Sex bias in celiac disease: XWAS and monocyte eQTLs in women identify TMEM187 as a functional candidate gene.

BACKGROUND: Celiac disease (CeD) is an immune-mediated disorder that develops in genetically predisposed individuals upon gluten consumption. HLA risk alleles explain 40% of the genetic component of CeD, so there have been continuing efforts to uncover non-HLA loci that can explain the remaining heritability. As in most autoimmune disorders, the prevalence of CeD is significantly higher in women. Here, we investigated the possible involvement of the X chromosome on the sex bias of CeD. METHODS: We performed a X chromosome-wide association study (XWAS) and a gene-based association study in women from the CeD Immunochip (7062 cases, 5446 controls). We also constructed a database of X chromosome cis-expression quantitative trait loci (eQTLs) in monocytes from unstimulated (n = 226) and lipopolysaccharide (LPS)-stimulated (n = 130) female donors and performed a Summary-data-based MR (SMR) analysis to integrate XWAS and eQTL information. We interrogated the expression of the potentially causal gene (TMEM187) in peripheral blood mononuclear cells (PBMCs) from celiac patients at onset, on a gluten-free diet, potential celiac patients and non-celiac controls. RESULTS: The XWAS and gene-based analyses identified 13 SNPs and 25 genes, respectively, 22 of which had not been previously associated with CeD. The X chromosome cis-eQTL analysis found 18 genes with at least one cis-eQTL in naïve female monocytes and 8 genes in LPS-stimulated female monocytes, 2 of which were common to both situations and 6 were unique to LPS stimulation. SMR identified a potentially causal association of TMEM187 expression in naïve monocytes with CeD in women, regulated by CeD-associated, eQTL-SNPs rs7350355 and rs5945386. The CeD-risk alleles were correlated with lower TMEM187 expression. These results were replicated using eQTLs from LPS-stimulated monocytes. We observed higher levels of TMEM187 expression in PBMCs from female CeD patients at onset compared to female non-celiac controls, but not in male CeD individuals. CONCLUSION: Using X chromosome genotypes and gene expression data from female monocytes, SMR has identified TMEM187 as a potentially causal candidate in CeD. Further studies are needed to understand the implication of the X chromosome in the higher prevalence of CeD in women.

Celiac disease (CeD) is an immune-related condition triggered by gluten consumption in genetically susceptible individuals. Women present higher prevalence of CeD than men, but the biological explanation of such difference has not been elucidated. In this study, we investigated whether specific genetic variations on the X chromosome were associated with CeD in each sex. Surprisingly, we found 13 genetic variants and 25 genes significantly linked to CeD in women, but not in men. Additionally, we identified genetic variants on the X chromosome associated with gene expression of monocytes, a type of immune cells that is activated in CeD after gluten intake. Integrating these data with our previous findings, we found that lower expression of a gene termed TMEM187 might be associated with a potential increase in CeD risk in women. Finally, validation experiments confirmed higher TMEM187 levels in blood cells from female CeD patients compared to non-celiac women, while no such difference was seen in males. In summary, our study suggests that the X-chromosome gene TMEM187 may play a key role in CeD development, providing insights into the higher prevalence of CeD in females.

Two-Sample Mendelian Randomization detects bidirectional causality between gut microbiota and celiac disease in individuals with high genetic risk.

Background: Celiac Disease (CeD) is an autoimmune disorder triggered by gluten intake in genetically susceptible individuals. Highest risk individuals are homozygous for the Human Leucocyte Antigen (HLA) DQ2.5 haplotype or DQ2.5/DQ2.2 heterozygous. Both the HLA-DQ2-positive high genetic risk individuals and those that have developed the disease have altered intestinal microbiota, but it remains unclear whether these alterations are a cause or a consequence of CeD. Objective: To investigate a potential bidirectional causality between gut microbiota (GM) and CeD in HLA-DQ2 high genetic risk individuals. Materials and Methods: We performed a bidirectional Two-Sample Mendelian Randomization (2SMR) test using summary statistics from the largest publicly available Genome-Wide Association Study (GWAS) of GM and the summary statistics of the Immunochip CeD study of those individuals with the HLA-DQ2 high-risk haplotype. To test whether changes in GM composition were causally linked to CeD, GM data were used as exposure and CeD data as outcome; to test for reverse causation, the exposure and outcome datasets were inverted. Results: We identified several bacteria from Ruminococcaceae and Lachnospiraceae families of the Firmicutes phylum as potentially causal in both directions. In addition, our results suggest that changes in the abundance of Veillonellaceae family might be causal in the development of CeD, while alterations in Pasteurellaceae family might be a consequence of the disease itself. Conclusion: Our results suggest that the relationship between GM and HLA-DQ2 high risk individuals is highly complex and bidirectional.

