TIRR regulates 53BP1 by masking its histone methyl-lysine binding function

53BP1 is a multi-functional double-strand break (DSB) repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumors to PARP inhibitors. Central to all 53BP1 activities is its recruitment to DSBs via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, TIRR (Tudor Interacting Repair Regulator) that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, ATM phosphorylates 53BP1 and recruits RIF1 to dissociate the 53BP1–TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to DSBs. Depletion of TIRR destabilizes 53BP1 in the nuclear soluble fraction and also alters the DSB-induced protein complex centering 53BP1. These findings identify TIRR as a new factor that influences DSB repair utilizing a unique mechanism of masking the histone methyl-lysine binding function of 53BP1

Biochemistry of hypoxia: Refulation of hypoxia inducible factor HIF-1α by casein kinase 1, CK1

Hypoxia inducible factor 1, HIF-1, is a heterodimeric transcription factor regulating the cellular response to hypoxia and implicated in many pathological conditions including cancer, ischemia and inflammation. Its inducible subunit, HIF-1α is controlled by oxygen levels as well as by various oxygen-independent mechanisms. Post-translational modifications like hydroxylation and phosphorylation play a very important role in HIF-1α regulation. The present study analyzes the phosphorylation of HIF-1α in its N-terminal domain and its role in the activity of HIF-1. In order to identify the phosphorylation sites, small parts of HIF-1α were constructed as GST-fusion proteins and subjected to in vitro phosphorylation by Hela cell nuclear extracts. The phosphorylation site was mapped between HIF-1α amino acids 229-251. Analysis of this sequence revealed serine²⁴⁷ as a potential target of phosphorylation by CK1. In vitro phosphorylation assays showed that HIF-1α was indeed phosphorylated by recombinant CK1. Conversion of Ser²⁴⁷ of HIF-1α into Ala (S247A) as well as inhibition of CK1 by chemical inhibitors impaired this phosphorylation. Moreover, overexpression of the mutant S247A form of HIF-1α in mammalian and yeast cells, or inhibition of CK1, or silencing of CK1δ by siRNA, caused an increase in HIF-1 transcriptional activity in different cell lines. On the other hand, overexpression of the active form of CK1δ or overexpression of HIF-1α mutant form carrying the ‘phosphomimetic’ mutation S247D, inhibited the activity of HIF-1. Phosphorylation of Ser²⁴⁷ by CK1 did not affect HIF-1α protein expression levels, its stability or its subcellular localization. However, co-immunoprecipitation and in vitro binding assays showed that CK1 inhibition enhanced interaction of HIF1α with ARNT and that mutation of Ser²⁴⁷ affects the heterodimer complex formation. A structural model of the complex between the PAS-B domains of HIF-1α and ARNT showed that Ser²⁴⁷ is accessible to kinases and its phosphorylation reduces heterodimeric complex stability. To conclude, a new regulatory mechanism of HIF-1α by phosphorylation was identified. HIF-1α is phosphorylated at serine²⁴⁷ by CK1 and this modification inhibits HIF-1 activity by preventing the formation of the heterodimeric complex between HIF-1α and ARNT. This new mechanism can be a thepapeutic target in pathological conditions that involve hypoxia and HIF-1.Ο επαγόµενος από την υποξία παράγοντας, HIF-1 είναι ένας ετεροδιµερής µεταγραφικός παράγοντας που ρυθµίζει την απόκριση στην υποξία και εµπλέκεται σε παθολογικές καταστάσεις όπως ο καρκίνος, η ισχαιµία και η φλεγµονή. Η επαγόµενη υποµονάδα HIF-1α ρυθµίζεται από το οξυγόνο αλλά και από ανεξάρτητους µηχανισµούς. Σηµαντικό ρόλο στη ρύθµιση του HIF-1α παίζουν η υδροξυλίωση και η φωσφορυλίωση. Στην παρούσα διατριβή µελετήθηκε η φωσφορυλίωση του HIF-1α στην αµινοτελική του επικράτεια και ο ρόλος της στη δράση του HIF-1. Προκειµένου να βρεθεί η αλληλουχία φωσφορυλίωσης κατασκευάστηκαν µικρά ανασυνδυασµένα τµήµατα τoυ HIF-1α ως πρωτεΐνες σύντηξης µε GST και φωσφορυλιώθηκαν in vitro µε πυρηνικά εκχυλίσµατα κυττάρων HeLa. Η αλληλουχία φωσφορυλίωσης περιορίστηκε ανάµεσα στα αµινοξέα 229-251. Η ανάλυση της υπέδειξε την Ser²⁴⁷ ως πιθανό στόχο φωσφορυλίωσης από την κινάση της καζεΐνης 1, CK1. Από πειράµατα in vitro βρέθηκε ότι πράγµατι ο HIF-1α φωσφορυλιώνεται από τη CK1. Η µετατροπή της Ser²⁴⁷ του HIF-1α σε Ala (S247A) µέσω στοχευµένης µεταλλαξιγένεσης αλλά και χηµικοί αναστολείς της CK1 ανέστειλαν τη φωσφορυλίωση τόσο από τη CK1 όσο και τα πυρηνικά εκχυλίσµατα. Επιπλέον, η υπερέκφραση της µορφής S247A του HIF-1α σε κύτταρα θηλαστικών και ζύµης, η αναστολή της CK1 και η αποσιώπηση της CK1δ µε siRNA, αύξησαν τη µεταγραφική ενεργότητα του HIF-1 σε διαφορετικές κυτταρικές σειρές. Αντίθετα, η υπερέκφραση της ενεργής µορφής της CK1δ ή της µορφής του HIF-1α που περιέχει τη «φωσφοµιµητική» µετάλλαξη Ser²⁴⁷ σε Asp (S247D) ανέστειλαν τη δράση του HIF1. Η φωσφορυλίωση της Ser²⁴⁷ του HIF-1α από τη CK1 δεν επηρέασε την έκφραση και σταθερότητά του ούτε τον υποκυτταρικό του εντοπισµό. Ωστόσο, έπειτα από πειράµατα ανοσοκατακρήµνισης και in vitro δέσµευσης, παρατηρήθηκε ότι η αναστολή της CK1 ενίσχυσε την αλληλεπίδραση του HIF-1α µε τον ARNT και ότι η τροποποίηση της Ser²⁴⁷ επηρεάζει το σχηµατισµό του ετεροδιµερούς. Ένα δοµικό µοντέλο του συµπλόκου των περιοχών PAS-B του HIF-1α και του ARNT έδειξε ότι η Ser²⁴⁷ είναι προσβάσιµη και ότι η φωσφορυλίωσή της επηρεάζει αρνητικά τη σταθερότητα του ετεροδιµερούς. Συµπερασµατικά, βρέθηκε ένας νέος µηχανισµός ρύθµισης του HIF-1α µέσω της φωσφορυλίωσής στη Ser²⁴⁷ από τη CK1. Η φωσφορυλίωση αυτή αναστέλλει τη δράση του HIF-1 µέσω παρεµπόδισης του σχηµατισµού ετεροδιµερούς µεταξύ του HIF-1α µε τον ARNT. Ο νέος αυτός µηχανισµός µπορεί να αποτελέσει θεραπευτικό στόχο σε παθολογικές καταστάσεις στις οποίες εµπλέκεται η υποξία και ο HIF-1

