Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export.

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway lead to R-loop accumulation, which contributes to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export.

A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes.

The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the bioUb approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field.

The dystrophia myotonica WD repeat-containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase.

Despite its potential clinical relevance, the product of the DMWD (dystrophia myotonica, WD repeat containing) gene is a largely uncharacterized protein. The DMWD amino acid sequence is similar to that of WDR20, a known regulator of the USP12 and USP46 deubiquitinases (DUBs). Here, we apply a combination of in silico and experimental methods to investigate several aspects of DMWD biology. Molecular evolution and phylogenetic analyses reveal that WDR20 and DMWD, similar to USP12 and USP46, arose by duplication of a common ancestor during the whole genome duplication event in the vertebrate ancestor lineage. The analysis of public human gene expression datasets suggests that DMWD expression is positively correlated with USP12 expression in normal tissues and negatively correlated with WDR20 expression in tumors. Strikingly, a survey of the annotated interactome for DMWD and WDR20 reveals a largely nonoverlapping set of interactors for these proteins. Experimentally, we first confirmed that DMWD binds both USP12 and USP46 through direct coimmunoprecipitation of epitope-tagged proteins. We found that DMWD and WDR20 share the same binding interface in USP12, suggesting that their interaction with the DUB may be mutually exclusive. Finally, we show that both DMWD and WDR20 promote USP12 enzymatic activity, but they differentially modulate the subcellular localization of the DUB. Altogether, our findings suggest a model whereby mutually exclusive binding of DMWD and WDR20 to USP12 may lead to formation of deubiquitinase complexes with distinct subcellular localization, potentially targeting different substrate repertoires.

WDR20 regulates shuttling of the USP12 deubiquitinase complex between the plasma membrane, cytoplasm and nucleus.

The human deubiquitinases USP12 and USP46 are very closely related paralogs with critical functions as tumor suppressors. The catalytic activity of these enzymes is regulated by two cofactors: UAF1 and WDR20. USP12 and USP46 show nearly 90% amino acid sequence identity and share some cellular activities, but have also evolved non-overlapping functions. We hypothesized that, correlating with their functional divergence, the subcellular localization of USP12 and USP46 might be differentially regulated by their cofactors. We used confocal and live microscopy analyses of epitope-tagged proteins to determine the effect of UAF1 and WDR20 on the localization of USP12 and USP46. We found that WDR20 differently modulated the localization of the DUBs, promoting recruitment of USP12, but not USP46, to the plasma membrane. Using site-directed mutagenesis, we generated a large set of USP12 and WDR20 mutants to characterize in detail the mechanisms and sequence determinants that modulate the subcellular localization of the USP12/UAF1/WDR20 complex. Our data suggest that the USP12/UAF1/WDR20 complex dynamically shuttles between the plasma membrane, cytoplasm and nucleus. This shuttling involved active nuclear export mediated by the CRM1 pathway, and required a short N-terminal motif (1MEIL4) in USP12, as well as a novel nuclear export sequence (450MDGAIASGVSKFATLSLHD468) in WDR20. In conclusion, USP12 and USP46 have evolved divergently in terms of cofactor binding-regulated subcellular localization. WDR20 plays a crucial role in as a "targeting subunit" that modulates CRM1-dependent shuttling of the USP12/UAF1/WDR20 complex between the plasma membrane, cytoplasm and nucleus.

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.

Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport.

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

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.