Potentially causal associations between placental DNA methylation and schizophrenia and other neuropsychiatric disorders.

Increasing evidence supports the role of placenta in neurodevelopment and potentially, in the later onset of neuropsychiatric disorders. Recently, methylation quantitative trait loci (mQTL) and interaction QTL (iQTL) maps have proven useful to understand SNP-genome wide association study (GWAS) relationships, otherwise missed by conventional expression QTLs. In this context, we propose that part of the genetic predisposition to complex neuropsychiatric disorders acts through placental DNA methylation (DNAm). We constructed the first public placental cis-mQTL database including nearly eight million mQTLs calculated in 368 fetal placenta DNA samples from the INMA project, ran cell type- and gestational age-imQTL models and combined those data with the summary statistics of the largest GWAS on 10 neuropsychiatric disorders using Summary-based Mendelian Randomization (SMR) and colocalization. Finally, we evaluated the influence of the DNAm sites identified on placental gene expression in the RICHS cohort. We found that placental cis-mQTLs are highly enriched in placenta-specific active chromatin regions, and useful to map the etiology of neuropsychiatric disorders at prenatal stages. Specifically, part of the genetic burden for schizophrenia, bipolar disorder and major depressive disorder confers risk through placental DNAm. The potential causality of several of the observed associations is reinforced by secondary association signals identified in conditional analyses, regional pleiotropic methylation signals associated to the same disorder, and cell type-imQTLs, additionally associated to the expression levels of relevant immune genes in placenta. In conclusion, the genetic risk of several neuropsychiatric disorders could operate, at least in part, through DNAm and associated gene expression in placenta.

Gluten-induced RNA methylation changes regulate intestinal inflammation via allele-specific XPO1 translation in epithelial cells.

OBJECTIVES: Coeliac disease (CD) is a complex autoimmune disorder that develops in genetically susceptible individuals. Dietary gluten triggers an immune response for which the only available treatment so far is a strict, lifelong gluten free diet. Human leucocyte antigen (HLA) genes and several non-HLA regions have been associated with the genetic susceptibility to CD, but their role in the pathogenesis of the disease is still essentially unknown, making it complicated to develop much needed non-dietary treatments. Here, we describe the functional involvement of a CD-associated single-nucleotide polymorphism (SNP) located in the 5'UTR of XPO1 in the inflammatory environment characteristic of the coeliac intestinal epithelium. DESIGN: The function of the CD-associated SNP was investigated using an intestinal cell line heterozygous for the SNP, N6-methyladenosine (m6A)-related knock-out and HLA-DQ2 mice, and human samples from patients with CD. RESULTS: Individuals harbouring the risk allele had higher m6A methylation in the 5'UTR of XPO1 RNA, rendering greater XPO1 protein amounts that led to downstream nuclear factor kappa B (NFkB) activity and subsequent inflammation. Furthermore, gluten exposure increased overall m6A methylation in humans as well as in in vitro and in vivo models. CONCLUSION: We identify a novel m6A-XPO1-NFkB pathway that is activated in CD patients. The findings will prompt the development of new therapeutic approaches directed at m6A proteins and XPO1, a target under evaluation for the treatment of intestinal disorders.

Genetic Contribution of Endometriosis to the Risk of Developing Hormone-Related Cancers.

