CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity

CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity Valeria Catena, Tiziana Bruno, Simona Iezzi, Silvia Matteoni, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-459016/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2021 Read the published version in Journal of Experimental & Clinical Cancer Research → Version 1 posted 11 You are reading this latest preprint version Abstract Background Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in Multiple Myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding. Methods Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three-serine residue (Ser 316 , Ser 320 and Ser 321 ) in 308–325 AA region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser 316 , Ser 320 and Ser 321 were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis. Results Here, we confirm that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser 316 , Ser 320 and Ser 321 and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation. Conclusions Che-1 phosphorylation at Ser 316 , Ser 320 and Ser 321 by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability. Cancer Biology Systems and Networking Laboratory Diagnostics Che-1 cancer cells cell proliferation histone acetylation phosphorylation CK2 SV40 LT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Che-1/AATF (Che-1) is a highly conserved nuclear protein overexpressed in several cancerous tissues, such as prostate, lung, colon, lymphomas as well as in multiple myeloma (MM) and in hepatocellular carcinoma (HCC), where its levels increase during disease progression ( 1 – 5 ). It has been previously demonstrated Che-1’s involvement in important cellular activities, including proliferation, regulation of cell cycle, apoptosis and stress response ( 6 – 10 ). Many studies have highlighted the ability of Che-1 to affect the transcriptional machinery by its association with several proteins, such as RNA Polymerase II, the retinoblastoma protein (Rb), p65 and STAT3 ( 7 , 11 , 12 ). Importantly, Che-1 contains an acidic region spanning from 308 to 325 aa highly homologous to HDAC proteins and several viral oncoproteins (SV40 Large T, E1A) by which it contacts Rb, thus inhibiting its binding to HDAC 1 and promoting cell proliferation ( 11 , 13 ). More recently, we have demonstrated that this region is required for displacing HDAC proteins from histones, increasing chromatin accessibility and MM cell proliferation ( 14 ). Che-1 half-life and functions are modulated by several post-translational modifications ( 12 , 15 , 16 ), and its phosphorylation at specific serine or threonine residues has been associated with Che-1 activity in response to different stimuli, including induction of DNA damage or apoptosis ( 6 , 17 ). Casein kinase II (CK2) is a ubiquitous highly pleiotropic serine/threonine protein kinase present in cells in a tetrameric form consisting of two catalytic subunits (CK2α and its isoform CK2α’) and two regulatory β subunits ( 18 ). It is expressed in all tissues of eukaryotic organisms and localized in different cell compartments ( 19 ). Due to its ability to phosphorylate a wide variety of substrates, CK2 is involved in many cellular pathways, including cell growth, proliferation and survival ( 20 – 22 ). Several studies highlighted that CK2 is up-regulated in different tumors, including kidney, head and neck, lung and prostate ( 23 ) and it is considered an attractive target to counteract drug resistance in cancer ( 18 ). Indeed, a rising number of CK2 inhibitors have been developed and their effectiveness proven alone or in combination with conventional anticancer drugs supporting their possible application in clinical therapy ( 18 ). Moreover, several studies have demonstrated that this kinase can interact with and regulate many factors involved in chromatin remodelling ( 24 , 25 ). In this study, we demonstrate that Che-1 ability to promote histone acetylation and cell proliferation is not only restricted to MM cells, and that the phosphorylation at three serine residues, Ser 316 , Ser 320 and Ser 321 , is required for these activities. Notably, we identified CK2 kinase as the enzyme responsible for these modifications. Finally, we produce evidence that also in the SV40 Large T protein the phosphorylation of conserved serine residues, within the region of homology with Che-1, is required for its activity, thus underlining the importance of these modifications in the regulation of cell proliferation. Methods Cell culture and transfections Human HCT116, HeLa, 293T and U2OS cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Euroclone) high glucose supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 2 mM glutamine (Thermo Fisher Scientific) and 40 µg/ml gentamicin. All cell lines were cultured at 37°C, in a humidified atmosphere with 5% CO 2 . Mycoplasma contamination was periodically tested by RT–PCR analysis, using the following primers: Forward: 5’ - ACT CCT ACG GGA GGC AGC AGT A − 3’ Reverse: 5’ - TCG ACC ATC TGT CAC TCT GTT AAC − 3’ Transfection experiments were carried out by using Lipofectamine 3000 Transfection System (Thermo Fisher Scientific) following the manufacturer's instructions. Cells were analysed 48 h after transfection by western blot (WB) or immunofluorescence. Cell number and viability were assessed by Trypan Blue staining using Countess automated cell counter (Thermo Fisher Scientific). Recombinant plasmids and reagents Myc-Che-1 has already been described ( 11 , 26 ). pCI-HDAC 1 was a kind gift from Dr. Sartorelli, while pSG5 Large T (pSG5 SV40 LT) plasmid was a gift from William Hahn (Addgene plasmid #9053; http://n2t.net/addgene:9053 ; RRID: Addgene_9053). Myc-Che-1 3S and pSG5 SV40 LT 3S were generated by in vitro mutagenesis using the QuikChange site-directed Mutagenesis system (Agilent Technologies) following the manufacturer’s instructions. PCR reactions were achieved using the following primers: Myc- Che-1 3S: Forward 5’ - GCCCAATGCGGGA GGT GAGGAGATT GCTGGT GAAGATGATGAGC − 3’ Reverse 5’ - GCTCATCATCTTCACCAGCAATCTCCTCACCTCCCGCATTGGGC − 3’ pSG5 SV 40 LT 3S: Forward 5’ - AACCTGTTTTGC GCA GAAGAAATGCCA GCTGGT GATGATGAGGCT − 3’ Reverse 5’ - AGCCTCATCATCACCAGCTGGCATTTCTTCTGCGCAAAACAGGTT − 3’ All mutations were confirmed by sequencing realized by Eurofins Genomics. Stealth siRNA oligonucleotides targeting Che-1 (siChe-1), CSNK2A (siCK2), control sequence (siControl) and custom Che-1 3’UTR (sense 5’-CCCGCCUUUAAACGCCACAAAUAAA-3’; antisense 5’-UUUAUUUGUGGCGUUUAAAGGCGGG-3’) were purchased from Thermo Fisher Scientific. TBB (4,5,6,7 – Tetrabromobenzotriazole) was purchased from SelleckChem. Casein kinase II (CK2 - P60105) recombinant protein and Adenosine 5’-triphosphate (ATP - P07565) were purchased from New England BioLabs. Cell extracts, immunoprecipitation and RNA isolation Total, cytoplasmic and nuclear extracts were prepared as previously described ( 12 , 27 , 28 ). For immunoprecipitation (IP) experiments, nuclear extracts were resuspended in dilution buffer (50 mM TRIS pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 0.5% NP40, protease and phosphatase inhibitors). Dynabeads magnetic beads (Thermo Fisher Scientific) were incubated for 1 h at room temperature on a rotating wheel with the indicated antibodies. Nuclear extracts were then added to the beads and the incubation was continued for 2 additional hours. Immuno-complexes were purified by using a magnet (DynaMag-2, Thermo Fisher Scientific), the beads were washed five times with dilution buffer and eluted in 4X lithium dodecyl sulphate (LDS) sample buffer (Thermo Fisher Scientific) for WB analysis. Detailed information for all antibodies is provided in Supplementary Table S1. Total RNA was isolated from cells using EuroGOLD TriFast reagent (Euroclone) according to the manufacturer's instructions. The amount of RNA was measured by using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Western blot analysis Samples were prepared as previously described ( 27 ), resolved on Bolt pre-cast 4–12% gels (Thermo Fisher Scientific), run using Bolt MES SDS running buffer (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes. After a blocking step in 5% non fat-dried milk in phosphate-buffered saline (PBS)-0.1% Tween, membranes were incubated with primary antibodies overnight at 4°C. After three washes in PBS − 0.1% Tween, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad Laboratories) for 45 min at room temperature, washed with PBS-0.1% Tween and analysed by chemi-luminescence (GE Healthcare Life Science). Images were acquired and quantified using Alliance Mini HD6 system by UVITEC Ltd, Cambridge, equipped with UVI1D Software (UVITEC, 14–630275). Detailed information for all antibodies is provided in the Supplementary Table S1. Immunofluorescence Detection of both heterochromatic foci and chromatin modifications was performed as described in Bruno et al. ( 14 ). Briefly, HCT116 cells were grown on cover-lips, washed twice in PBS, fixed in 100% methanol for 5 min and then permeabilized for one hour with 1% BSA, 10% normal goat serum, 0.3 M glycine in 0.1% PBS-Tween. Cells were incubated with anti-H3K9me3 overnight at 4°C and stained 45 min with Alexa-Fluor-594-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific). For anti-Myc tag and anti-Che-1 immunofluorescences, cells were fixed in 4% formaldehyde for 10 min and then permeabilized with PBS − 0.2% Triton X-100 for 5 min at room temperature. Cells were stained for two hours with the primary antibody, rinsed three times with PBS and stained respectively with Alexa-Fluor-594-conjugated anti-mouse or with Alexa-Fluor-488-conjugated anti-rabbit secondary antibodies (Thermo Fisher Scientific) 45 min at room temperature. Nuclei were visualized by staining with 1 µg/ml Hoechst dye 33258 (Sigma-Aldrich) for 10 min. Images were acquired using a fluorescence microscope with a 40X objective (Zeiss, Germany) and processed with AxioVision 4.7.1 software. In vitro kinase assay GST-Che-1 fusion protein was cloned into pGEX-4T-3 vector as already described ( 12 ). For in vitro kinase assay, GST-Che-1 was incubated with or without CK2 constitutively active for 1 h at 30°C in slowly rotation on a rotating wheel in CK2 Reaction buffer (New England Biolabs) in presence of 200 µM ATP. Adding 4X LDS stopped reactions and the proteins were resolved by Bolt pre-cast 4–12% gels. The gel was stained with SimplyBlue SafeStain (Thermo Fisher Scientific) for three hours at room temperature, and bands of interest were cut and then analysed by Nano - LC Mass spectrometry (MS). Nano - LC Mass spectrometry After kinase assay the bands of interest were cut from the gel, de-stained, reduced, alkylated and digested with trypsin following the procedure described by Shevchenko et al. ( 29 ). The analysis of tryptic peptides was carried out by mean of nano-HPLC-MS/MS using an Ultimate 3000 nano-HPLC system and the obtained data were processed using an in-house software. The peptide pellets were re-suspended immediately before analysis and firstly loaded, from the sample loop, onto a trapping column (Acclaim PepMap C18; 2 cm × 100 µm × 5 µm, 100 Å- Thermo Fisher Scientific) using the loading eluents composition (95 − 5% ACN/H2O + 0.05% trifluoroacetic acid). The separation of peptides was performed at the flow rate of 300 nL/min and at a temperature of 35°C using an Easy spray column (15 cm x75 µm PepMap C18 3 µm, Thermo Fisher Scientific). A linear gradient of solution B (95 − 5% ACN/H2O + 0.08% formic acid) from 4–95% in 55 min was applied. All the analyses were performed in the positive ion mode. Single MS survey scans were performed in the Orbitrap, recording a mass window between 395 and 2000 m/z using a maximal ion injection time of 100 ms. The resolution was set to 70,000 and the automatic gain control was set to 3x10 6 ions. The experiments were performed in data-dependent acquisition mode with alternating MS and MS/MS experiments. For MS/MS analysis an isolation window of 2Da was used. CID was done with a target value of 5000 ions, a maximal ion injection time of 50ms, normalized collision energy of 35%. Raw MS files were processed with the Thermo Scientific Proteome Discoverer software version 1.4. Peak list files were obtained by the SEQUEST search engine against the Human protein database containing both forward and reversed protein sequences. The resulting peptide hits were filtered for a maximum 1% FDR (false discovery rate) using the percolator tool. The database search parameters were: mass tolerance precursor 20ppm, mass tolerance fragment CID 0.8Da, dynamic modification of deamidation (N, Q), oxidation (M) and static modification of alkylation with IAM (C). Phosphorylation of Ser were set as variable modifications. The software phosphoRS was used to validate the correct assignment of the phosphorylation sites. In any case, the option trypsin with two missed cleavages was selected ( 30 ). Identification by nano–HPLC–ESI-MS/MS The data obtained by the HPLC separation and MS/MS analyses, carried out on digested samples, were submitted to the SEQUEST search engine against Uniprot Human protein database using the phosphoRS tool. This approach allowed the specific identification of Tau-F of Apoptosis-antagonizing transcription factor (AATF/Che-1) . Fifty-three high confidence peptides, corresponding to sequence coverage of 65.36% and high sequest score of 2391.39, were identified. From the MS/MS analysis it was possible to identify four phosphopeptides derived from Che-1, three of which were unambiguously assigned as phosphopeptides (Table 1). In a few cases it was not possible to assign the phosphate to specific serine residue(s), thus hindering the precise localization of the phospho-residue(s). The analysis of peptides obtained from Che-1 protein untreated allowed the identification fifty-four high confidence peptides corresponding to sequence coverage of 64.64% and high sequest score of 2082.81. As expected no phosphopeptides were found in this sample. 