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

International audienceDNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation

Tankyrase1/2 plays a role in RAD51 recruitment to DSBs and efficient HR.

<p><b>(A)</b> Cells depleted of TNKS1/2 show defective foci formation of RAD51. U2OS cells were treated with NCS and fixed 6 hours later. Frequency of RAD51 foci positive cells was determined in three independent experiments (N = 100). Results are shown as relative frequencies compared to control. <b>(B)</b> Representative confocal microscopy images corresponding to panel (A). <b>(C)</b> Quantification of RAD51 on pure DSBs in U2OS17 cells reveals a drop in siTNKS conditions. U2OS17 cells were transfected with the indicated siRNAs, and then co-transfected with mCherry-lacR and ISce-I. Cells were fixed and stained using an antibody against RAD51. Colocalization frequency of the lacR and the RAD51 signal was determined. Relative frequencies compared to the control are results of three independent experiments with SD. <b>(D)</b> Forced binding of TNKS1 to a DSB increases RAD51 recruitment efficiency. U2OS17 cells were transfected with GFP-lacR or GFP-lacR-TNKS1 and ISce-I. The percentage of cells having RAD51 signal on the array was determined after immunofluorescence staining. Results from three independent experiments are shown with SD (N = 100). <b>(E)</b> MDC1-mediated TNKS recruitment is necessary for efficient RAD51 binding to DSBs <i>in vivo</i>. U2OS17 cells were transfected with mCherry-lacR-MDC1 wt or its TBD mutant version and ISce-I. Percent of cells harboring RAD51 signal on the array was determined. Results from three independent experiments are shown with SD (N = 100). <b>(F)</b> HR efficiency is decreasing in TNKS1/2 depleted HRind cells measured in a stable cellular system [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005791#pgen.1005791.ref049" target="_blank">49</a>]. Results of three independent experiments are shown with SD. N>10⁵ cells were observed by FACS for GFP expression. Western blot analysis of rescue-efficiency is shown on the right. “res” stands for siRNA rersistant version of the construct, “mut” stands for mutation in the PARP activity.</p

TNKS loads the BRCA1A complex on chromatin.