Endometriosis is a common gynecological disorder that has been associated with endometrial, breast and epithelial ovarian cancers in epidemiological studies. Since complex diseases are a result of multiple environmental and genetic factors, we hypothesized that the biological mechanism underlying their comorbidity might be explained, at least in part, by shared genetics. To assess their potential genetic relationship, we performed a two-sample mendelian randomization (2SMR) analysis on results from public genome-wide association studies (GWAS). This analysis confirmed previously reported genetic pleiotropy between endometriosis and endometrial cancer. We present robust evidence supporting a causal genetic association between endometriosis and ovarian cancer, particularly with the clear cell and endometrioid subtypes. Our study also identified genetic variants that could explain those associations, opening the door to further functional experiments. Overall, this work demonstrates the value of genomic analyses to support epidemiological data, and to identify targets of relevance in multiple disorders.

Celiac disease susceptibility: The genome and beyond.

Celiac Disease (CeD) is an immune-mediated complex disease that is triggered by the ingestion of gluten and develops in genetically susceptible individuals. It has been known for a long time that the Human Leucocyte Antigen (HLA) molecules DQ2 and DQ8 are necessary, although not sufficient, for the disease development, and therefore other susceptibility genes and (epi)genetic events must participate in CeD pathogenesis. The advances in Genomics during the last 15 years have made CeD one of the immune-related disorders with the best-characterized genetic component. In the present work, we will first review the main Genome-Wide Association Studies (GWAS) carried out in the disorder, and emphasize post-GWAS discoveries, including diverse integrative strategies, SNP prioritization approaches, and insights into the Microbiome through the host Genomics. Second, we will explore CeD-related Epigenetics and Epigenomics, mostly focusing on the emerging knowledge of the celiac methylome, and the vast but yet under-explored non-coding RNA (ncRNA) landscape. We conclude that much has been done in the field although there are still completely unvisited areas in the post-Genomics of CeD. Chromatin conformation and accessibility, and Epitranscriptomics are promising domains that need to be unveiled to complete the big picture of the celiac Genome.

Sustained CHK2 activity, but not ATM activity, is critical to maintain a G1 arrest after DNA damage in untransformed cells.

BACKGROUND: The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized. RESULTS: Here we examine the impact of ATM and CHK2 activities on G1 checkpoint maintenance in untransformed cells after DNA damage caused by DSBs. We show that ATM becomes dispensable for G1 checkpoint maintenance as early as 1 h after DSB induction. In contrast, CHK2 kinase activity is necessary to maintain the G1 arrest, independently of ATM, ATR, and DNA-PKcs, implying that the G1 arrest is maintained in a lesion-independent manner. Sustained CHK2 activity is achieved through auto-activation and its acute inhibition enables cells to abrogate the G1-checkpoint and enter into S-phase. Accordingly, we show that CHK2 activity is lost in cells that recover from the G1 arrest, pointing to the involvement of a phosphatase with fast turnover. CONCLUSION: Our data indicate that G1 checkpoint maintenance relies on CHK2 and that its negative regulation is crucial for G1 checkpoint recovery after DSB induction.

A Systematic Two-Sample Mendelian Randomization Analysis Identifies Shared Genetic Origin of Endometriosis and Associated Phenotypes.

Endometriosis, one of the most common gynecological disorders, is a complex disease characterized by the growth of endometrial-like tissue in extra-uterine locations and is a cause of pelvic pain and infertility. Evidence from observational studies indicate that endometriosis usually appears together with several other phenotypes. These include a list of autoimmune diseases, most of them more prevalent in women, anthropometric traits associated with leanness in the adulthood, as well as female reproductive traits, including altered hormone levels and those associated with a prolonged exposure to menstruation. However, the biological mechanisms underlying their co-morbidity remains unknown. To explore whether those phenotypes and endometriosis share a common genetic origin, we performed a systematic Two-Sample Mendelian Randomization (2SMR) analysis using public GWAS data. Our results suggest potential common genetic roots between endometriosis and female anthropometric and reproductive traits. Particularly, our data suggests that reduced weight and BMI might be mediating the genetic susceptibility to suffer endometriosis. Furthermore, data on female reproductive traits strongly suggest that genetic variants that predispose to a more frequent exposure to menstruation, through earlier age at menarche and shorter menstrual cycles, might also increase the risk to suffer from endometriosis.