2D Gel electrophoresis For 2D-Gel, cell pellets were resuspended in 2-D lysis buffer (2 M thiourea, 7 M urea, 4% CHAPS, 1% DTT, protease inhibitors cocktail) and sonicated. Total extracts were treated, where indicated, with Lambda Protein Phosphatase (ʎ-PP) (New England BioLabs) following the protocol described in Yagamata et al., with minor modifications ( 31 ). In short, cells pellets were re-suspended and 20 mM MnCl 2 was added to 150 µg of protein extract in order to obtain a final concentration of 2 mM ʎ-PP buffer and brought to a final volume of 20 µl with deionized water. The mixture was divided into two aliquots and 200 units of ʎ-PP were added to one of the aliquots. After mixing, aliquots were incubated overnight at 30°C under constant shaking and subsequently 100 µl of 2-D lysis buffer was added togheter with 1% sulfobetaine SB 4–7. Isoelectric focusing was performed using 70 mm 4.0–7.0 linear Immobilized pH Gradient gel strips (IPG strips) (Bio-Rad Laboratories) on an electrophoresis unit (Ettan IPGphor 3; GE Healthcare Life Science). Equal amounts of proteins (70 µg) were loaded by passive in-gel rehydration for 12 hours then run as follows: 1 hour at 50 V, 30 min at 200 V, voltage gradient 30 min up to 1,000 V, 20 min at 1,000 V, voltage gradient 30 min up to 5,000 V and 1hour at 5,000 V. Before performing the 2nd electrophoresis dimension, IPG strips were equilibrated for 15 min at room temperature in 1% DTT to reduce the proteins, and sulfhydryl groups were subsequently derivatized using 4% iodoacetamide (both solutions were prepared in 50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 2% bromophenol blue) for 15 min. Strips were transferred to 1.0-mm-thick 4–12% pre-casted polyacrylamide mini-gels (Thermo Fisher Scientific) for the 2nd electrophoresis dimension and then gels were run at 130 V for 2 h. 2D gels were then transferred onto PVDF filters. In vitro GST pull down assay Full length H3 open reading frame was cloned into EcoRI/XhoI sites of pGEX-4T-1 in order to generate GST-H3 fusion protein. GST-H3 or GST control proteins were prepared following standard procedures and used for in vitro binding assay. Briefly, GST proteins were pre-incubated with Che-1 308–325, Che-1 3S or control peptides (Biomatik Corporation) for 1 h at 4°C on a rotating wheel in interaction buffer (20 mM Tris pH 7.9, 300 mM NaCl, 0.2 mM EDTA, 0.1% NP40, 10 µg/ml BSA). Total extracts from HCT116 cells over-expressing pCI-HDAC 1 were then added to the GST/peptides complexes and the incubation was continued for three additional hours. Samples were eluted in 4X LDS sample buffer by heating for 10 min at 70°C and then subjected to WB analysis. Peptides sequence commercially synthesized are: Control: LKGAKPIRPVVVKAPPA Che-1 308–325: DGTKPNAGSEEISSEDDEL Che-1 3S: DGTKPNAGAEEIAAEDDEL Micrococcal nuclease assay Micrococcal nuclease (MNase) assay was performed using the protocol described in Zaret et al. ( 32 ). Briefly, HCT116 cells were seeded in 100mm plate and transiently transfected with Myc-Che-1 wt, Myc-Che-1 3S or empty vector. 24 h post transfection cells were subjected to the protocol described in Bruno et al. ( 14 ). Quantification and Statistical Analysis Data are presented as the mean of three independent experiments ± standard deviation (SD). Statistical analyses were performed using R software. Two-tailed Student's t -tests with Benjamini–Hochberg correction were performed to compare one parameter between two groups. For the multiple group comparison, one-way ANOVA with Tukey HSD test was used. P < 0.05 was considered significant. Statistical significance is indicated by asterisks as follows: * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001, n.s. = not significant. Results Che-1 depletion reduces proliferation in several cancer cell lines As previously observed, Che-1 displayed to have a key role in MM cells proliferation ( 14 ). To assess whether this effect could also be observed in other tumor cells of different origins, we performed cell proliferation analysis in HCT116, HeLa, 293T and U2OS cells, depleted or not for Che-1 expression. As shown in Fig. 1 A, Che-1 inhibition produced a significant reduction of cell proliferation when compared to control cells in all tested cell lines. Since Che-1 depletion induces heterochromatin accumulation in MM cells ( 14 ), we evaluated whether this was a general phenomenon. To this aim, we analysed the formation of heterochromatin foci by immunofluorescence in HCT116 cells, observing that Che-1 depleted cells exhibited a strong increase of Hoechst-positive heterochromatic foci (from 9 to 32 foci/cell) (Fig. 1 B). Consistent with these findings, WB analysis showed that Che-1 knockdown produced a significant increase of H3K9me3 levels, an important hallmark of heterochromatin (Fig. 1 C). These results were confirmed through immunofluorescence experiments in which a marked increase of H3K9me3 positive dots was observed following Che-1 downregulation (Fig. 1 D). It has been shown that Che-1 depletion produces a global reduction of histone acetylation in MM cells ( 14 ). To extend these findings, we performed WB analyses in several tumor cell lines depleted or not for Che-1 expression, observing that the loss of Che-1 expression produced a strong reduction in both H3 and H4 histone acetylation (Fig. 1 E). In line with the obtained results, we observed a reduction of about 50% of total RNA in Che-1 depleted cells when compared to control cells (Fig. 1 F). Overall, these findings reinforce the role of Che-1 in sustaining cell proliferation by maintaining an active state of chromatin. Che-1 is highly phosphorylated Che-1 affects multiple cellular processes thanks to its ability to interact with several transcription factors ( 7 , 11 ). These interactions are modulated by post-translational modifications in response to different stimuli, including phosphorylation at specific serine or threonine residues ( 6 ). In this context, we have previously shown that Che-1 contains a highly acidic sequence (308–325 aa) involved in Che-1-histone binding. Indeed, Che-1 mutant lacking this region was no longer able to bind histone H3 ( 14 ). Interestingly, in this region are present three highly conserved serine residues (in bold; Fig. 2 A), whose phosphorylation has been reported by many phospho-proteomic studies ( 33 – 35 ). To investigate the relevance of this Che-1 region, we performed a 2D gel electrophoresis analysis of total extracts from HeLa cells treated or not with lambda phosphatase (λ-PP), and observed that Che-1 is constitutively and highly phosphorylated (Fig. 2 B). Next, we produced a Che-1 mutant (Che-1 3S) in which the serine residues in position 316, 320 and 321 were replaced with glycine or alanine (Fig. 2 C). A bidimensional gel electrophoresis analysis of HeLa cells over-expressing Che-1 wild type (Myc-Che-1) or 3S mutant (Myc-Che-1 3S) showed that this mutant was less phosphorylated than wild-type (wt) protein (Fig. 2 D), indicating that these residues contribute significantly to total protein phosphorylation. Next, we started to characterize the functional role of these modifications investigating whether the 3S mutant had a different cellular localization. Immunofluorescence experiments showed that the mutant retained the peculiar nuclear signal of the wt protein (Fig. 2 E). In agreement with these data, a subcellular fractionation of HCT116 cells transiently transfected with Myc-Che-1 wt or 3S mutant, detected a strong accumulation in the nuclear fraction of both molecules (Fig. 2 F). Che-1 phosphorylation is required for its pro-proliferative ability The data shown above demonstrate that three serine residues contained in the 308–325 region are phosphorylated. This region plays an important role in Che-1's functions ( 11 , 14 ). To shed light on the relevance of these phosphorylations, we evaluated the effect of the over-expression of Myc-Che-1 wt or 3S mutant. As shown in Fig. 3 A and Supplementary Fig. 1A, Myc-Che-1 3S over-expression in HCT116 cells did not induce cell proliferation when compared to Myc-Che-1 wt. These results were confirmed in MM cells (KMS27 cells) and in other cell lines (Supplementary Figs. 1B-D). The relevance of these three serine residues in the pro-proliferative role of Che-1 was further confirmed by evaluating the effect of the overexpression of these molecules on the levels of cyclin B1. In fact, as shown in Fig. 3 B, only Myc-Che-1 wt was able to increase the levels of this protein. Next, we verified whether the over-expression of Myc-Che-1 3S had any impact on global transcription by measuring the level of total RNA. As shown in Fig. 3 C, the Myc-Che-1 3S mutant was completely unable to activate transcription. In order to evaluate whether Che-1 phosphorylation in Ser 316 , Ser 320 and Ser 321 was required to sustain histone acetylation, we analysed the effect of Myc-Che-1 wt or 3S mutant over-expression on H3 and H4 histone acetylation. As shown in Fig. 3 D, Myc-Che-1 3S mutant showed no effect on histone acetylation and remarkably it was unable to rescue the effects of endogenous Che-1 depletion (Fig. 3 E). These results prompted us to investigate the ability of the Che-1 3S mutant to interact with histone H3. To this aim, we performed co-immunoprecipitation experiments with anti-Myc antibody using nuclear extracts from HCT116 cells over-expressing Myc-Che-1 wt or its 3S mutant. As shown in Fig. 3 F and in Supplementary Fig. 1E, the replacement of the three serine residues prevented Che-1/histone H3 interaction. Moreover, an in vitro competition assay demonstrated that only the Che-1 308–325 peptide containing phosphorylated serines was able to displace HDAC 1 from histone H3 (Fig. 3 G). Notably, KMS27 cells transfected with Che-1 308–325, but not with Che-1 3S peptide, displayed a reduction in cell proliferation (Supplementary Fig. 1F). Consistent with these results, an MNase susceptibility assay performed in HCT116 cells transiently transfected with Myc-Che-1 wt or 3S mutant demonstrated that the mutant overexpression produced a more compacted chromatin structure (Fig. 3 H). Overall, these results clearly indicate that the presence of the three serine residues in 308–325 aa region represents an essential requirement for Che-1 activity, promoting the Che-1/histone binding and histone acetylation. Che-1 and SV40 Large T antigen display similar functions SV40 LT is a multifunctional viral oncoprotein implicated in a wide range of cellular processes including transcriptional activation and repression, stimulation of the cell cycle and cell transformation ( 36 ). Interestingly, SV40 LT expression increases global histone acetylation and CREB-binding protein (CBP) histone acetyltransferase (HAT) activity ( 36 ). As already described, Che-1 308–325 aa region is highly conserved among mammalian species and shared with several viral and HDAC 1–3 proteins ( 11 ). Among viral oncoproteins, SV40 Large T antigen (SV40 LT) contains three particular serine residues, in position 106, 110 and 111, corresponding to those of Che-1 (in red; Fig. 4 A). These observations prompted us to verify whether SV40 LT phosphorylation on Ser 106 , Ser 110 and Ser 111 residues, was required for its ability to regulate cellular proliferation. To this aim, we produced a SV40 LT mutant (SV40 LT 3S) in which Ser 106 , Ser 110 and Ser 111 residues were replaced with alanine or glycine (Fig. 4 B). Notably, HCT116 cells over-expressing SV40 LT 3S mutant didn’t exhibit the induction of cellular proliferation observed in SV40 LT wt cells (Fig. 4 C). Furthermore, this mutant was unable to induce the histone acetylation increase observed in cells transfected with SV40 LT wt (Fig. 4 D). Since Che-1 phosphorylation in the 308–325 region is required for the interaction with histone H3 (Fig. 3 F), we evaluated whether SV40 LT wt was also able to interact with histone H3 and whether phosphorylation of Ser 106 , Ser 110 and Ser 111 residues was involved in this binding. To this aim, we performed co-immunoprecipitation experiments with anti-SV40 antibody in nuclear extracts from HCT116 transiently transfected with SV40 LT wt or its 3S mutant. Remarkably, we observed that the SV40 LT wt protein showed the ability to bind histone H3 whereas, in line with results obtained with Che-1, the mutant lost this ability (Fig. 4 E). Taken together, these data reinforce the importance of this highly acidic region in several proteins for binding to histones and cell proliferation. CK2 