Subcellular Localization of the Human Proto-oncogene Protein DEK

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture. Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture. serine/arginine-repeat polyacrylamide gel electrophoresis monoclonal antibody radioimmune precipitation buffer phosphate-buffered saline bovine serum albumin 1,4-piperazinediethanesulfonic acid minichromosome maintenance DNA in the nucleus is organized into a hierarchy of structures with the nucleosome as the basic building block. It has become widely accepted that modification of nucleosome structure is an important mechanism that regulates the accessibility of chromatin to DNA binding factors (1Kingston R.E. Narlikar G.J. Genes Dev. 1999; 13: 2339-2352Crossref PubMed Scopus (609) Google Scholar, 2Peterson C.L. Logie C. J. Cell. Biochem. 2000; 78: 179-185Crossref PubMed Scopus (67) Google Scholar). In the search for factors that change the structure of chromatin and the replicational activity of chromatin templates, we recently identified the proto-oncogene protein DEK as a candidate protein that changes the topology of DNA in chromatin in vitro (3Alexiadis V. Waldmann T. Andersen J. Mann M. Knippers R. Gruss C. Genes Dev. 2000; 14: 1308-1312PubMed Google Scholar). DEK is a 43-kDa phosphoprotein that was first isolated as part of a fusion protein expressed in a subtype of acute myeloid leukemias with (t6;9) chromosomal translocations (4von Lindern M. Fornerod M. van Baal S. Jaegle M. de Wit T. Buijs A. Grosveld G. Mol. Cell. Biol. 1992; 12: 1687-1697Crossref PubMed Scopus (332) Google Scholar). DEK was later identified as an autoimmune antigen in patients with pauciarticular onset juvenile rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune diseases (5Szer I.S. Sierakowska H. Szer W. J. Rheumatol. 1994; 21: 2136-2142PubMed Google Scholar, 6Dong X. Michelis M.A. Wang J. Bose R. DeLange T. Reeves W.H. Arthritis Rheum. 1998; 41: 1505-1510Crossref PubMed Scopus (38) Google Scholar, 7Wichmann I. Respaldiza N. Garcia-Lozano J.R. Montes M. Sanchez-Roman J. Nunez-Roldan A. Clin. Exp. Immunol. 2000; 119: 530-532Crossref PubMed Scopus (27) Google Scholar). In addition, DEK has been reported to be a site-specific DNA binding factor, which recognizes a specific DNA element in the human immunodeficiency virus enhancer (8Fu G.K. Grosveld G. Markovitz D.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1811-1815Crossref PubMed Scopus (78) Google Scholar). In a recent study, it was demonstrated that DEK associates with splicing complexes through interactions promoted by SR1 proteins. It was shown that DEK associates with mRNA in a splicing-dependent manner, indicating that it could function to coordinate splicing with subsequent steps in gene expression (9McGarvey T. Rosonina E. McCracken S. Li Q. Arnaout R. Mientjes E. Nickerson J.A. Awrey D. Greenblatt J. Grosveld G. Blencowe B.J. J. Cell Biol. 2000; 150: 309-320Crossref PubMed Scopus (110) Google Scholar). In addition DEK was found in a ∼335-kDa five-component complex at a conserved position 20–24 nucleotides upstream of exon-exon junctions (10Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (715) Google Scholar). Our recent experiments have identified DEK as a protein that induces alterations in the superhelical density of DNA in chromatin (3Alexiadis V. Waldmann T. Andersen J. Mann M. Knippers R. Gruss C. Genes Dev. 2000; 14: 1308-1312PubMed Google Scholar). The change in topology was only observed with chromatin but not with naked DNA and depends on the presence of histone H2A/H2B dimers. In addition we could show that DEK inhibits the replication efficiency of chromatin templates but not of naked DNA in vitro, demonstrating that DEK acts in a chromatin-specific manner. Association with chromatin has already been reported by Fornerod et al. (11Fornerod M. Boer J. van Baal S. Jaegle M. von Lindern M. Murti K.G. Davis D. Bonten J. Buijs A. Grosveld G. Oncogene. 