A Two-Sample Mendelian Randomization Analysis Investigates Associations Between Gut Microbiota and Celiac Disease.

Celiac disease (CeD) is a complex immune-mediated inflammatory condition triggered by the ingestion of gluten in genetically predisposed individuals. Literature suggests that alterations in gut microbiota composition and function precede the onset of CeD. Considering that microbiota is partly determined by host genetics, we speculated that the genetic makeup of CeD patients could elicit disease development through alterations in the intestinal microbiota. To evaluate potential causal relationships between gut microbiota and CeD, we performed a two-sample Mendelian randomization analysis (2SMR). Exposure data were obtained from the raw results of a previous genome-wide association study (GWAS) of gut microbiota and outcome data from summary statistics of CeD GWAS and Immunochip studies. We identified a number of putative associations between gut microbiota single nucleotide polymorphisms (SNPs) associated with CeD. Regarding bacterial composition, most of the associated SNPs were related to Firmicutes phylum, whose relative abundance has been previously reported to be altered in CeD patients. In terms of functional units, we linked a number of SNPs to several bacterial metabolic pathways that seemed to be related to CeD. Overall, this study represented the first 2SMR approach to elucidate the relationship between microbiome and CeD.

Chromosomes trapped in micronuclei are liable to segregation errors.

DNA in micronuclei is likely to get damaged. When shattered DNA from micronuclei gets reincorporated into the primary nucleus, aberrant rearrangements can take place, a phenomenon referred to as chromothripsis. Here, we investigated how chromatids from micronuclei act in subsequent divisions and how this affects their fate. We observed that the majority of chromatids derived from micronuclei fail to establish a proper kinetochore in mitosis, which is associated with problems in chromosome alignment, segregation and spindle assembly checkpoint activation. Remarkably, we found that, upon their formation, micronuclei already display decreased levels of important kinetochore assembly factors. Importantly, these defects favour the exclusion of the micronucleus over the reintegration into the primary nucleus over several divisions. Interestingly, the defects observed in micronuclei are likely overcome once micronuclei are reincorporated into the primary nuclei, as they further propagate normally. We conclude that the formation of a separate small nuclear entity represents a mechanism for the cell to delay the stable propagation of excess chromosome(s) and/or damaged DNA, by inducing kinetochore defects.

An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability.

The cellular response to DNA damage is essential for maintaining the integrity of the genome. Recent evidence has identified E2F7 as a key player in DNA damage-dependent transcriptional regulation of cell-cycle genes. However, the contribution of E2F7 to cellular responses upon genotoxic damage is still poorly defined. Here we show that E2F7 represses the expression of genes involved in the maintenance of genomic stability, both throughout the cell cycle and upon induction of DNA lesions that interfere with replication fork progression. Knockdown of E2F7 leads to a reduction in 53BP1 and FANCD2 foci and to fewer chromosomal aberrations following treatment with agents that cause interstrand crosslink (ICL) lesions but not upon ionizing radiation. Accordingly, E2F7-depleted cells exhibit enhanced cell-cycle re-entry and clonogenic survival after exposure to ICL-inducing agents. We further report that expression and functional activity of E2F7 are p53-independent in this context. Using a cell-based assay, we show that E2F7 restricts homologous recombination through the transcriptional repression of RAD51. Finally, we present evidence that downregulation of E2F7 confers an increased resistance to chemotherapy in recombination-deficient cells. Taken together, our results reveal an E2F7-dependent transcriptional program that contributes to the regulation of DNA repair and genomic integrity.

Doxorubicin-induced DNA Damage Causes Extensive Ubiquitination of Ribosomal Proteins Associated with a Decrease in Protein Translation.