phosphorylates Che-1 Next, we focused on identifying the kinase responsible for the phosphorylation of Che-1 in Ser 316 , Ser 320 and Ser 321 residues. Previous studies have demonstrated that protein kinase Casein Kinase II (CK2) phosphorylates SV40 LT at Ser 111 and Ser 112 producing an augment of 50-fold of the rate of nuclear import of its protein ( 37 ). In the Che-1 308–325 aa region, two consecutive CK2 consensus sites (S/T-x-x-D/E/pS) are located in the proximity of its NLS ( 38 , 39 ). To investigate whether Che-1 could be a new CK2 target, we started testing if Che-1 physically interacts with CK2. As shown in Fig. 5 A and Supplementary Fig. 2A, immunoprecipitation experiments revealed that Che-1 co-precipitates with CK2. Next, we analysed the role of CK2 in Che-1 phosphorylation by performing 2D-electrophoresis experiments in HeLa cells treated or not with the CK2 inhibitor, 4,5,6,7–Tetrabromobenzotriazole (TBB). From this experiment, we observed a strong decrease in Che-1 phosphorylation in the absence of CK2 kinase activity (Fig. 5 B and Supplementary Fig. 2B). Consistent with these findings, the depletion of CK2 expression by using specific siRNA stealth oligonucleotides (siCK2) induced a remarkable reduction of Che-1 phosphorylation when compared to control cells (Fig. 5 C and Supplementary Fig. 2C). Next, we sought to determine the possibility that Che-1 was a direct substrate of CK2. To this aim, GST-Che-1 fusion protein was incubated or not with recombinant CK2 enzyme and subjected to in vitro kinase assay. The kinase reaction products were analysed by nano-HPLC-MS/MS and the results obtained were then submitted to the SEQUEST search engine against the Uniprot Human protein database using the phosphoRS tool. This approach allowed identifying four Che-1 phosphopeptides, including those containing Ser 316 , Ser 320 and Ser 321 (Table 1 and Supplementary Fig. 2D). As expected, no phosphopeptides were detected when Che-1 was incubated in the absence of CK2 (data not shown). Next, we verified the effect of CK2 silencing on Che-1 cellular localization by performing immunofluorescence experiments. As shown in Fig. 5 D and Supplementary Fig. 2E, the absence of protein kinase does not affect nuclear localization of Che-1. Then, in order to confirm the role of these phosphorylations in Che-1 activity, we tested whether the absence of CK2 could affect Che-1/histone H3 binding. Co-immunoprecipitation experiments in HCT116 cells treated or not with TBB, demonstrated that the inhibition of CK2 kinase activity induces a drastic reduction of histone H3 interaction with Che-1 (Supplementary Fig. 2F), and similar results were obtained following CK2 depletion (Fig. 5 E). Altogether, these data clearly demonstrate that CK2 phosphorylates Che-1 at Ser 316 , Ser 320 and Ser 321 , and that these modifications are essential for Che-1 activity. Discussion Thanks to its ability to interact with several transcription factors, Che-1 represent an important cofactor involved in multiple cellular processes ( 7 , 11 , 12 ). In this context, post-translational modifications play a crucial role in controlling Che-1 co-transcriptional activity in response to different stimuli ( 6 , 15 ). We recently described Che-1 involvement in MM proliferation by affecting chromatin structure and sustaining global gene transcription. Also, we demonstrated that 308–325 aa region of Che-1 is required for these activities ( 14 ). In this study, we extended these results, confirming the roles played by Che-1 in the control of cellular proliferation and chromatin remodelling, in other cancer cell lines. More importantly, our results highlighted that the phosphorylations at Ser 316 , Ser 320 and Ser 321 , contained in the Che-1 308–325 aa region, are essential for Che-1 activity. Consistent with these findings, a Che-1 mutant lacking these residues (Che-1 3S) is unable to bind histones and to regulate chromatin structure and transcription. Finally, we identified CK2 as the protein kinase responsible for Che-1 phosphorylation (Fig. 6 ). The relevance of 308–325 aa region in controlling Che-1’s functions has already been highlighted ( 11 , 14 ). This region is highly conserved among several species and is present in many proteins involved in transcriptional regulation ( 11 , 13 ). In addition, phosphoproteomic studies have demonstrated Ser 316 , Ser 320 and Ser 321 phosphorylation in all species analysed and that this modification is strongly abolished by the inhibition of different protein kinases implicated in cell cycle control ( 33 – 35 ). In this study, we demonstrate the functional relevance of these modifications, since the replacement of these three serines produces a protein unable to modulate chromatin structure and gene transcription. This mutant does not bind histone proteins, and presumably serine phosphorylation is necessary to increase the acidic components of this region, thus enhancing the binding with the basic structure of histones. The importance of this region and its modifications is further confirmed by the analysis of the SV40 Large T protein (SV40 LT). This oncoprotein contains a region of high homology with the 308–325 aa region of Che-1, and also it is able to bind histones, sustaining their acetylation, and to activate cell proliferation by regulating gene transcription ( 36 ). By producing a SV40 LT mutant protein, we were able to demonstrate that even in this case, the phosphorylation of these serines is necessary for the functionality of the protein, thus supporting the hypothesis of a close correlation between viral oncoproteins and Che-1. Finally, we demonstrated that Che-1 is phosphorylated by CK2 at Ser 316 , Ser 320 and Ser 321 in proliferating cells although in which phase of the cycle these modifications occurs, remain to be clarified. CK2 is highly expressed in several tumor cells, and its activity sustains cell proliferation ( 20 – 23 ), therefore it is considered an attractive target to counteract drug resistance in cancer. Indeed, specific CK2 inhibitors have shown proven efficacy on tumor cell proliferation and drug resistance ( 18 ). Since Che-1 silencing is able to induce apoptosis or cell cycle arrest and sensitize MM cells to treatment with proteasome and BET inhibitors ( 2 , 14 ), targeting Che-1 by using CK2 inhibitors could represent an interesting novel therapeutic approach. Conclusion In conclusion, our findings clearly confirmed the global pro-proliferative ability of Che-1 in tumor cells. We highlighted the relevance of the phosphorylation at Ser 316 , Ser 320 and Ser 321 for its functions and identified CK2 as the protein kinase responsible for this modification. Abbreviations MM multiple myeloma; HCC hepatocellular carcinoma; CK2 Casein Kinase II; SV40 LT Simian virus 40 Large T; Rb retinoblastoma; WB western blot; IP immunoprecipitation; MNase Micrococcal nuclease; MS Mass Spectrometry; ATP Adenosine 5’-triphosphate; λ-PP lambda protein phosphate; wt wild type; Ser serine; aa aminoacid; NLS nuclear localization signal. Declarations FUNDINGS This work was supported by the Italian Association for Cancer Research (15255 to M.F.) and Ministry of Health (RF-2019-12368737 to M.F). AUTHOR’S CONTRIBUTIONS V.C., T.B and S.I. performed most of the experiments and analysed data; S.M. performed 2D gel electrophoresis assays; C.S. conducted immunofluorescence analyses; A.S. performed MS/MS analyses; G.D. participated in data analyses; M.F. conceived the projects, obtained funding and supervised the experiments; M.F and V.C. discussed the experiments and wrote the manuscript. COMPETING INTERESTS The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ACKNOWLEDGEMENT We thank Mrs. Tania Merlino for the English language revision of the manuscript and Dr. Francesca Fabretti for technical and scientific support. References Bacalini MG, Tavolaro S, Peragine N, Marinelli M, Santangelo S, Del Giudice I, et al. A subset of chronic lymphocytic leukemia patients display reduced levels of PARP1 expression coupled with a defective irradiation-induced apoptosis. Exp Hematol. 2012;40(3):197-206 e1. Desantis A, Bruno T, Catena V, De Nicola F, Goeman F, Iezzi S, et al. Che-1-induced inhibition of mTOR pathway enables stress-induced autophagy. EMBO J. 2015;34(9):1214-30. Kaul D. Cellular AATF gene: armour against HIV-1. Indian J Biochem Biophys. 2007;44(5):276-8. Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347(6220):1260419. Kumar DP, Santhekadur PK, Seneshaw M, Mirshahi F, Tuculescu CU, Sanyal AJ. A Novel Regulatory Role of Apoptosis Antagonizing Transcription Factor in the Pathogenesis of NAFLD and HCC. Hepatology. 2018. Iezzi S, Fanciulli M. Discovering Che-1/AATF: a new attractive target for cancer therapy. Front Genet. 2015;6:141. Ishigaki S, Fonseca SG, Oslowski CM, Jurczyk A, Shearstone JR, Zhu LJ, et al. AATF mediates an antiapoptotic effect of the unfolded protein response through transcriptional regulation of AKT1. Cell Death Differ. 2010;17(5):774-86. Caliskan G, Baris IC, Ayaydin F, Dobson MJ, Senarisoy M, Boros IM, et al. Che1/AATF interacts with subunits of the histone acetyltransferase core module of SAGA complexes. PLoS One. 2017;12(12):e0189193. Welcker D, Jain M, Khurshid S, Jokic M, Hohne M, Schmitt A, et al. AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer. Oncogene. 2018;37(11):1503-18. Bruno T, Valerio M, Casadei L, De Nicola F, Goeman F, Pallocca M, et al. Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1 alpha stabilization. J Exp Clin Canc Res. 2017;36. Bruno T, De Angelis R, De Nicola F, Barbato C, Di Padova M, Corbi N, et al. Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb. Cancer Cell. 2002;2(5):387-99. Bruno T, De Nicola F, Iezzi S, Lecis D, D'Angelo C, Di Padova M, et al. Che-1 phosphorylation by ATM/ATR and Chk2 kinases activates p53 transcription and the G2/M checkpoint. Cancer Cell. 2006;10(6):473-86. Di Padova M, Bruno T, De Nicola F, Iezzi S, D'Angelo C, Gallo R, et al. Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter. J Biol Chem. 2003;278(38):36496-504. Bruno T, De Nicola F, Corleone G, Catena V, Goeman F, Pallocca M, et al. Che-1/AATF-induced transcriptionally active chromatin promotes cell proliferation in multiple myeloma. Blood Adv. 2020;4(22):5616-30. De Nicola F, Catena V, Rinaldo C, Bruno T, Iezzi S, Sorino C, et al. HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation. Cell Death Dis. 2014;5:e1414. Hopker K, Hagmann H, Khurshid S, Chen S, Hasskamp P, Seeger-Nukpezah T, et al. AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. EMBO J. 2012;31(20):3961-75. Bruno T, Iezzi S, Fanciulli M. Che-1/AATF: A Critical Cofactor for Both Wild-Type- and Mutant-p53 Proteins. Front Oncol. 2016;6:34. Borgo C, Ruzzene M. Role of protein kinase CK2 in antitumor drug resistance. J Exp Clin Canc Res. 2019;38. Seldin DC, Lou DY, Toselli P, Landesman-Bollag E, Dominguez I. Gene targeting of CK2 catalytic subunits. Mol Cell Biochem. 2008;316(1-2):141-7. Homma MK, Homma Y. Cell cycle and activation of CK2. Mol Cell Biochem. 2008;316(1-2):49-55. Coccetti P, Tripodi F, Tedeschi G, Nonnis S, Marin O, Fantinato S, et al. The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G(1) to S transition in Saccharomyces cerevisiae. Cell Cycle. 2008;7(10):1391-401. Li HC, Liu XS, Yang XM, Wang YM, Wang Y, Turner JR, et al. Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formation of kinetochore-microtubule attachments. Embo Journal. 2010;29(17):2953-65. Chua MMJ, Ortega CE, Sheikh A, Lee M, Abdul-Rassoul H, Hartshorn KL, et al. CK2 in Cancer: Cellular and Biochemical Mechanisms and Potential Therapeutic Target. Pharmaceuticals-Base. 2017;10(1). Deplus R, Blanchon L, Rajavelu A, Boukaba A, Defrance M, Luciani J, et al. Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a. Cell Rep. 2014;8(3):743-53. Wu SY, Lee AY, Lai HT, Zhang H, Chiang CM. Phospho Switch Triggers Brd4 Chromatin Binding and Activator Recruitment for Gene-Specific Targeting. Mol Cell. 2013;49(5):843-57. Fanciulli M, Bruno T, Di Padova M, De Angelis R, Iezzi S, Iacobini C, et al. Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb. FASEB J. 2000;14(7):904-12. Sorino C, Catena V, Bruno T, De Nicola F, Scalera S, Bossi G, et al. Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription. Nucleic Acids Res. 2020;48(11):5891-906. Sorino C, Bruno T, Desantis A, Di Certo MG, Iezzi S, De Nicola F, et al. Centrosomal Che-1 protein is involved in the regulation of mitosis and DNA damage response by mediating pericentrin (PCNT)-dependent Chk1 protein localization. J Biol Chem. 2013;288(32):23348-57. Marques-Santos LF, Grassi G, Bergami E, Faleri C, Balbi T, Salis A, et al. Cationic polystyrene nanoparticle and the sea urchin immune system: biocorona formation, cell toxicity, and multixenobiotic resistance phenotype. Nanotoxicology. 2018;12(8):847-67. Taus T, Kocher T, Pichler P, Paschke C, Schmidt A, Henrich C, et al. Universal and Confident Phosphorylation Site Localization Using phosphoRS. Journal of Proteome Research. 2011;10(12):5354-62. Yamagata A KD, Takeda Y, Miyamoto Y, Okada K, Inamatsu M, Yoshizato K. Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics. 2002;2(9):1267-76. Zaret K. Micrococcal nuclease analysis of chromatin structure. Curr Protoc Mol Biol. 2005;Chapter 21:Unit 21 1. Alayev A, Doubleday PF, Berger SM, Ballif BA, Holz MK. Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling. J Proteome Res. 2014;13(12):5734-42. Dephoure N, Zhou C, Villen J, Beausoleil SA, Bakalarski CE, Elledge SJ, et al. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 2008;105(31):10762-7. Santamaria A, Wang B, Elowe S, Malik R, Zhang F, Bauer M, et al. The Plk1-dependent phosphoproteome of the early mitotic spindle. Mol Cell Proteomics. 2011;10(1):M110 004457. Valls E, de la Cruz X, Martinez-Balbas MA. The SV40 T antigen modulates CBP histone acetyltransferase activity. Nucleic Acids Res. 2003;31(12):3114-22. Rihs HP, Jans DA, Fan H, Peters R. The Rate of Nuclear Cytoplasmic Protein-Transport Is Determined by the Casein Kinase-Ii Site Flanking the Nuclear-Localization Sequence of the Sv40 T-Antigen. Embo Journal. 1991;10(3):633-9. Lindfors K, Halttunen T, Nupponen N, Vihinen M, Visakorpi T, Maki M, et al. Identification of a novel transcription factor-like gene repressed during TGF-beta induced human intestinal epithelial cell differentiation. Gastroenterology. 2000;118(4):A289-A. Xiao CY, Jans P, Jans DA. Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T-antigen NLS by importin: effect of conformation. Febs Lett. 1998;440(3):297-301. Tables Table 1. CK2-phosphorylated Che-1 residues identified by nano-LC-mass spectrometry . MS/MS analysis of in vitro phosphorylation assay showing Che-1 phosphopeptides including Ser 316 , Ser 320 and Ser 321 . For each peptide the following details are reported: aminoacid sequence, phosphorylation site(s); pRS peptide score (cumulative binomal probability that the observed match is a random events); pRS isoform probability (probability that the isoform is correct); pRS site probability (probability of each site being truly phosphorylated); q -value (minimal false discovery rate at which the identification is considered correct); PEP score (probability that the peptide-spectrum match is incorrect); number of missed-cleavages. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-459016","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":23892150,"identity":"63d644f6-1cfc-41b5-a064-e1ab81b41537","order_by":0,"name":"Valeria Catena","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIie2RMUsDMRTHXwjklleyprT4GSI3CaX3VTwCzh0PFIwcpJtd67fpcWCXw65CpyOL4xVBEEXMYa9IISduIvkNjwTej/d/CUAg8CehGkAebhOM9NcJ/QrpFNqWC8TVXvE7RHfj2lJCp3jHyHVxY7PZFPh8U9jZ1WbsktGnnYFx4lOqND+tpAJRKRov77fokrGzO+MPNtSpGWpJ3TqKjZBtMRHnLMaqR1nU81ctr0FyG73hxwPiTwoXqSFaliCFYnRgVq1CLWZ9Sp27YGsUjzYeDW6V26UwZJmJw2sfw7gqdvr98oQv0voZX6YJRnnZNHKSdH/q43sKYgSA6O8/hja/6w8EAoF/zif+IkpvSt1GcwAAAABJRU5ErkJggg==","orcid":"","institution":"Regina Elena National Cancer Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Catena","suffix":""},{"id":23892151,"identity":"542fc4ee-8aae-442d-9e70-32d61f79ad9f","order_by":1,"name":"Tiziana Bruno","email":"","orcid":"","institution":"National Cancer Institute: Istituto Regina Elena","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tiziana","middleName":"","lastName":"Bruno","suffix":""},{"id":23892152,"identity":"7fd85f00-3db5-4821-9d82-f6b8810a2543","order_by":2,"name":"Simona Iezzi","email":"","orcid":"","institution":"National Cancer Institute: Istituto Regina Elena","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Simona","middleName":"","lastName":"Iezzi","suffix":""},{"id":23892153,"identity":"a7341c70-ae60-4f9c-b6fe-db90129c862e","order_by":3,"name":"Silvia Matteoni","email":"","orcid":"","institution":"National Cancer Institute: Istituto Regina Elena","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Matteoni","suffix":""},{"id":23892154,"identity":"843327b2-cc07-4ec7-8005-1e7eb1d89f6d","order_by":4,"name":"Annalisa Salis","email":"","orcid":"","institution":"Universita degli Studi di Genova","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Annalisa","middleName":"","lastName":"Salis","suffix":""},{"id":23892155,"identity":"fbe64215-377f-4c1e-8ff0-7dec74ca5ad6","order_by":5,"name":"Cristina Sorino","email":"","orcid":"","institution":"National Cancer Institute: Istituto Regina Elena","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Sorino","suffix":""},{"id":23892156,"identity":"0757eced-7ab2-4f75-a176-95088c4a5aa2","order_by":6,"name":"Gianluca Damonte","email":"","orcid":"","institution":"Universita degli Studi di Genova","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gianluca","middleName":"","lastName":"Damonte","suffix":""},{"id":23892157,"identity":"fd185085-b601-4f16-aa49-3998ab2e7e37","order_by":7,"name":"Maurizio Fanciulli","email":"","orcid":"","institution":"National Cancer Institute: Istituto Regina Elena","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maurizio","middleName":"","lastName":"Fanciulli","suffix":""}],"badges":[],"createdAt":"2021-04-24 15:26:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-459016/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-459016/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13046-021-02038-x","type":"published","date":"2021-07-15T15:02:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":8652914,"identity":"3c296417-b2b6-45c6-a092-3c714a11607d","added_by":"auto","created_at":"2021-04-30 17:56:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1115222,"visible":true,"origin":"","legend":"Che-1 depletion reduces proliferation in several cancer cell lines. A: Cell proliferation analysis (top) and WB analysis of total cell extracts (bottom) from the indicated cell lines transiently transfected with stealth siRNA Control (siControl) or siRNA Che-1 (siChe-1) for 48 hours. Bar plot shows the average of number of cells observed in these experiments (n=3). B: Two images representing nuclear morphology (left), corresponding fluorescence intensity analysis (middle) and WB analysis (right) of HCT116 cells transiently transfected with siControl or siChe-1. Nuclei were visualized by staining with Hoechst dye. Scale bar, 10 μm. Error bars represent the SD after combining the results of three different experiments. C: Representative WB analysis with the indicated antibodies of nuclear extracts from HCT116 cells transfected as in B. D: Representative immunofluorescence analysis (left) and corresponding fluorescence intensity analysis (right) of H3K9me3 expression in HCT116 cells transfected as in B. Nuclei were visualized by staining with Hoechst dye. Scale bar, 10 μm. E: Representative WB analysis with the indicated antibodies of total cell extracts from the indicated cell lines and transfected as in A. F: Cell number-normalized quantification of total RNA extracted from the cell lines used in A. Statistical significance is indicated by asterisks as follow: *P\u003c0.05, **P\u003c0.01, ***P\u003c0.005, ****P\u003c0.001, n.s. = not significant.","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/8326f7eb403c0af5b0d005e9.png"},{"id":8652916,"identity":"811f20aa-6409-45ee-bbce-cef63addcd26","added_by":"auto","created_at":"2021-04-30 17:56:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":984967,"visible":true,"origin":"","legend":"Che-1 is highly phosphorylated. A: A multiple sequence alignment of five mammalian Che-1 proteins. Below each position of the protein sequence alignment is present a key denoting conserved sites (*), sites with conservative replacements (:), sites with semi-conservative replacements (.) and sites with non-conservative replacements ( ). B: Total cell extracts from HeLa cells treated or not with λ-PP (see Methods) were separated onto parallel 2D-Gel electrophoresis. C: Schematic representation of Che-1 point mutations (see Methods) at the indicated sites. D: 2-D gel electrophoresis (top) and representative WB (bottom) of HeLa cells transiently transfected with Myc-Che-1 or Myc-Che-1 3S. E: Representative immunofluorescence images (left) and WB analysis (right) of HCT116 cells transfected with the indicated expression vectors. Scale bar, 10 μm. F: WB analysis with the indicated antibodies of total, cytoplasmic and nuclear fractions from HCT116 cells transfected as in D.","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/3596f656d86526ee3fb6c632.png"},{"id":8652915,"identity":"5caf83c9-bb7a-442b-884a-355149156933","added_by":"auto","created_at":"2021-04-30 17:56:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1228935,"visible":true,"origin":"","legend":"Che-1 phosphorylation is required for its pro-proliferative ability. A: Cell proliferation analysis of HCT116 cells transiently transfected with Myc-Che-1, Myc-Che-1 3S or control vector (pCS2MT). Bar plot shows the average number of cells observed in these experiments (n=6). B: Representative WB analysis with the indicated antibodies of total cell extracts from HCT116 cells transfected as in A. C: Cell number-normalized total RNA quantification of the indicated cell line transiently transfected with Myc-Che-1 wt, 3S mutant or control vector. Error bars represent the SD of triplicate experiments (n=3). D: WB analysis with the indicated antibodies of total cell extracts from HCT116 cells transfected as in C. E: WB analysis of total cell extracts of HCT116 cells transfected with siControl or siChe-1 3’UTR alone or in combination with empty vector or with Che-1 expressing vectors (Myc-Che-1 and Myc-Che-1 3S). F: Nuclear extracts from HCT116 cells transiently transfected with Myc-Che-1 wt or 3S mutant, were subjected to IP with anti-Myc monoclonal antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP (left). Relative H3 binding was calculated from three different experiments by densitometry. H3 intensity was normalized to the one of Myc-Che-1 wt or 3S mutant (right). G: WB analysis of GST-pull down experiment conducted using total extracts from HCT116 cells over-expressing pCI-HDAC1 incubated with purified GST-H3 fusion protein or control GST agarose beads, in presence or absence of the indicated peptides. GST fusion proteins expression is shown by Comassie blue staining. H: MNase digestion pattern of nuclei (left) corresponding WB analysis (right) with the indicated antibodies of total cell extracts from HCT116 cells transfected as in A. Statistical significance is indicated by asterisks as follow: *P\u003c 0.05, **P\u003c0.01, ***P\u003c0.005, ****P\u003c0.001, n.s. = not significant.","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/799c9b82d3748ffcee12dc61.png"},{"id":8652511,"identity":"6ada26b3-52f5-4a8c-af47-3463a0c8620b","added_by":"auto","created_at":"2021-04-30 17:53:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":386042,"visible":true,"origin":"","legend":"Che-1 and SV40 Large T antigen display similar functions. A: Sequence alignment of Che-1 and SV40 Large T antigen (SV40 LT) proteins. Conserved serine residues are in red. B: Schematic representation of SV40 LT point mutations (see Methods) at the indicated sites. C: Cell proliferation analysis (left) and relative representative WB analysis (right) of HCT116 cells transfected with pSG5-SV40 LT wt, 3S mutant or empty vector (pcDNA3). Bar plot shows the average number of cells observed in these experiments (n=3). D: WB analysis with the indicated antibodies of total cell extracts from HCT116 cells transfected as in C. E: Nuclear extracts from HCT116 cells transfected as in C and subjected to IP with SV40 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP (left). Relative H3 binding was calculated from three different experiments by densitometry. H3 intensity was normalized to the one of SV40 wt or 3S mutant (right). Statistical significance is indicated by asterisks as follow: *P\u003c0.05, **P\u003c0.01, ***P\u003c0.005, ****P\u003c0.001, n.s. = not significant.","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/64a73f385aa08b41425e8c40.png"},{"id":8652509,"identity":"9058290c-9f47-439a-bfcd-e79daacfc100","added_by":"auto","created_at":"2021-04-30 17:53:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1615066,"visible":true,"origin":"","legend":"CK2 phosphorylates Che-1. A: Nuclear extracts from HCT116 cells subjected to IP with Che-1 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. B and C: 2D-Gel electrophoresis of total cell extracts from HeLa cells treated or not with 80 μM TBB for 4 hours (B) or transfected with siControl or siCK2 (C). D: Representative immunofluorescence analysis of Che-1 expression in the indicated cell lines transiently transfected with siControl or siCK2. Scale bar, 10 μm. E: Nuclear extracts from HCT116 cells transiently transfected as in D and subjected to IP with H3 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies (left). Input corresponds to 10% of the nuclear extracts used for IP. Relative Che-1 binding was calculated from three different experiments by densitometry. Che-1 intensity was normalized to the one of H3 (right). Statistical significance is indicated by asterisks as follow: *P\u003c0.05, **P\u003c0.01, ***P\u003c0.005, ****P\u003c0.001, n.s. = not significant.","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/ac21b11176912f8bdfb465c7.png"},{"id":8652513,"identity":"7062939a-8ff8-45c8-b2a3-89aa2fdd728a","added_by":"auto","created_at":"2021-04-30 17:53:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":364536,"visible":true,"origin":"","legend":"Role of CK2 phosphorylation in regulating