<p><b>(A)</b> TNKS tethering to the lacO array leads to MERIT40 and RAP80 binding in a PARP activity independent manner even in the absence of DSBs. U2OS17 cells were transfected with GFP-lacR, GFP-lacR-TNKS1 or the PARP activity mutant version: GFP-lacR-TNKS1mut. ISce-I was co-transfected to induce pure DSBs at the lacO array. Immunofluorescence staining was performed to visualize the localization pattern of MERIT40 or RAP80 in the cells. The values represent the results of three independent experiments with SD (N = 100). On the right representative confocal microscopy pictures are shown. <b>(B)</b> The recruitment of MERIT40 and RAP80 is affected in TNKS1/2 knock down cells. U2OS17 cells were transfected with the indicated siRNAs. After depletion, cells were transfected with mCherry-lacR and ISce-I where indicated. Immunofluorescence staining was performed to visualize Merit40 or RAP80 in the cells. Relative values compared to the control are the results of three independent experiments with SD (N = 100 cells in each condition). <b>(C)</b> TNKS depleted cells are deficient for RAP80 foci formation. U2OS cells were transfected with siRNAs as shown and treated with NCS 48 hours later. The frequency of foci-positive cells was determined in three independent experiments (N = 100) and is shown as relative to the control <b>(D)</b> MDC1-mediated TNKS recruitment is necessary for efficient binding of the BRCA1 complex to DSBs <i>in vivo</i>. U2OS17 cells were transfected with mCherry-lacR-MDC1 wt or its TBD mutant version and ISce-I. Percent of cells harboring MERIT40 or RAP80 signal on the array was determined after immunofuorescence staining. Results from three independent experiments are shown with SD (N = 100) as relative frequencies compared to control. <b>(E)</b> MERIT40 stabilizes BRCA1 on the TNKS-bound chromatin. Control or MERIT40 depleted U2OS17 cells were transfected with GFP-lacR, GFP-lacR-TNKS1, GFP-lacR-TNKS1mut and ISce-I. Percent of cells harboring BRCA1 signal on the array was determined. Results from three independent experiments are shown with SD (N = 100).</p

TNKSs stabilize the BRCA1A complex at DSBs and activate the G2/M checkpoint.

<p><b>(A)</b> RNF8 mediated ubiquitination is dispensable for BRCA1A complex’s loading onto the chromatin. U2OS17 cells were transfected with siSCR control or siRNF8, and GFP-lacR, GFP-lacR-TNKS1 or its PARP activity mutant version. The frequency of MERIT40, RAP80 and BRCA1 signal on the lacO array was analyzed after immunofluorescence staining with the corresponding antibodies. Results of three independent experiments are shown with SD (N = 100). <b>(B)</b> RAP80 is recruited to TNKS1 bound chromatin in an ubiquitin-binding independent manner. U2OS17 cells were transfected with GFP-lacR or GFP-lacR-TNKS1 together with mCherry-RAP80 (wt or UIM mutant). Pure DSBs were inflicted with the cotransfection of ISce-I. Colocalization frequency of the GFP and mCherry signal is shown from three independent experiments (N = 100) with SD. <b>(C)</b> TNKS depleted cells show RAP80 foci-forming deficiency at later timepoints after DSB induction. U2OS cells were transfected with the indicated siRNAs, treated with NCS and fixed at the indicated timepoints. Results from three independent experiments are shown with SD (N = 100). <b>(D)</b> Knockdown of TNKS1/2 leads to G2/M checkpoint escape and elevated number of mitotic cells after DSB induction compared to the control. U2OS cells were transfected with the indicated siRNAs. 48 hours later cells were treated with Phleomycin and released for 6 hours. Mitotic cells were detected by an anti- histone H3 S10P fluorescence staining and their frequency determined. Values are represented on a Whisker box plot as relative to the control from three independent experiments. <b>(E)</b> Schematic action of TNKSs in DSB repair is shown.</p

Mapping of the Tankyrase interacting domain of MDC1.

<p><b>(A)</b> Confocal microscopy images of U2OS17 cells transfected with different mCherry-lacR-MDC1 domain constructs and flag tagged TNKS1 (FN-TNKS1). The contour of nuclei is shown. Colocalization of the two proteins can be observed in the merged images. <b>(B)</b> Quantification of the colocalization frequency of flag-TNKS1 (FN-TNKS1) or myc-TNKS2 (MN-TNKS2) and the different tethered MDC1 domains. Values represent mean ± SD from three independent experiments (N = 100 cells in each experiment). Statistical significance in all relevant Figs was calculated using the t-test (P<0,05 *, P<0,01 **, P<0,001 ***) <b>(C)</b> The PST repeats of MDC1 regulate the interaction between Tankyrases and MDC1. U2OS17 cells were transfected with different constructs expressing cherry-lacR fused versions of MDC1 (marked on the X axis) and Tankyrase (TNKS1 or TNKS2). % of cells having Tankyrase on the array was determined as for panel (B).</p

TNKSs recruit the CtIP-BRCA1 complex to chromatin.