Protein posttranslational modifications (PTMs) play a central role in the DNA damage response. In particular, protein phosphorylation and ubiquitination have been shown to be essential in the signaling cascade that coordinates break repair with cell cycle progression. Here, we performed whole-cell quantitative proteomics to identify global changes in protein ubiquitination that are induced by DNA double-strand breaks. In total, we quantified more than 9,400 ubiquitin sites and found that the relative abundance of ∼10% of these sites was altered in response to DNA double-strand breaks. Interestingly, a large proportion of ribosomal proteins, including those from the 40S as well as the 60S subunit, were ubiquitinated in response to DNA damage. In parallel, we discovered that DNA damage leads to the inhibition of ribosome function. Taken together, these data uncover the ribosome as a major target of the DNA damage response.

A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K.

The exportin CRM1 binds nuclear export signals (NESs), and mediates active transport of NES-bearing proteins from the nucleus to the cytoplasm. Structural and biochemical analyses have uncovered the molecular mechanisms underlying CRM1/NES interaction. CRM1 binds NESs through a hydrophobic cleft, whose open or closed conformation facilitates NES binding and release. Several cofactors allosterically modulate the conformation of the NES-binding cleft through intramolecular interactions involving an acidic loop and a C-terminal helix in CRM1. This current model of CRM1-mediated nuclear export has not yet been evaluated in a cellular setting. Here, we describe SRV100, a cellular reporter to interrogate CRM1 nuclear export activity. Using this novel tool, we provide evidence further validating the model of NES binding and release by CRM1. Furthermore, using both SRV100-based cellular assays and in vitro biochemical analyses, we investigate the functional consequences of a recurrent cancer-related mutation, which targets a residue near CRM1 NES-binding cleft. Our data indicate that this mutation does not necessarily abrogate the nuclear export activity of CRM1, but may increase its affinity for NES sequences bearing a more negatively charged C-terminal end.

Mutations in the 'Fingers' subdomain of the deubiquitinase USP1 modulate its function and activity.

Ubiquitin-specific protease (USP)1 is a member of the USP family of deubiquitinating enzymes. Efficient USP1 activity requires binding to its cofactor USP1-associated factor 1 (UAF1), and the USP1-UAF1 deubiquitinase complex has important roles in regulating DNA damage-related processes. USPs show common folding of their catalytic domain, with three subdomains termed Thumb, Palm, and Fingers. The Fingers subdomain appears to be the primary site for ubiquitin binding. In USP1, the Fingers subdomain also mediates its interaction with UAF1, and thus represents a crucial, but poorly characterized, motif in USP1. To explore the role of USP1-UAF1 in ubiquitin-dependent nuclear processes, we tested the effect of modulating USP1-UAF1 activity on the level and/or localization of conjugated ubiquitin and the DNA damage-related proteins phosphorylated histone H2AX, Lys56-acetylated histone H3, and p53-binding protein 1 (53BP1). Small interfering RNA-mediated USP1 knockdown or treatment with the novel USP1-UAF1 inhibitor ML323 increased the recruitment of conjugated ubiquitin and 53BP1 into nuclear foci. Strikingly, ectopic coexpression of USP1 and UAF1 depleted conjugated ubiquitin in the nucleus and blocked the recruitment of 53BP1 to DNA damage foci. In a direct comparison with other overexpressed USPs, USP1-UAF1 behaved as a relatively promiscuous deubiquitinase. Experimental and cancer-related mutations in the USP1 The Fingers subdomain abrogated substrate deubiquitination without interfering with other USP1 activities, such as UAF1 binding or autocleavage. These results provide new insights into the function and regulation of the USP1-UAF1 complex.

Structure-function analysis of USP1: insights into the role of Ser313 phosphorylation site and the effect of cancer-associated mutations on autocleavage.

BACKGROUND: In complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage. METHODS: We have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage. RESULTS: A non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420-520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420-520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency. CONCLUSIONS: USP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420-520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.

USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy.

Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

Two nuclear localization signals in USP1 mediate nuclear import of the USP1/UAF1 complex.

The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex.

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family.

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP-USP21 and, to a lesser extent, GFP-OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.