Che-1 functions. Phosphorylation of Che-1 by CK2 is required for displacing HDACs from chromatin and sustaining cancer cell proliferation.","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/2fcb78f853bf885d3b76ecb6.png"},{"id":13689610,"identity":"943e4ce5-f2b4-417c-b841-0561d6f56212","added_by":"auto","created_at":"2021-09-17 12:30:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6239702,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/7836dccb-dfd4-45ed-aeec-67c7ba2de15c.pdf"},{"id":8652917,"identity":"7c41fd4a-5e5d-4254-a673-2f2d7c21e7e1","added_by":"auto","created_at":"2021-04-30 17:56:29","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3410284,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-459016/v1/3281ed87d70fce3e6e5eb652.pdf"}],"financialInterests":"","formattedTitle":"CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity","fulltext":[{"header":"Background","content":" \u003cp\u003eChe-1/AATF (Che-1) is a highly conserved nuclear protein overexpressed in several cancerous tissues, such as prostate, lung, colon, lymphomas as well as in multiple myeloma (MM) and in hepatocellular carcinoma (HCC), where its levels increase during disease progression (\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). It has been previously demonstrated Che-1\u0026rsquo;s involvement in important cellular activities, including proliferation, regulation of cell cycle, apoptosis and stress response (\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Many studies have highlighted the ability of Che-1 to affect the transcriptional machinery by its association with several proteins, such as RNA Polymerase II, the retinoblastoma protein (Rb), p65 and STAT3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Importantly, Che-1 contains an acidic region spanning from 308 to 325 aa highly homologous to HDAC proteins and several viral oncoproteins (SV40 Large T, E1A) by which it contacts Rb, thus inhibiting its binding to HDAC 1 and promoting cell proliferation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). More recently, we have demonstrated that this region is required for displacing HDAC proteins from histones, increasing chromatin accessibility and MM cell proliferation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Che-1 half-life and functions are modulated by several post-translational modifications (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and its phosphorylation at specific serine or threonine residues has been associated with Che-1 activity in response to different stimuli, including induction of DNA damage or apoptosis (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCasein kinase II (CK2) is a ubiquitous highly pleiotropic serine/threonine protein kinase present in cells in a tetrameric form consisting of two catalytic subunits (CK2α and its isoform CK2α\u0026rsquo;) and two regulatory β subunits (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). It is expressed in all tissues of eukaryotic organisms and localized in different cell compartments (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Due to its ability to phosphorylate a wide variety of substrates, CK2 is involved in many cellular pathways, including cell growth, proliferation and survival (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Several studies highlighted that CK2 is up-regulated in different tumors, including kidney, head and neck, lung and prostate (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) and it is considered an attractive target to counteract drug resistance in cancer (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Indeed, a rising number of CK2 inhibitors have been developed and their effectiveness proven alone or in combination with conventional anticancer drugs supporting their possible application in clinical therapy (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Moreover, several studies have demonstrated that this kinase can interact with and regulate many factors involved in chromatin remodelling (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that Che-1 ability to promote histone acetylation and cell proliferation is not only restricted to MM cells, and that the phosphorylation at three serine residues, Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e, is required for these activities. Notably, we identified CK2 kinase as the enzyme responsible for these modifications. Finally, we produce evidence that also in the SV40 Large T protein the phosphorylation of conserved serine residues, within the region of homology with Che-1, is required for its activity, thus underlining the importance of these modifications in the regulation of cell proliferation.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfections\u003c/h2\u003e \u003cp\u003eHuman HCT116, HeLa, 293T and U2OS cell lines were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM, Euroclone) high glucose supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 2 mM glutamine (Thermo Fisher Scientific) and 40 \u0026micro;g/ml gentamicin. All cell lines were cultured at 37\u0026deg;C, in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. Mycoplasma contamination was periodically tested by RT\u0026ndash;PCR analysis, using the following primers:\u003c/p\u003e \u003cp\u003eForward: 5\u0026rsquo; - ACT CCT ACG GGA GGC AGC AGT A \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026rsquo; - TCG ACC ATC TGT CAC TCT GTT AAC \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e \u003cp\u003eTransfection experiments were carried out by using Lipofectamine 3000 Transfection System (Thermo Fisher Scientific) following the manufacturer's instructions. Cells were analysed 48 h after transfection by western blot (WB) or immunofluorescence. Cell number and viability were assessed by Trypan Blue staining using Countess automated cell counter (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant plasmids and reagents\u003c/h2\u003e \u003cp\u003eMyc-Che-1 has already been described (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). pCI-HDAC 1 was a kind gift from Dr. Sartorelli, while pSG5 Large T (pSG5 SV40 LT) plasmid was a gift from William Hahn (Addgene plasmid #9053; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:9053\u003c/span\u003e\u003c/span\u003e; RRID: Addgene_9053). Myc-Che-1 3S and pSG5 SV40 LT 3S were generated by in vitro mutagenesis using the QuikChange site-directed Mutagenesis system (Agilent Technologies) following the manufacturer\u0026rsquo;s instructions. PCR reactions were achieved using the following primers:\u003c/p\u003e \u003cp\u003eMyc- Che-1 3S:\u003c/p\u003e \u003cp\u003eForward 5\u0026rsquo; - GCCCAATGCGGGA\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGGT\u003c/span\u003eGAGGAGATT\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGCTGGT\u003c/span\u003eGAAGATGATGAGC \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo; - GCTCATCATCTTCACCAGCAATCTCCTCACCTCCCGCATTGGGC \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e \u003cp\u003epSG5 SV 40 LT 3S:\u003c/p\u003e \u003cp\u003eForward 5\u0026rsquo; - AACCTGTTTTGC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGCA\u003c/span\u003eGAAGAAATGCCA\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGCTGGT\u003c/span\u003eGATGATGAGGCT \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo; - AGCCTCATCATCACCAGCTGGCATTTCTTCTGCGCAAAACAGGTT \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e \u003cp\u003eAll mutations were confirmed by sequencing realized by Eurofins Genomics.\u003c/p\u003e \u003cp\u003eStealth siRNA oligonucleotides targeting Che-1 (siChe-1), CSNK2A (siCK2), control sequence (siControl) and custom Che-1 3\u0026rsquo;UTR (sense 5\u0026rsquo;-CCCGCCUUUAAACGCCACAAAUAAA-3\u0026rsquo;; antisense 5\u0026rsquo;-UUUAUUUGUGGCGUUUAAAGGCGGG-3\u0026rsquo;) were purchased from Thermo Fisher Scientific. TBB (4,5,6,7 \u0026ndash; Tetrabromobenzotriazole) was purchased from SelleckChem. Casein kinase II (CK2 - P60105) recombinant protein and Adenosine 5\u0026rsquo;-triphosphate (ATP - P07565) were purchased from New England BioLabs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell extracts, immunoprecipitation and RNA isolation\u003c/h2\u003e \u003cp\u003eTotal, cytoplasmic and nuclear extracts were prepared as previously described (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). For immunoprecipitation (IP) experiments, nuclear extracts were resuspended in dilution buffer (50 mM TRIS pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 0.5% NP40, protease and phosphatase inhibitors). Dynabeads magnetic beads (Thermo Fisher Scientific) were incubated for 1 h at room temperature on a rotating wheel with the indicated antibodies. Nuclear extracts were then added to the beads and the incubation was continued for 2 additional hours. Immuno-complexes were purified by using a magnet (DynaMag-2, Thermo Fisher Scientific), the beads were washed five times with dilution buffer and eluted in 4X lithium dodecyl sulphate (LDS) sample buffer (Thermo Fisher Scientific) for WB analysis. Detailed information for all antibodies is provided in Supplementary Table S1. Total RNA was isolated from cells using EuroGOLD TriFast reagent (Euroclone) according to the manufacturer's instructions. The amount of RNA was measured by using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eSamples were prepared as previously described (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), resolved on Bolt pre-cast 4\u0026ndash;12% gels (Thermo Fisher Scientific), run using Bolt MES SDS running buffer (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes. After a blocking step in 5% non fat-dried milk in phosphate-buffered saline (PBS)-0.1% Tween, membranes were incubated with primary antibodies overnight at 4\u0026deg;C. After three washes in PBS \u0026minus;\u0026thinsp;0.1% Tween, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad Laboratories) for 45 min at room temperature, washed with PBS-0.1% Tween and analysed by chemi-luminescence (GE Healthcare Life Science). Images were acquired and quantified using Alliance Mini HD6 system by UVITEC Ltd, Cambridge, equipped with UVI1D Software (UVITEC, 14\u0026ndash;630275). Detailed information for all antibodies is provided in the Supplementary Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eDetection of both heterochromatic foci and chromatin modifications was performed as described in Bruno et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Briefly, HCT116 cells were grown on cover-lips, washed twice in PBS, fixed in 100% methanol for 5 min and then permeabilized for one hour with 1% BSA, 10% normal goat serum, 0.3 M glycine in 0.1% PBS-Tween. Cells were incubated with anti-H3K9me3 overnight at 4\u0026deg;C and stained 45 min with Alexa-Fluor-594-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific). For anti-Myc tag and anti-Che-1 immunofluorescences, cells were fixed in 4% formaldehyde for 10 min and then permeabilized with PBS \u0026minus;\u0026thinsp;0.2% Triton X-100 for 5 min at room temperature. Cells were stained for two hours with the primary antibody, rinsed three times with PBS and stained respectively with Alexa-Fluor-594-conjugated anti-mouse or with Alexa-Fluor-488-conjugated anti-rabbit secondary antibodies (Thermo Fisher Scientific) 45 min at room temperature. Nuclei were visualized by staining with 1 \u0026micro;g/ml Hoechst dye 33258 (Sigma-Aldrich) for 10 min. Images were acquired using a fluorescence microscope with a 40X objective (Zeiss, Germany) and processed with AxioVision 4.7.1 software.