1995; 10: 1739-1748PubMed Google Scholar), who demonstrated that DEK is associated with condensed chromosomes during metaphase. Thus, DEK seems to be a factor with dual RNA and DNA binding properties. In order to elucidate the localization of DEK in the cell, we performed fractionation studies. We found that DEK is an abundant protein in the cell and is eluted from the nuclei with 250 mm salt. Treatment of nuclei with RNase released only ∼10% of the DEK protein, whereas DNase treatment released most of the DEK protein, indicating that most of DEK is associated with chromatin in vivo. Treatment of nuclei with micrococcal nuclease followed by glycerol gradient sedimentation revealed that DEK is associated with oligonucleosomes. Chromatin fractionation studies demonstrated that DEK is more or less equally distributed on transcriptional active and inactive chromatin regions. The amount and localization of DEK does not change during the cell cycle. Human HeLa S3 cells were grown on plastic dishes in Dulbecco's modified Eagle's medium with 5% fetal calf serum. Cells were synchronized by a double thymidine block at the beginning of S phase and released into thymidine-free medium (12Ritzi M. Baack M. Musahl C. Romanowski P. Laskey R.A. Knippers R. J. Biol. Chem. 1998; 273: 24543-24549Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). S phase was determined by cell counting and by pulse-labeling with [3H]thymidine and mitosis by mitotic indexes exactly as described recently (Fig. 1) (12Ritzi M. Baack M. Musahl C. Romanowski P. Laskey R.A. Knippers R. J. Biol. Chem. 1998; 273: 24543-24549Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Immunoblotting was carried out according to standard procedures. Proteins were separated by SDS-PAGE (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and transferred to nitrocellulose. The membrane was blocked in Rotiblock solution (Roth) and incubated with different antibodies. Enhanced chemiluminescence reagents (ECL, Amersham Pharmacia Biotech) were used for detection. The polyclonal DEK antibodies (raised against His-DEK) were kindly provided by Gerald Grosveld (St. Jude Hospital, Memphis, TN) and the anti-SR antibodies mAb NM4 were a gift from Benjamin Blencowe (University of Toronto, Toronto, Ontario, Canada). The MCM5 antibodies have been described (12Ritzi M. Baack M. Musahl C. Romanowski P. Laskey R.A. Knippers R. J. Biol. Chem. 1998; 273: 24543-24549Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Cells were washed three times on the plate with ice cold hypotonic buffer A (20 mm HEPES pH 7.4, 20 mm NaCl, 5 mm MgCl2, 1 mm ATP) and lysed by Dounce homogenization. After 15 min on ice, the cytosolic supernatant was separated from the nuclear pellet by centrifugation (5 min, 600 × g). Nuclei were resuspended in buffer A supplemented with 0.5% Nonident P40 (Nonidet P-40) and kept on ice for 15 min to lyse the nuclear envelope. Centrifugation separated the free nucleosolic proteins from the pelleted nuclei (5 min, 1000 × g). The pellet was extracted for 15 min on ice in buffer B (20 mm HEPES, pH 7.4, 0.5 mm MgCl2, 1 mm ATP, 0.3m sucrose) plus NaCl in concentrations from 0.1 to 0.45m to release structure-bound proteins. The final pellet was extracted in RIPA (50 mm Tris-HCl, pH 8, 150 mmNaCl, 1% Nonidet P-40, 0.5% sodium desoxycholate). Proteins of each fraction were precipitated according to Wessel and Flügge (14Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar) and analyzed by SDS-PAGE and Western was isolated from the by acid as described Gruss C. 1995; PubMed Scopus Google Scholar) and by Nuclei were as described and washed in buffer (20 mm HEPES, pH 7.4, 0.5 mm MgCl2, 1 mm ATP, 0.3m sucrose) mm Nuclei of were incubated for min at with of DNase of cell nuclei or of of cell were and The was on ice with mm and pellet were separated by centrifugation micrococcal nuclease nuclei of were to mm and with micrococcal nuclease with the and The was on ice with mm chromatin were separated from by centrifugation min, × Proteins were from the DNA for min at in extracted as described by Wessel and Flügge (14Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar), and analyzed by SDS-PAGE and Western DNA was on by The supernatant from the micrococcal nuclease digestion was separated for on glycerol (20 mm HEPES, pH 7.4, 0.5 mm MgCl2, mm NaCl, 1 mm Proteins and DNA from the were analyzed as described The of and chromatin was based on the described by and J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar) with nuclei were isolated in buffer 15 pH mm mm pH 8, mm 0.5 mm 1 mm 