CCDC6 inhibits the phosphatase activity of PP4c.

<p>(<b>a</b>) PP4 complex immunopurified from HeLa cells transfected with CCDC6-specific shRNAs (shCCDC6) or with non-targeting control shRNAs (shCTRL), was incubated for 30 minutes at 30°C with pH2AX S139-enriched chromatin purified from irradiated cells. The phosphatase reactions were followed by western blot and probed with the indicated antibodies. (<b>b</b>) PP4c phosphatase was immunoprecipitated from shCCDC6 or shCTRL. 1, 0,7, 0,3 voulmes of total PP4c immunoprecipitated from 3 mg of total cell extract were mixed with 3 µg histones purified from cells exposed to 10 Gy IR and incubated in phosphatase buffer at 30°C for 30 minutes. Phosphatase reaction was terminated by the addition of 100 µl of Malachite Green solution and absorbance was measured at 630 nm. After the phosphatase assay, the actual amount of PP4c in each immunoprecipitate was determined by Western Blotting with the indicated antibody. PP4c activity is represented in arbitrary units (a.u.) calculated as the ratio between released free phosphate (absorbance at 630 nm) and PP4c densitometric signal at western blot. (<b>c</b>) Enzimatic activity of PP4c immunopurified from HeLa cells transfected with CCDC6-specific shRNA (shCCDC6) or with non-targeting control sh-RNAs (shCTRL) was assessed by Malachite Green phosphatase assay. 1, 0,7 and 0,3 volumes of total PP4c immunoprecipitated from 3 mg of total cell extract were incubated with 175 µM of RKpTIRR synthetic peptide for 30 minutes at 30°C. Phosphatase reaction was terminated by the addition of 100 µl of Malachite Green solution and absorbance was measured at 630 nm. After the phosphatase assay, the actual amount of PP4c in each immunoprecipitate was determined by Western Blotting with the indicated antibody. PP4c activity is represented in arbitrary units (a.u.) calculated as the ratio between released free phosphate (absorbance at 630 nm) and PP4c densitometric signal at western blot. (<b>d</b>) PP4 complex immunopurified from TPC-1 cells transfected with CCDC6 wt, CCDC6 (1–223) and (1–101) truncated mutants, was incubated for 30 minutes at 30°C with pH2AX S139-enriched chromatin, purified from irradiated cells. The phosphatase reactions were followed by immunoblotting and probed with the indicated antibodies. (<b>e</b>) (<b>f</b>) Enzimatic activity of PP4c immunopurified from TPC-1 cells transfected with epitope-tagged CCDC6 wt or empty vector, was determined as described in (<b>b</b>) and (<b>c</b>).</p

Loss of CCDC6 increases cell growth and confers resistance to genotoxic stress.

<p>(<b>a</b>) HeLa CCDC6 depleted clones were obtained after transfection of a plasmid pool of mission ShRNA (pLKo.1 puro ShCCDC6 NM_005436, Sigma-Aldrich) after two weeks puromycin selection. Immunoblot with anti CCDC6 and a-tubulin were shown. (<b>b</b>) CCDC6-depleted HeLa clones (shCCDC6 #1 and #2) and control HeLa cells (shCTRL) were plated at 10×10⁵/dish in triplicate and counted at the indicated times (<b>c</b>) Cell cycle distribution of a stable HeLa CCDC6 silenced clone (shCCDC6 #1) and control HeLa cells (shCTRL) after release from double thymidine block (TT-block) (<b>d</b>) CCDC6-depleted HeLa clones (shCCDC6 #1 and #2) and control HeLa cells (shCTRL) were plated at 10×10⁵, treated with the indicated doses of Etoposide and collected at 48 hours. The histograms are representative of three independent experiments and error bars indicate the standard error mean.</p

Loss of CCDC6 affects H2AX phosphorylation after DSBs.