<p><b>(A)</b> TNKS depletion leads to defects in CtIP loading and RPA phosphorylation. U2OS19 cells were transfected with the indicated siRNAs and pure DSBs were generated by transfecting ISce-I together with mCherry-lacR. Colocalization frequency of RPA-P or CtIP with the lacO array was established in 100 cells in three independent experiments. Values represent mean ± SD. <b>(B)</b> TNKS is required for the recruitment of the BRCA1 to pure DSBs <i>in vivo</i>. U2OS17 cells were transfected with the indicated siRNAs, and then co-transfected with mCherry-lacR and ISce-I. Immunofluorescence staining with an anti-BRCA1 antibody was performed and the colocalization frequency of the lacR and BRCA1 signal quantified. Relative values compared to the control are the results of three independent experiments with SD (N = 100 cells in each condition). <b>(C)</b> TNKS depleted cells are deficient for BRCA1 foci formation. U2OS cells were transfected with siRNAs as shown and treated with NCS 48 hours later. Cells were fixed 6 hours after treatment, immunofluorescence staining detecting BRCA1 was performed. The frequency of foci-positive cells was determined in three independent experiments (N = 100). Representative confocal microscopy pictures for BRCA1 staining are shown on the bottom. <b>(D)</b> TNKS tethering to the chromatin results in BRCA1 recruitment in a partially PARP activity dependent manner. U2OS17 cells were transfected with the indicated plasmids and immunofluorescence staining was performed to establish colocalization frequencies between GFP-lacR-TNKS1 and BRCA1. Representative confocal microscopy pictures are shown on the bottom. <b>(E)</b> MDC1-mediated TNKS recruitment is necessary for efficient binding of BRCA1 to DSBs <i>in vivo</i>. U2OS17 cells were transfected with mCherry-lacR-MDC1 wt or its TBD mutant version and ISce-I. Percent of cells harboring BRCA1 signal on the array was determined. Results from three independent experiments are shown as relative frequencies compared to control with SD (N = 100).</p

An association study between hypoxia inducible factor-1alpha (HIF-1α) polymorphisms and osteonecrosis.

Bone hypoxia resulting from impaired blood flow is the final pathway for the development of osteonecrosis (ON). The aim of this study was to evaluate if HIF-1α, the major transcription factor triggered by hypoxia, is genetically implicated in susceptibility to ON. For this we analyzed frequencies of three known HIF-1α polymorphisms: one in exon 2 (C111A) and two in exon 12 (C1772T and G1790A) and their association with ON in a Greek population. Genotype analysis was performed using PCR-RFLP and rare alleles were further confirmed with sequencing. We found that genotype and allele frequency of C1772T and G1790A SNP of HIF-1α (SNPs found in our cohort) were not significantly different in ON patients compared to control patients. Furthermore these SNPs could not be associated with the different subgroups of ON. At the protein level we observed that the corresponding mutations (P582S and A588T, respectively) are not significant for protein function since the activity, expression and localization of the mutant proteins is practically indistinguishable from wt in HEK293 and Saos-2 cells. These results suggest that these missense mutations in the HIF-1α gene are not important for the risk of developing ON

MDC1 interacts with Tankyrase 1 and Tankyrase 2.

<p><b>(A)</b> Schematic representation of MDC1 and its domains. The different deletion constructs used in our study are depicted below the full-length protein. The domains marked are: Forkhead associated domain (FHA), SDT-repeats, RNF8 Binding motif (RBM), PST repeats, BRCA1 C-terminal domains (BRCTs). We also placed three regions with so far unknown functions, named”inter I-II-III”. <b>(B)</b> Schematic representation of the two human Tankyrase proteins with their domains; the ankyrin repeats (ANK), the sterile alpha motif (SAM) and the PARP enzymatic domain (PARP). The clones obtained in our yeast-two-hybrid screen are depicted in black below the full-length proteins. <b>(C)</b> Endogenous MDC1 and TNKS1 interact <i>in vivo</i>. HEK 293T cells were treated or not with 100 ng/ml NCS and the cell extracts subjected to immunoprecipitation using an anti-MDC1 antibody. TNKS1 is detected in the immunoprecipitate and the interaction is enhanced after DSB induction. <b>(D)</b> Schematic representation of the tethering system used in our study. <b>(E)</b> Both TNKS1 and TNKS2 interact with chromatin bound MDC1 <i>in vivo</i>. Full length MDC1 was tethered to chromatin in U2OS17 cells (for the system used see panel D) while cells were co-transfected with tagged versions of either TNKS1 or TNKS2. Colocalization of FN-TNKS1 or MN-TNKS2 with mCherry-lacR-MDC1 is detected by immunofluorescence staining.</p