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003ekinase assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGST-Che-1 fusion protein was cloned into pGEX-4T-3 vector as already described (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). For \u003cem\u003ein vitro\u003c/em\u003e kinase assay, GST-Che-1 was incubated with or without CK2 constitutively active for 1 h at 30\u0026deg;C in slowly rotation on a rotating wheel in CK2 Reaction buffer (New England Biolabs) in presence of 200 \u0026micro;M ATP. Adding 4X LDS stopped reactions and the proteins were resolved by Bolt pre-cast 4\u0026ndash;12% gels. The gel was stained with SimplyBlue SafeStain (Thermo Fisher Scientific) for three hours at room temperature, and bands of interest were cut and then analysed by Nano - LC Mass spectrometry (MS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNano - LC Mass spectrometry\u003c/h2\u003e \u003cp\u003eAfter kinase assay the bands of interest were cut from the gel, de-stained, reduced, alkylated and digested with trypsin following the procedure described by Shevchenko et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The analysis of tryptic peptides was carried out by mean of nano-HPLC-MS/MS using an Ultimate 3000 nano-HPLC system and the obtained data were processed using an in-house software. The peptide pellets were re-suspended immediately before analysis and firstly loaded, from the sample loop, onto a trapping column (Acclaim PepMap C18; 2 cm \u0026times; 100 \u0026micro;m \u0026times; 5 \u0026micro;m, 100 \u0026Aring;- Thermo Fisher Scientific) using the loading eluents composition (95\u0026thinsp;\u0026minus;\u0026thinsp;5% ACN/H2O\u0026thinsp;+\u0026thinsp;0.05% trifluoroacetic acid). The separation of peptides was performed at the flow rate of 300 nL/min and at a temperature of 35\u0026deg;C using an Easy spray column (15 cm x75 \u0026micro;m PepMap C18 3 \u0026micro;m, Thermo Fisher Scientific). A linear gradient of solution B (95\u0026thinsp;\u0026minus;\u0026thinsp;5% ACN/H2O\u0026thinsp;+\u0026thinsp;0.08% formic acid) from 4\u0026ndash;95% in 55 min was applied. All the analyses were performed in the positive ion mode. Single MS survey scans were performed in the Orbitrap, recording a mass window between 395 and 2000 m/z using a maximal ion injection time of 100 ms. The resolution was set to 70,000 and the automatic gain control was set to 3x10\u003csup\u003e6\u003c/sup\u003e ions. The experiments were performed in data-dependent acquisition mode with alternating MS and MS/MS experiments. For MS/MS analysis an isolation window of 2Da was used. CID was done with a target value of 5000 ions, a maximal ion injection time of 50ms, normalized collision energy of 35%. Raw MS files were processed with the Thermo Scientific Proteome Discoverer software version 1.4. Peak list files were obtained by the SEQUEST search engine against the \u003cem\u003eHuman protein database\u003c/em\u003e containing both forward and reversed protein sequences. The resulting peptide hits were filtered for a maximum 1% FDR (false discovery rate) using the percolator tool. The database search parameters were: mass tolerance precursor 20ppm, mass tolerance fragment CID 0.8Da, dynamic modification of deamidation (N, Q), oxidation (M) and static modification of alkylation with IAM (C). Phosphorylation of Ser were set as variable modifications. The software phosphoRS was used to validate the correct assignment of the phosphorylation sites. In any case, the option trypsin with two missed cleavages was selected (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIdentification by nano\u0026ndash;HPLC\u0026ndash;ESI-MS/MS\u003c/h2\u003e \u003cp\u003eThe data obtained by the HPLC separation and MS/MS analyses, carried out on digested samples, were submitted to the SEQUEST search engine against \u003cem\u003eUniprot Human\u003c/em\u003e protein database using the phosphoRS tool. This approach allowed the specific identification of \u003cem\u003eTau-F of Apoptosis-antagonizing transcription factor (AATF/Che-1)\u003c/em\u003e. Fifty-three high confidence peptides, corresponding to sequence coverage of 65.36% and high sequest score of 2391.39, were identified. From the MS/MS analysis it was possible to identify four phosphopeptides derived from Che-1, three of which were unambiguously assigned as phosphopeptides (Table\u0026nbsp;1). In a few cases it was not possible to assign the phosphate to specific serine residue(s), thus hindering the precise localization of the phospho-residue(s). The analysis of peptides obtained from Che-1 protein untreated allowed the identification fifty-four high confidence peptides corresponding to sequence coverage of 64.64% and high sequest score of 2082.81. As expected no phosphopeptides were found in this sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2D Gel electrophoresis\u003c/h2\u003e \u003cp\u003eFor 2D-Gel, cell pellets were resuspended in 2-D lysis buffer (2 M thiourea, 7 M urea, 4% CHAPS, 1% DTT, protease inhibitors cocktail) and sonicated. Total extracts were treated, where indicated, with Lambda Protein Phosphatase (ʎ-PP) (New England BioLabs) following the protocol described in Yagamata et al., with minor modifications (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In short, cells pellets were re-suspended and 20 mM MnCl\u003csub\u003e2\u003c/sub\u003e was added to 150 \u0026micro;g of protein extract in order to obtain a final concentration of 2 mM ʎ-PP buffer and brought to a final volume of 20 \u0026micro;l with deionized water. The mixture was divided into two aliquots and 200 units of ʎ-PP were added to one of the aliquots. After mixing, aliquots were incubated overnight at 30\u0026deg;C under constant shaking and subsequently 100 \u0026micro;l of 2-D lysis buffer was added togheter with 1% sulfobetaine SB 4\u0026ndash;7.\u003c/p\u003e \u003cp\u003eIsoelectric focusing was performed using 70 mm 4.0\u0026ndash;7.0 linear Immobilized pH Gradient gel strips (IPG strips) (Bio-Rad Laboratories) on an electrophoresis unit (Ettan IPGphor 3; GE Healthcare Life Science). Equal amounts of proteins (70 \u0026micro;g) were loaded by passive in-gel rehydration for 12 hours then run as follows: 1 hour at 50 V, 30 min at 200 V, voltage gradient 30 min up to 1,000 V, 20 min at 1,000 V, voltage gradient 30 min up to 5,000 V and 1hour at 5,000 V. Before performing the 2nd electrophoresis dimension, IPG strips were equilibrated for 15 min at room temperature in 1% DTT to reduce the proteins, and sulfhydryl groups were subsequently derivatized using 4% iodoacetamide (both solutions were prepared in 50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 2% bromophenol blue) for 15 min. Strips were transferred to 1.0-mm-thick 4\u0026ndash;12% pre-casted polyacrylamide mini-gels (Thermo Fisher Scientific) for the 2nd electrophoresis dimension and then gels were run at 130 V for 2 h. 2D gels were then transferred onto PVDF filters.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003eGST pull down assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFull length H3 open reading frame was cloned into EcoRI/XhoI sites of pGEX-4T-1 in order to generate GST-H3 fusion protein. GST-H3 or GST control proteins were prepared following standard procedures and used for in vitro binding assay. Briefly, GST proteins were pre-incubated with Che-1 308\u0026ndash;325, Che-1 3S or control peptides (Biomatik Corporation) for 1 h at 4\u0026deg;C on a rotating wheel in interaction buffer (20 mM Tris pH 7.9, 300 mM NaCl, 0.2 mM EDTA, 0.1% NP40, 10 \u0026micro;g/ml BSA). Total extracts from HCT116 cells over-expressing pCI-HDAC 1 were then added to the GST/peptides complexes and the incubation was continued for three additional hours. Samples were eluted in 4X LDS sample buffer by heating for 10 min at 70\u0026deg;C and then subjected to WB analysis. Peptides sequence commercially synthesized are:\u003c/p\u003e \u003cp\u003eControl: LKGAKPIRPVVVKAPPA\u003c/p\u003e \u003cp\u003eChe-1 308\u0026ndash;325: DGTKPNAGSEEISSEDDEL\u003c/p\u003e \u003cp\u003eChe-1 3S: DGTKPNAGAEEIAAEDDEL\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicrococcal nuclease assay\u003c/h2\u003e \u003cp\u003eMicrococcal nuclease (MNase) assay was performed using the protocol described in Zaret et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Briefly, HCT116 cells were seeded in 100mm plate and transiently transfected with Myc-Che-1 wt, Myc-Che-1 3S or empty vector. 24 h post transfection cells were subjected to the protocol described in Bruno et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and Statistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as the mean of three independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using R software. Two-tailed Student's \u003cem\u003et\u003c/em\u003e-tests with Benjamini\u0026ndash;Hochberg correction were performed to compare one parameter between two groups. For the multiple group comparison, one-way ANOVA with Tukey HSD test was used. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003cp\u003eStatistical significance is indicated by asterisks as follows: *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n.s. = not significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChe-1 depletion reduces proliferation in several cancer cell lines\u003c/h2\u003e \u003cp\u003eAs previously observed, Che-1 displayed to have a key role in MM cells proliferation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To assess whether this effect could also be observed in other tumor cells of different origins, we performed cell proliferation analysis in HCT116, HeLa, 293T and U2OS cells, depleted or not for Che-1 expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Che-1 inhibition produced a significant reduction of cell proliferation when compared to control cells in all tested cell lines. Since Che-1 depletion induces heterochromatin accumulation in MM cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), we evaluated whether this was a general phenomenon. To this aim, we analysed the formation of heterochromatin foci by immunofluorescence in HCT116 cells, observing that Che-1 depleted cells exhibited a strong increase of Hoechst-positive heterochromatic foci (from 9 to 32 foci/cell) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistent with these findings, WB analysis showed that Che-1 knockdown produced a significant increase of H3K9me3 levels, an important hallmark of heterochromatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results were confirmed through immunofluorescence experiments in which a marked increase of H3K9me3 positive dots was observed following Che-1 downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). It has been shown that Che-1 depletion produces a global reduction of histone acetylation in MM cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To extend these findings, we performed WB analyses in several tumor cell lines depleted or not for Che-1 expression, observing that the loss of Che-1 expression produced a strong reduction in both H3 and H4 histone acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In line with the obtained results, we observed a reduction of about 50% of total RNA in Che-1 depleted cells when compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Overall, these findings reinforce the role of Che-1 in sustaining cell proliferation by maintaining an active state of chromatin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChe-1 is highly phosphorylated\u003c/h2\u003e \u003cp\u003eChe-1 affects multiple cellular processes thanks to its ability to interact with several transcription factors (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These interactions are modulated by post-translational modifications in response to different stimuli, including phosphorylation at specific serine or threonine residues (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In this context, we have previously shown that Che-1 contains a highly acidic sequence (308\u0026ndash;325 aa) involved in Che-1-histone binding. Indeed, Che-1 mutant lacking this region was no longer able to bind histone H3 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Interestingly, in this region are present three highly conserved serine residues (in bold; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whose phosphorylation has been reported by many phospho-proteomic studies (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). To investigate the relevance of this Che-1 region, we performed a 2D gel electrophoresis analysis of total extracts from HeLa cells treated or not with lambda phosphatase (λ-PP), and observed that Che-1 is constitutively and highly phosphorylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, we produced a Che-1 mutant (Che-1 3S) in which the serine residues in position 316, 320 and 321 were replaced with glycine or alanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). A bidimensional gel electrophoresis analysis of HeLa cells over-expressing Che-1 wild type (Myc-Che-1) or 3S mutant (Myc-Che-1 3S) showed that this mutant was less phosphorylated than wild-type (wt) protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating that these residues contribute significantly to total protein phosphorylation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we started to characterize the functional role of these modifications investigating whether the 3S mutant had a different cellular localization. Immunofluorescence experiments showed that the mutant retained the peculiar nuclear signal of the wt protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In agreement with these data, a subcellular fractionation of HCT116 cells transiently transfected with Myc-Che-1 wt or 3S mutant, detected a strong accumulation in the nuclear fraction of both molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChe-1 phosphorylation is required for its pro-proliferative ability\u003c/h2\u003e \u003cp\u003eThe data shown above demonstrate that three serine residues contained in the 308\u0026ndash;325 region are phosphorylated. This region plays an important role in Che-1's functions (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To shed light on the relevance of these phosphorylations, we evaluated the effect of the over-expression of Myc-Che-1 wt or 3S mutant. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;1A, Myc-Che-1 3S over-expression in HCT116 cells did not induce cell proliferation when compared to Myc-Che-1 wt. These results were confirmed in MM cells (KMS27 cells) and in other cell lines (Supplementary Figs.