<p>(<b>a</b>) In the WCL of two representative CCDC6-depleted HeLa clones (shCCDC6 #1 and #2) and control HeLa cells (shCTRL), thirty minutes after 1–5 Gy IR exposure, the phosphorylation of H2AX was detected with the mouse anti-pH2AX S139 by western blot. Anti-total H2AX was used as a loading control. The immunoblots with anti-CCDC6 and α-tubulin antibodies were shown in the bottom. (<b>b</b>) H2AX phosphorylation detection with mouse anti-pH2AX S139 by WCL analysis of CCDC6-depleted Hela clone #1 (shCCDC6) and control cells (shCTRL) at several time points as indicated after exposure to 1Gy of IR. Anti total H2AX is shown as loading control. (<b>c</b>) Immunofluorescence analysis of pH2AX S139 foci in CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL), thirty minutes after 1, and 5 Gy IR exposure. Nuclei were counterstained with DAPI. Magnification_ was at 63x. (<b>d</b>) Quantification of pH2AX S139 foci number. At least 300 cells were analysed per experiment. Error bars indicate the standard mean error. (<b>e)</b> CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL) transfected with expression vectors encoding CCDC6wt, CCDC6T434A or the empty vector were treated with etoposide at 1, 2,5 and 5 µM for 8 h and western blot analysis of pH2AX S139 and myc-tagged proteins were performed. (<b>f</b>) H2AX phosphorylation detection with mouse anti-pH2AX S139 by WCL analysis of CCDC6-depleted Hela clone #1 (shCCDC6) and control cells (shCTRL) at several time points as indicated after exposure to 1Gy of IR. Anti total H2AX is shown as loading control. The anti-pSer-1981-ATM and the ATM hybridization are shown at bottom of the figure.</p

CCDC6 in the genome stability control.

<p>(<b>a</b>) shCCDC6 and shCTRL HeLa cells were depleted of PP4c by shRNA (48 hours) and were exposed to 1 Gy of IR, as indicated (−/+). Phosphorylation of H2AX, PP4c, CCDC6, total H2AX and tubulin amount were revealed at IB of WCL. (<b>b</b>) In the cell extract of CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL), after double thymidine block (TT block) and release in presence of 1 µM Etoposide at several time point, as indicated, phosphorylation levels of H2AX and of RPA2 were revealed with anti-pH2AX S139 and with anti-p-RPA2 by western blot. Anti-total H2AX and anti-total RPA were shown as loading control. (<b>c</b>) Schematic diagram of CCDC6 function in modulating PP4c activity on the phosphorylation status of H2AX in the DNA-damage response.</p

Loss of CCDC6 affects the DNA damage induced G2 arrest.

<p>(<b>a</b>) Mitotic entry of the stable HeLa CCDC6 silenced clone #1 (shCCDC6) and control HeLa cells (shCTRL) after TT-block and release in 1 µM Etoposide for one hour where indicated, in presence of 50 ng/ml Nocodazole was monitored by western blot using the anti-p-S/T-MPM2 antibody. Sketch of the cells treatment is shown in the bottom panel. <b>(b)</b> Percentage of mitotic cells was monitored, by FACS analysis, with anti-p-Ser10-histone H3 staining, in stable CCDC6 silenced and control HeLa cells treated as in (a) at 8 hours, as indicated. <b>(c)</b> in HeLa CCDC6 silenced clone #1 (shCCDC6) and control HeLa cells (shCTRL) growth on coverslips and collected at several time points following G1/S syncronization by double thymidine block (TT-block) in the presence of 1 µM Etoposide, as indicated, mitotic figures were counted after nuclear counterstaining with Dapi. Magnification_ was at 40x. The histograms are representative of three independent experiments and error bars indicate the standard error mean. (<b>d</b>) After TT-block and release in 1 µM Etoposide stable HeLa CCDC6 silenced clone #1 (shCCDC6) and control HeLa cells (shCTRL) were collected at several time points as indicated. Checkpoint activity was monitored by western blot using the anti-pSer317-chk1 antibody. Total chk1 is shown at bottom of the figure.</p

Loss of CCDC6 affects DSBs repair.

<p>(<b>a</b>) Detection of DSBs by PFGE. After 10 Gy IR exposure CCDC6-depleted (shCCDC6) and CCDC6-proficient (shCTRL) HeLa cells have been collected at different time points (1, 2, 4, 24 hours). Densitometric analysis of DSBs bands were plotted as percentage of total DNA. <b>(b</b>) The percentages of GFP positive cells, compared to controls, have been plotted on the histograms that are representative of three independent experiments. Error bars indicate the standard error mean. The anti-HA-I-Sce1 and anti-tubulin immunoblots are shown at bottom of the figure. <b>(c</b>) HeLa cells, bearing the doxycycline-inducible I-Sce1 DNA repair construct, have been transfected with control shRNAs (shCTRL) or sh-CCDC6 by Microporator MP-100 transfection system (Digital Bio, Korea). The CCDC6 protein depletion was assessed by western blot analysis for every rate of transfection (#1, #2, #3, #4). The percentages of protein expression, compared to controls, have been plotted on the histogram below.</p

Identification of Sumoylation Sites in CCDC6, the First Identified RET Partner Gene in Papillary Thyroid Carcinoma, Uncovers a Mode of Regulating CCDC6 Function on CREB1 Transcriptional Activity

<div><p>CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424) which are highly conserved in vertebrates. We demonstrate that the post-translational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.</p> </div

CCDC6 is modified by SUMO1 and SUMO2 in vitro.

