Coilin interacts with Ku proteins and inhibits in vitro non-homologous DNA end joining

AbstractCoilin is a nuclear protein that plays a role in Cajal body formation. The function of nucleoplasmic coilin is unknown. Here we report that coilin interacts with Ku70 and Ku80, which are major players in the DNA repair process. Ku proteins compete with SMN and SmB′ proteins for coilin interaction sites. The binding domain on coilin for Ku proteins cannot be localized to one discrete region, and only full-length coilin is capable of inhibiting in vitro non-homologous DNA end joining (NHEJ). Since Ku proteins do not accumulate in CBs, these findings suggest that nucleoplasmic coilin participates in the regulation of DNA repair.Structured summaryMINT-8052983:coilin (uniprotkb:P38432) physically interacts (MI:0915) with SmB′ (uniprotkb:P14678) by pull down (MI:0096)MINT-8052941:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku70 (uniprotkb:P12956) by competition binding (MI:0405)MINT-8052765:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by pull down (MI:0096)MINT-8052971:coilin (uniprotkb:P38432) physically interacts (MI:0915) with SMN (uniprotkb:Q16637) by pull down (MI:0096)MINT-8052957:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by competition binding (MI:0405)MINT-8052894, MINT-8052908:coilin (uniprotkb:P38432) binds (MI:0407) to Ku80 (uniprotkb:P13010) by pull down (MI:0096)MINT-8052804:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by anti bait coimmunoprecipitation (MI:0006)MINT-8052925:coilin (uniprotkb:P38432) binds (MI:0407) to Ku70 (uniprotkb:P12956) by pull down (MI:0096)MINT-8052786:Ku80 (uniprotkb:P13010) physically interacts (MI:0914) with coilin (uniprotkb:P38432) and Ku70 (uniprotkb:P12956) by anti bait coimmunoprecipitation (MI:0006)MINT-8052776:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku70 (uniprotkb:P12956) by pull down (MI:0096

Coilin levels modulate cell cycle progression and γH2AX levels in etoposide treated U2OS cells

AbstractCoilin is considered the Cajal body (CB) marker protein. In this report, we investigated the role of coilin in the DNA damage response and found that coilin reduction correlated with significantly increased levels of soluble γH2AX in etoposide treated U2OS cells. Additionally, coilin levels influenced the proliferation rate and cell cycle distribution of cells exposed to etoposide. Moreover, coilin overexpression inhibited nucleolar localization of endogenous coilin in etoposide treated U2OS cells. Collectively, these data provide additional evidence for coilin and CBs in the DNA damage response

Mutant p53 protects ETS2 from non-canonical COP1/DET1 dependent degradation

Mutations in the tumor suppressor gene TP53 contribute to the development of approximately half of all human cancers. One mechanism by which mutant p53 (mtp53) acts is through interaction with other transcription factors, which can either enhance or repress the transcription of their target genes. Mtp53 preferentially interacts with the erythroblastosis virus E26 oncogene homologue 2 (ETS2), an ETS transcription factor, and increases its protein stability. To study the mechanism underlying ETS2 degradation, we knocked down ubiquitin ligases known to interact with ETS2. We observed that knockdown of the constitutive photomorphogenesis protein 1 (COP1) and its binding partner De-etiolated 1 (DET1) significantly increased ETS2 stability, and conversely, their ectopic expression led to increased ETS2 ubiquitination and degradation. Surprisingly, we observed that DET1 binds to ETS2 independently of COP1, and we demonstrated that mutation of multiple sites required for ETS2 degradation abrogated the interaction between DET1 and ETS2. Furthermore, we demonstrate that mtp53 prevents the COP1/DET1 complex from ubiquitinating ETS2 and thereby marking it for destruction. Mechanistically, we show that mtp53 destabilizes DET1 and also disrupts the DET1/ETS2 complex thereby preventing ETS2 degradation. Our study reveals a hitherto unknown function in which DET1 mediates the interaction with the substrates of its cognate ubiquitin ligase complex and provides an explanation for the ability of mtp53 to protect ETS2

Regression-based Deep-Learning predicts molecular biomarkers from pathology slides

Deep Learning (DL) can predict biomarkers from cancer histopathology. Several clinically approved applications use this technology. Most approaches, however, predict categorical labels, whereas biomarkers are often continuous measurements. We hypothesized that regression-based DL outperforms classification-based DL. Therefore, we developed and evaluated a new self-supervised attention-based weakly supervised regression method that predicts continuous biomarkers directly from images in 11,671 patients across nine cancer types. We tested our method for multiple clinically and biologically relevant biomarkers: homologous repair deficiency (HRD) score, a clinically used pan-cancer biomarker, as well as markers of key biological processes in the tumor microenvironment. Using regression significantly enhances the accuracy of biomarker prediction, while also improving the interpretability of the results over classification. In a large cohort of colorectal cancer patients, regression-based prediction scores provide a higher prognostic value than classification-based scores. Our open-source regression approach offers a promising alternative for continuous biomarker analysis in computational pathology

