) and HA tagged ubiquitin in the presence of shRNA vectors targeting USP26.
To understand how Usp26 expression is regulated, we assessed the promoter binding activity of Usp26 by PRC1 subunits using the ChIP-qPCR assay during RA induced ESC differentiation, and observed that PRC1 components PCGF2 and Cbx7 occupation as well as H2A-ubi1 modifications decreased significantly in Usp26 promoter, thus indicating that CBX7 containing PRC1 complex is a potential repressor of Usp26.