<p>A) and B): 200 nM of CCDC6 were incubated with recombinant E1, E2 and either SUMO1 or SUMO2 in presence of Mg-ATP solution. In control reactions the Mg-ATP was omitted. Detection of SUMO1- and SUMO2- CCDC6 conjugates at western blot are indicated by arrows. Immunoblot anti-CCDC6 and anti-Tubulin are shown as loading controls. C) Endogenous CCDC6 is conjugated to SUMO1 and to SUMO2: HEK293 cells were transfected with plasmids expressing His-tagged SUMO2 or SUMO1. SUMO-conjugated proteins were isolated by nickel affinity chromatography and separated by SDS-PAGE. Endogenous CCDC6 was detected by immunoblotting using anti-CCDC6 antibody (top panel). Star (*) indicates the unsumoylated CCDC6 bands. The His SUMO1 and His-SUMO2 were revealed by anti-His hybridization. The expression of CCDC6 in crude extract was also detected using an anti-CCDC6 antibody (right panel).The filter was stripped and reprobed using anti-his antibody to assess comparable amount of His-SUMO proteins. The anti-tubulin is shown as loading control. Mock in C indicates mock transfection control.</p

SUMO2 limits CCDC6 inhibition on CREB1 transcriptional activity.

<p>A) ChIP was performed in B-CPAP cells, which express endogenous CCDC6 protein. The cells were crosslinked and immunoprecipitated with anti-CCDC6. The precipitated DNA was subjected to qRT-PCR with specific primers, which amplify CRE element of human AREG promoter. B) CRE promoter activity was analysed in B-CPAP cells in presence of CREB1, CCDC6 and SUMO2 expression vectors. Luciferase activity was determined 24 hours after transfection. All transfections were performed in duplicate; C) the same as in B, except that the CRE promoter activity analysed in B-CPAP cells was revealed in presence of CREB1, SUMO2 and CCDC6 wild type (WT) or triple mutant KKK74, 266, 424RRR (TM). Data are mean +/− SD of three independent experiments. D) E) CCNA2 and AREG relative expression in PC CL3 cells, starved for 24 hours and treated with 10 µM Forskolin for 6 hours, analysed by qRT-PCR. Data are the mean +/− SD of three independent experiments. Transfections normalization is shown at bottom of B, C, D, and E. In C, D and E, p values are shown for each panel.</p

Identification of SUMO modification sites in CCDC6.

<p>A) Schematic representation of the three putative consensus sumoylation sites (L<b>K</b><i>X</i>E) in human CCDC6, as revealed by the SUMO-plot Analysis program (<a href="http://www.abgent.com/tools/" target="_blank">www.abgent.com/tools/</a>). B) HEK293 cells were transfected with Myc-CCDC6 wt, myc-CCDC6K74R, myc-CCDC6K266R, myc-CCDC6K424R, myc-CCDC6 TM (KKK74, 266, 424RRR) and FLAG-SUMO2. Whole cell lysates were prepared and equal amount of proteins were immunoprecipitated with anti-myc antibody. Then, the immunocomplexes were analysed by western blotting using the anti-FLAG antibody. Immunoblotting with anti-myc showed comparable amount of myc- CCDC6 transfected proteins. On the right of the same panel in the WCL the anti-Flag and the anti-myc hybridization are shown. C) HeLa YFP-SUMO2 cells were transfected with pECFP-CCDC6 wt or pECFP-CCDC6TM. FRET was measured by using the acceptor photo-bleaching technique as described in materials and methods. Briefly, CFP fluorescence images were recorded before and after 2 minutes of photobleaching of YFP fluorescence by 514 nm laser line. FRET efficiency (measured as increase of pre-bleach CFP fluorescence after YFP photobleaching in cytosolic random regions of interest [ROIs, little squares]) is expressed in % as mean of 3 independent experiments. Error bars, +/− SD. * p≤0,001. The images magnification (large square) shows an example of ROIs. To note that in the nucleus there was no energy transfer between the two fluorophors.</p