\u0026nbsp;1B-D). The relevance of these three serine residues in the pro-proliferative role of Che-1 was further confirmed by evaluating the effect of the overexpression of these molecules on the levels of cyclin B1. In fact, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, only Myc-Che-1 wt was able to increase the levels of this protein. Next, we verified whether the over-expression of Myc-Che-1 3S had any impact on global transcription by measuring the level of total RNA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the Myc-Che-1 3S mutant was completely unable to activate transcription. In order to evaluate whether Che-1 phosphorylation in Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e was required to sustain histone acetylation, we analysed the effect of Myc-Che-1 wt or 3S mutant over-expression on H3 and H4 histone acetylation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, Myc-Che-1 3S mutant showed no effect on histone acetylation and remarkably it was unable to rescue the effects of endogenous Che-1 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results prompted us to investigate the ability of the Che-1 3S mutant to interact with histone H3. To this aim, we performed co-immunoprecipitation experiments with anti-Myc antibody using nuclear extracts from HCT116 cells over-expressing Myc-Che-1 wt or its 3S mutant. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and in Supplementary Fig.\u0026nbsp;1E, the replacement of the three serine residues prevented Che-1/histone H3 interaction. Moreover, an \u003cem\u003ein vitro\u003c/em\u003e competition assay demonstrated that only the Che-1 308\u0026ndash;325 peptide containing phosphorylated serines was able to displace HDAC 1 from histone H3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Notably, KMS27 cells transfected with Che-1 308\u0026ndash;325, but not with Che-1 3S peptide, displayed a reduction in cell proliferation (Supplementary Fig.\u0026nbsp;1F). Consistent with these results, an MNase susceptibility assay performed in HCT116 cells transiently transfected with Myc-Che-1 wt or 3S mutant demonstrated that the mutant overexpression produced a more compacted chromatin structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, these results clearly indicate that the presence of the three serine residues in 308\u0026ndash;325 aa region represents an essential requirement for Che-1 activity, promoting the Che-1/histone binding and histone acetylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChe-1 and SV40 Large T antigen display similar functions\u003c/h2\u003e \u003cp\u003eSV40 LT is a multifunctional viral oncoprotein implicated in a wide range of cellular processes including transcriptional activation and repression, stimulation of the cell cycle and cell transformation (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Interestingly, SV40 LT expression increases global histone acetylation and CREB-binding protein (CBP) histone acetyltransferase (HAT) activity (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). As already described, Che-1 308\u0026ndash;325 aa region is highly conserved among mammalian species and shared with several viral and HDAC 1\u0026ndash;3 proteins (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Among viral oncoproteins, SV40 Large T antigen (SV40 LT) contains three particular serine residues, in position 106, 110 and 111, corresponding to those of Che-1 (in red; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These observations prompted us to verify whether SV40 LT phosphorylation on Ser\u003csup\u003e106\u003c/sup\u003e, Ser\u003csup\u003e110\u003c/sup\u003e and Ser\u003csup\u003e111\u003c/sup\u003e residues, was required for its ability to regulate cellular proliferation. To this aim, we produced a SV40 LT mutant (SV40 LT 3S) in which Ser\u003csup\u003e106\u003c/sup\u003e, Ser\u003csup\u003e110\u003c/sup\u003e and Ser\u003csup\u003e111\u003c/sup\u003e residues were replaced with alanine or glycine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Notably, HCT116 cells over-expressing SV40 LT 3S mutant didn\u0026rsquo;t exhibit the induction of cellular proliferation observed in SV40 LT wt cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, this mutant was unable to induce the histone acetylation increase observed in cells transfected with SV40 LT wt (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Since Che-1 phosphorylation in the 308\u0026ndash;325 region is required for the interaction with histone H3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), we evaluated whether SV40 LT wt was also able to interact with histone H3 and whether phosphorylation of Ser\u003csup\u003e106\u003c/sup\u003e, Ser\u003csup\u003e110\u003c/sup\u003e and Ser\u003csup\u003e111\u003c/sup\u003e residues was involved in this binding. To this aim, we performed co-immunoprecipitation experiments with anti-SV40 antibody in nuclear extracts from HCT116 transiently transfected with SV40 LT wt or its 3S mutant. Remarkably, we observed that the SV40 LT wt protein showed the ability to bind histone H3 whereas, in line with results obtained with Che-1, the mutant lost this ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these data reinforce the importance of this highly acidic region in several proteins for binding to histones and cell proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCK2 phosphorylates Che-1\u003c/h2\u003e \u003cp\u003eNext, we focused on identifying the kinase responsible for the phosphorylation of Che-1 in Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e residues. Previous studies have demonstrated that protein kinase Casein Kinase II (CK2) phosphorylates SV40 LT at Ser\u003csup\u003e111\u003c/sup\u003e and Ser\u003csup\u003e112\u003c/sup\u003e producing an augment of 50-fold of the rate of nuclear import of its protein (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In the Che-1 308\u0026ndash;325 aa region, two consecutive CK2 consensus sites (S/T-x-x-D/E/pS) are located in the proximity of its NLS (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). To investigate whether Che-1 could be a new CK2 target, we started testing if Che-1 physically interacts with CK2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;2A, immunoprecipitation experiments revealed that Che-1 co-precipitates with CK2. Next, we analysed the role of CK2 in Che-1 phosphorylation by performing 2D-electrophoresis experiments in HeLa cells treated or not with the CK2 inhibitor, 4,5,6,7\u0026ndash;Tetrabromobenzotriazole (TBB). From this experiment, we observed a strong decrease in Che-1 phosphorylation in the absence of CK2 kinase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;2B). Consistent with these findings, the depletion of CK2 expression by using specific siRNA stealth oligonucleotides (siCK2) induced a remarkable reduction of Che-1 phosphorylation when compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;2C). Next, we sought to determine the possibility that Che-1 was a direct substrate of CK2. To this aim, GST-Che-1 fusion protein was incubated or not with recombinant CK2 enzyme and subjected to \u003cem\u003ein vitro\u003c/em\u003e kinase assay. The kinase reaction products were analysed by nano-HPLC-MS/MS and the results obtained were then submitted to the SEQUEST search engine against the Uniprot Human protein database using the phosphoRS tool. This approach allowed identifying four Che-1 phosphopeptides, including those containing Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e (Table\u0026nbsp;1 and Supplementary Fig.\u0026nbsp;2D). As expected, no phosphopeptides were detected when Che-1 was incubated in the absence of CK2 (data not shown).\u003c/p\u003e \u003cp\u003eNext, we verified the effect of CK2 silencing on Che-1 cellular localization by performing immunofluorescence experiments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;2E, the absence of protein kinase does not affect nuclear localization of Che-1. Then, in order to confirm the role of these phosphorylations in Che-1 activity, we tested whether the absence of CK2 could affect Che-1/histone H3 binding. Co-immunoprecipitation experiments in HCT116 cells treated or not with TBB, demonstrated that the inhibition of CK2 kinase activity induces a drastic reduction of histone H3 interaction with Che-1 (Supplementary Fig.\u0026nbsp;2F), and similar results were obtained following CK2 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eAltogether, these data clearly demonstrate that CK2 phosphorylates Che-1 at Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e, and that these modifications are essential for Che-1 activity.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eThanks to its ability to interact with several transcription factors, Che-1 represent an important cofactor involved in multiple cellular processes (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In this context, post-translational modifications play a crucial role in controlling Che-1 co-transcriptional activity in response to different stimuli (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). We recently described Che-1 involvement in MM proliferation by affecting chromatin structure and sustaining global gene transcription. Also, we demonstrated that 308\u0026ndash;325 aa region of Che-1 is required for these activities (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we extended these results, confirming the roles played by Che-1 in the control of cellular proliferation and chromatin remodelling, in other cancer cell lines. More importantly, our results highlighted that the phosphorylations at Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e, contained in the Che-1 308\u0026ndash;325 aa region, are essential for Che-1 activity. Consistent with these findings, a Che-1 mutant lacking these residues (Che-1 3S) is unable to bind histones and to regulate chromatin structure and transcription. Finally, we identified CK2 as the protein kinase responsible for Che-1 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relevance of 308\u0026ndash;325 aa region in controlling Che-1\u0026rsquo;s functions has already been highlighted (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This region is highly conserved among several species and is present in many proteins involved in transcriptional regulation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition, phosphoproteomic studies have demonstrated Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e phosphorylation in all species analysed and that this modification is strongly abolished by the inhibition of different protein kinases implicated in cell cycle control (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In this study, we demonstrate the functional relevance of these modifications, since the replacement of these three serines produces a protein unable to modulate chromatin structure and gene transcription. This mutant does not bind histone proteins, and presumably serine phosphorylation is necessary to increase the acidic components of this region, thus enhancing the binding with the basic structure of histones. The importance of this region and its modifications is further confirmed by the analysis of the SV40 Large T protein (SV40 LT). This oncoprotein contains a region of high homology with the 308\u0026ndash;325 aa region of Che-1, and also it is able to bind histones, sustaining their acetylation, and to activate cell proliferation by regulating gene transcription (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). By producing a SV40 LT mutant protein, we were able to demonstrate that even in this case, the phosphorylation of these serines is necessary for the functionality of the protein, thus supporting the hypothesis of a close correlation between viral oncoproteins and Che-1.\u003c/p\u003e \u003cp\u003eFinally, we demonstrated that Che-1 is phosphorylated by CK2 at Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e in proliferating cells although in which phase of the cycle these modifications occurs, remain to be clarified. CK2 is highly expressed in several tumor cells, and its activity sustains cell proliferation (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), therefore it is considered an attractive target to counteract drug resistance in cancer. Indeed, specific CK2 inhibitors have shown proven efficacy on tumor cell proliferation and drug resistance (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Since Che-1 silencing is able to induce apoptosis or cell cycle arrest and sensitize MM cells to treatment with proteasome and BET inhibitors (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), targeting Che-1 by using CK2 inhibitors could represent an interesting novel therapeutic approach.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn conclusion, our findings clearly confirmed the global pro-proliferative ability of Che-1 in tumor cells. We highlighted the relevance of the phosphorylation at Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e for its functions and identified CK2 as the protein kinase responsible for this modification.