Coilin Phosphomutants Disrupt Cajal Body Formation, Reduce Cell Proliferation and Produce a Distinct Coilin Degradation Product

Coilin is a nuclear phosphoprotein that accumulates in Cajal bodies (CBs). CBs participate in ribonucleoprotein and telomerase biogenesis, and are often found in cells with high transcriptional demands such as neuronal and cancer cells, but can also be observed less frequently in other cell types such as fibroblasts. Many proteins enriched within the CB are phosphorylated, but it is not clear what role this modification has on the activity of these proteins in the CB. Coilin is considered to be the CB marker protein and is essential for proper CB formation and composition in mammalian cells. In order to characterize the role of coilin phosphorylation on CB formation, we evaluated various coilin phosphomutants using transient expression. Additionally, we generated inducible coilin phosphomutant cell lines that, when used in combination with endogenous coilin knockdown, allow for the expression of the phosphomutants at physiological levels. Transient expression of all coilin phosphomutants except the phosphonull mutant (OFF) significantly reduces proliferation. Interestingly, a stable cell line induced to express the coilin S489D phosphomutant displays nucleolar accumulation of the mutant and generates a N-terminal degradation product; neither of which is observed upon transient expression. A N-terminal degradation product and nucleolar localization are also observed in a stable cell line induced to express a coilin phosphonull mutant (OFF). The nucleolar localization of the S489D and OFF coilin mutants observed in the stable cell lines is decreased when endogenous coilin is reduced. Furthermore, all the phosphomutant cells lines show a significant reduction in CB formation when compared to wild-type after endogenous coilin knockdown. Cell proliferation studies on these lines reveal that only wild-type coilin and the OFF mutant are sufficient to rescue the reduction in proliferation associated with endogenous coilin depletion. These results emphasize the role of coilin phosphorylation in the formation and activity of CBs

Phosphorylation regulates coilin activity and RNA association

Summary The Cajal body (CB) is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation

Transient expression of various GFP-coilin proteins in the presence of endogenous coilin.

<p>Expression denotes the ratio GFP-coilin or mutants thereof to endogenous coilin.</p><p>The data for the localization of WT, ON and OFF taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025743#pone.0025743-Hearst1" target="_blank">[16]</a>.</p><p>Proliferation impact reflects the change in proliferation rate when compared to the rate of cells expressing WT GFP-coilin.</p

Characterization of doxycycline inducible coilin phosphomutant cell lines.

<p>(A and B) Stable cell lines expressing GFP-coilin of GFP-coilin phosphomutant proteins. Non-induced (−) and 24 h doxycycline induced (1 µg/mL, +) cell lysates of GFP-coilin (WT) and GFP-coilin phosphomutants (T122E, ON, S489D, S271/272D or OFF) were subjected to SDS-PAGE, followed by western transfer. The blots were probed with mouse monoclonal anti-GFP antibodies for specific detection of the GFP-tagged coilin proteins (upper panel). The blots were re-probed with rabbit polyclonal anti-coilin antibodies to detect both endogenous coilin and the GFP-tagged WT and coilin phosphomutants (lower panel). Note that S489D and OFF expression generates an approximately 42 kDa degradation product. (C) Transiently transfected GFP-coilin S489D and GFP-coilin OFF phosphomutant proteins do not have a specific degradation product. HeLa cells were transiently transfected with GFP-coilin S489D and GFP-coilin OFF DNA constructs for 24 h followed by lysate generation, SDS-PAGE and western transfer. The blot was probed with antibodies to GFP. (D) Full length GFP-coilin S489D can be detected after doxycycline induction by immunoprecipitation. Doxycycline (0.33 or 1 µg/mL) induced and non-induced GFP-coilin-S489D stable cell extracts were immunoprecipitated with anti-GFP antibodies. The western blot was probed with anti-GFP antibodies for the detection of the GFP-coilin-S489D fragment (upper panel). The same blot was probed with anti-coilin antibodies for endogenous coilin and full length GFP-coilin-S489D protein detection (lower panel). IgG(H) denotes the immunoglobulin heavy chain.</p

Expression of GFP-coilin and GFP coilin phosphomutant proteins in endogenous coilin depleted stable cell lines.

<p>(A and B) Stable cell lines were transfected with coilin siRNA (+) to deplete endogenous coilin, or control siRNA (−). 24 h post siRNA transfection, GFP-coilin or GFP-coilin phosphomutant expression was induced with doxycycline. 24 h later (48 h post siRNA transfection) cells were harvested and lysates were subjected to SDS-PAGE followed by western transfer and probing with anti-GFP antibodies (upper panel). The blots were then probed with anti-coilin antibodies (middle panel) followed by detection of beta-tubulin with anti-beta-tubulin antibodies (lower panel). KD – and + indicates transfection with control siRNA or coilin siRNA, respectively. Note that the GFP-coilin ON signal was too low to be detected with anti-GFP antibodies and could only be detected with anti-coilin antibodies (A, lanes 5 and 6).</p