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emultiple myeloma;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehepatocellular carcinoma;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCK2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCasein Kinase II;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSV40 LT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSimian virus 40 Large T;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRb\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eretinoblastoma;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewestern blot;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunoprecipitation;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMNase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicrococcal nuclease;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMass Spectrometry;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenosine 5\u0026rsquo;-triphosphate;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eλ-PP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elambda protein phosphate;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ewt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild type;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSer\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eserine;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eaa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eaminoacid;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear localization signal.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDINGS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Italian Association for Cancer Research (15255 to M.F.) and Ministry of Health (RF-2019-12368737 to M.F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR\u0026rsquo;S CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV.C., T.B and S.I. performed most of the experiments and analysed data; S.M. performed 2D gel electrophoresis assays; C.S. conducted immunofluorescence analyses; A.S. performed MS/MS analyses; G.D. participated in data analyses; M.F. conceived the projects, obtained funding and supervised the experiments; M.F and V.C. discussed the experiments and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mrs. Tania Merlino for the English language revision of the manuscript and Dr. Francesca Fabretti for technical and scientific support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBacalini MG, Tavolaro S, Peragine N, Marinelli M, Santangelo S, Del Giudice I, et al. A subset of chronic lymphocytic leukemia patients display reduced levels of PARP1 expression coupled with a defective irradiation-induced apoptosis. Exp Hematol. 2012;40(3):197-206 e1.\u003c/li\u003e\n\u003cli\u003eDesantis A, Bruno T, Catena V, De Nicola F, Goeman F, Iezzi S, et al. Che-1-induced inhibition of mTOR pathway enables stress-induced autophagy. EMBO J. 2015;34(9):1214-30.\u003c/li\u003e\n\u003cli\u003eKaul D. Cellular AATF gene: armour against HIV-1. Indian J Biochem Biophys. 2007;44(5):276-8.\u003c/li\u003e\n\u003cli\u003eUhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347(6220):1260419.\u003c/li\u003e\n\u003cli\u003eKumar DP, Santhekadur PK, Seneshaw M, Mirshahi F, Tuculescu CU, Sanyal AJ. A Novel Regulatory Role of Apoptosis Antagonizing Transcription Factor in the Pathogenesis of NAFLD and HCC. Hepatology. 2018.\u003c/li\u003e\n\u003cli\u003eIezzi S, Fanciulli M. Discovering Che-1/AATF: a new attractive target for cancer therapy. Front Genet. 2015;6:141.\u003c/li\u003e\n\u003cli\u003eIshigaki S, Fonseca SG, Oslowski CM, Jurczyk A, Shearstone JR, Zhu LJ, et al. AATF mediates an antiapoptotic effect of the unfolded protein response through transcriptional regulation of AKT1. Cell Death Differ. 2010;17(5):774-86.\u003c/li\u003e\n\u003cli\u003eCaliskan G, Baris IC, Ayaydin F, Dobson MJ, Senarisoy M, Boros IM, et al. Che1/AATF interacts with subunits of the histone acetyltransferase core module of SAGA complexes. PLoS One. 2017;12(12):e0189193.\u003c/li\u003e\n\u003cli\u003eWelcker D, Jain M, Khurshid S, Jokic M, Hohne M, Schmitt A, et al. AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer. Oncogene. 2018;37(11):1503-18.\u003c/li\u003e\n\u003cli\u003eBruno T, Valerio M, Casadei L, De Nicola F, Goeman F, Pallocca M, et al. 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Che-1/AATF-induced transcriptionally active chromatin promotes cell proliferation in multiple myeloma. Blood Adv. 2020;4(22):5616-30.\u003c/li\u003e\n\u003cli\u003eDe Nicola F, Catena V, Rinaldo C, Bruno T, Iezzi S, Sorino C, et al. HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation. Cell Death Dis. 2014;5:e1414.\u003c/li\u003e\n\u003cli\u003eHopker K, Hagmann H, Khurshid S, Chen S, Hasskamp P, Seeger-Nukpezah T, et al. AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. EMBO J. 2012;31(20):3961-75.\u003c/li\u003e\n\u003cli\u003eBruno T, Iezzi S, Fanciulli M. Che-1/AATF: A Critical Cofactor for Both Wild-Type- and Mutant-p53 Proteins. Front Oncol. 2016;6:34.\u003c/li\u003e\n\u003cli\u003eBorgo C, Ruzzene M. Role of protein kinase CK2 in antitumor drug resistance. J Exp Clin Canc Res. 2019;38.\u003c/li\u003e\n\u003cli\u003eSeldin DC, Lou DY, Toselli P, Landesman-Bollag E, Dominguez I. Gene targeting of CK2 catalytic subunits. Mol Cell Biochem. 2008;316(1-2):141-7.\u003c/li\u003e\n\u003cli\u003eHomma MK, Homma Y. Cell cycle and activation of CK2. Mol Cell Biochem. 2008;316(1-2):49-55.\u003c/li\u003e\n\u003cli\u003eCoccetti P, Tripodi F, Tedeschi G, Nonnis S, Marin O, Fantinato S, et al. The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G(1) to S transition in Saccharomyces cerevisiae. Cell Cycle. 2008;7(10):1391-401.\u003c/li\u003e\n\u003cli\u003eLi HC, Liu XS, Yang XM, Wang YM, Wang Y, Turner JR, et al. Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formation of kinetochore-microtubule attachments. Embo Journal. 2010;29(17):2953-65.\u003c/li\u003e\n\u003cli\u003eChua MMJ, Ortega CE, Sheikh A, Lee M, Abdul-Rassoul H, Hartshorn KL, et al. CK2 in Cancer: Cellular and Biochemical Mechanisms and Potential Therapeutic Target. Pharmaceuticals-Base. 2017;10(1).\u003c/li\u003e\n\u003cli\u003eDeplus R, Blanchon L, Rajavelu A, Boukaba A, Defrance M, Luciani J, et al. Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a. Cell Rep. 2014;8(3):743-53.\u003c/li\u003e\n\u003cli\u003eWu SY, Lee AY, Lai HT, Zhang H, Chiang CM. Phospho Switch Triggers Brd4 Chromatin Binding and Activator Recruitment for Gene-Specific Targeting. Mol Cell. 2013;49(5):843-57.\u003c/li\u003e\n\u003cli\u003eFanciulli M, Bruno T, Di Padova M, De Angelis R, Iezzi S, Iacobini C, et al. Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb. FASEB J. 2000;14(7):904-12.\u003c/li\u003e\n\u003cli\u003eSorino C, Catena V, Bruno T, De Nicola F, Scalera S, Bossi G, et al. Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription. Nucleic Acids Res. 2020;48(11):5891-906.\u003c/li\u003e\n\u003cli\u003eSorino C, Bruno T, Desantis A, Di Certo MG, Iezzi S, De Nicola F, et al. Centrosomal Che-1 protein is involved in the regulation of mitosis and DNA damage response by mediating pericentrin (PCNT)-dependent Chk1 protein localization. J Biol Chem. 2013;288(32):23348-57.\u003c/li\u003e\n\u003cli\u003eMarques-Santos LF, Grassi G, Bergami E, Faleri C, Balbi T, Salis A, et al. Cationic polystyrene nanoparticle and the sea urchin immune system: biocorona formation, cell toxicity, and multixenobiotic resistance phenotype. Nanotoxicology. 2018;12(8):847-67.\u003c/li\u003e\n\u003cli\u003eTaus T, Kocher T, Pichler P, Paschke C, Schmidt A, Henrich C, et al. Universal and Confident Phosphorylation Site Localization Using phosphoRS. Journal of Proteome Research. 2011;10(12):5354-62.\u003c/li\u003e\n\u003cli\u003eYamagata A KD, Takeda Y, Miyamoto Y, Okada K, Inamatsu M, Yoshizato K. Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics. 2002;2(9):1267-76.\u003c/li\u003e\n\u003cli\u003eZaret K. Micrococcal nuclease analysis of chromatin structure. Curr Protoc Mol Biol. 2005;Chapter 21:Unit 21 1.\u003c/li\u003e\n\u003cli\u003eAlayev A, Doubleday PF, Berger SM, Ballif BA, Holz MK. Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling. J Proteome Res. 2014;13(12):5734-42.\u003c/li\u003e\n\u003cli\u003eDephoure N, Zhou C, Villen J, Beausoleil SA, Bakalarski CE, Elledge SJ, et al. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 2008;105(31):10762-7.\u003c/li\u003e\n\u003cli\u003eSantamaria A, Wang B, Elowe S, Malik R, Zhang F, Bauer M, et al. The Plk1-dependent phosphoproteome of the early mitotic spindle. Mol Cell Proteomics. 2011;10(1):M110 004457.\u003c/li\u003e\n\u003cli\u003eValls E, de la Cruz X, Martinez-Balbas MA. The SV40 T antigen modulates CBP histone acetyltransferase activity. Nucleic Acids Res. 2003;31(12):3114-22.\u003c/li\u003e\n\u003cli\u003eRihs HP, Jans DA, Fan H, Peters R. The Rate of Nuclear Cytoplasmic Protein-Transport Is Determined by the Casein Kinase-Ii Site Flanking the Nuclear-Localization Sequence of the Sv40 T-Antigen. Embo Journal. 1991;10(3):633-9.\u003c/li\u003e\n\u003cli\u003eLindfors K, Halttunen T, Nupponen N, Vihinen M, Visakorpi T, Maki M, et al. Identification of a novel transcription factor-like gene repressed during TGF-beta induced human intestinal epithelial cell differentiation. Gastroenterology. 2000;118(4):A289-A.\u003c/li\u003e\n\u003cli\u003eXiao CY, Jans P, Jans DA. Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T-antigen NLS by importin: effect of conformation. Febs Lett. 1998;440(3):297-301.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. CK2-phosphorylated Che-1 residues identified by nano-LC-mass spectrometry\u003c/strong\u003e. MS/MS analysis of \u003cem\u003ein vitro\u003c/em\u003e phosphorylation assay showing Che-1 phosphopeptides including Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e. For each peptide the following details are reported:\u0026nbsp;aminoacid\u0026nbsp;sequence, phosphorylation site(s);\u0026nbsp;pRS\u0026nbsp;peptide score\u0026nbsp;(cumulative\u0026nbsp;binomal\u0026nbsp;probability that the observed match is a random events);\u0026nbsp;pRS\u0026nbsp;isoform probability (probability that\u0026nbsp;the isoform is correct);\u0026nbsp;pRS\u0026nbsp;site probability (probability of each site being truly phosphorylated);\u0026nbsp;\u003cem\u003eq\u003c/em\u003e-value (minimal\u0026nbsp;false discovery rate at which the identification is considered correct); PEP score (probability that the peptide-spectrum\u0026nbsp;match is incorrect); number of missed-cleavages.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg 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Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Che-1, cancer cells, cell proliferation, histone acetylation, phosphorylation, CK2, SV40 LT","lastPublishedDoi":"10.21203/rs.3.rs-459016/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-459016/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChe-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in Multiple Myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSite-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three-serine residue (Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e) in 308\u0026ndash;325 AA region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by \u003cem\u003ein vitro\u003c/em\u003e kinase, 2D gel electrophoresis assays and mass spectrometry analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, we confirm that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eChe-1 phosphorylation at Ser\u003csup\u003e316\u003c/sup\u003e, Ser\u003csup\u003e320\u003c/sup\u003e and Ser\u003csup\u003e321\u003c/sup\u003e by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.\u003c/p\u003e","manuscriptTitle":"CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-30 17:53:26","doi":"10.21203/rs.3.rs-459016/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-05-09T13:59:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-05-09T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-05-04T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-04-29T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-04-28T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-04-28T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-04-28T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2021-04-27T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-04-26T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-04-26T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Experimental \u0026 Clinical Cancer Research","date":"2021-04-23T09:21:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc041db8-cbf0-4890-aac5-d3778ff40f0b","owner":[],"postedDate":"April 30th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":4000542,"name":"Cancer Biology"},{"id":4000543,"name":"Systems and Networking"},{"id":4000544,"name":"Laboratory Diagnostics"}],"tags":[],"updatedAt":"2021-08-22T15:13:42+00:00","versionOfRecord":{"articleIdentity":"rs-459016","link":"https://doi.org/10.1186/s13046-021-02038-x","journal":{"identity":"journal-of-experimental-and-clinical-cancer-research","isVorOnly":false,"title":"Journal of Experimental \u0026 Clinical Cancer Research"},"publishedOn":"2021-07-15 15:02:30","publishedOnDateReadable":"July 15th, 2021"},"versionCreatedAt":"2021-04-30 17:53:26","video":"","vorDoi":"10.1186/s13046-021-02038-x","vorDoiUrl":"https://doi.org/10.1186/s13046-021-02038-x","workflowStages":[]},"version":"v1","identity":"rs-459016","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-459016","identity":"rs-459016","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}