0.5 mm supplemented with a and resuspended in of nuclear buffer (20 mm Tris-HCl, pH mm NaCl, 20 mm 5 mm supplemented with Nuclei was incubated with of micrococcal nuclease at The was on ice and min of and were min, The first supernatant was the The pellet was resuspended in of incubated for min on ice, and The supernatant and the pellet were the and of the were and the DNA was by gel electrophoresis and Proteins were extracted according to Wessel and Flügge (14Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar) and analyzed by SDS-PAGE and Western with antibodies. HeLa S3 cells grown on were for 15 min at in phosphate-buffered saline followed by a in Cells were blocked for min at in a in followed by the with the antibodies for 1 at After three steps in cells were incubated for min at with antibodies. antibodies were or After in the DNA was with in cell cells were first and extracted with pH 1 mm 5% different NaCl double thymidine were for 5 min in mm mm HEPES, pH 7.4, mm 0.5% and in followed by in with and with the first and antibodies. were from HeLa S3 cells for with cells were by centrifugation at 250 × for 5 min, resuspended in hypotonic buffer mm Tris-HCl, pH 7.4, mmNaCl, 5 mm and kept for 5 min at cells were at 250 × for 5 min, resuspended in buffer acid and incubated for min at After centrifugation the pellet was resuspended in a buffer and kept on The solution was on and at were washed for three times for min in mm pH mm mm NaCl, 1% 0.5% incubated with the antibodies for 1 in at After in mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% 1% chromosomes were for min at with the in DNA was with The DEK protein changes the topology of chromatin in vitro at a of to three of by with histone H2A/H2B (3Alexiadis V. Waldmann T. Andersen J. Mann M. Knippers R. Gruss C. Genes Dev. 2000; 14: 1308-1312PubMed Google Scholar). It was of to investigate the of the that we performed cell fractionation HeLa cells were in hypotonic buffer 5 mm R. 1992; PubMed Scopus Google Scholar) to a supernatant cytosolic proteins. The isolated nuclei were lysed in 0.5% Nonidet and to nucleosolic proteins. The nuclear structure chromatin and the nuclear was extracted with a buffer NaCl concentrations and with Most of the DEK protein at NaCl, and only small were also in the or extracted with or mm (Fig. 1 The of the DEK protein was with that of other nuclear proteins. We found that the DEK protein and protein J. Mol. Cell. Biol. 2000; PubMed Scopus Google S. M. Baack M. Knippers R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The factor found in the and only in the 250 mm and mm already most of the histone eluted from the nuclei with mm T. A. R. M. Nature. 2000; PubMed Scopus Google Scholar), a portion was also of the nuclei with The of nuclear proteins found by cell fractionation only the of a proteins to in the nucleus and to chromatin studies revealed a nuclear the were from (Fig. 1 The of nuclei with and most of the DEK protein was released 250 and mm (Fig. 1 in with the fractionation studies of A. DEK could be to nuclear structures RNA or To we nuclei with DNase or RNase. The of DEK was determined by Western and by (Fig. Treatment of nuclei with DNase released a fraction of DEK, whereas RNase released only a small portion of DEK (Fig. This was by A nuclear RNase treatment of nuclei, which was DNase digestion DEK was DNase treatment and could be to the of DEK with splicing complexes (9McGarvey T. Rosonina E. McCracken S. Li Q. Arnaout R. Mientjes E. Nickerson J.A. Awrey D. Greenblatt J. Grosveld G. Blencowe B.J. J. Cell Biol. 2000; 150: 309-320Crossref PubMed Scopus (110) Google Scholar). We found that the DEK DNase with by antibodies B.J. R. J. P. 1995; Google Scholar), indicating that fraction of DEK might be associated with splicing complexes in vivo. The cell fractionation and the data both that DEK is an abundant protein in the To the of DEK and the of the DEK protein to in we cell of HeLa The amount of DEK in a cell was determined by Western with antibodies in with of protein not The revealed that × of which to a of of Thus, DEK is an abundant protein in the To the of the DEK protein, we HeLa nuclei with of micrococcal nuclease an that the DNA We DNA of the of and Proteins were extracted from the and chromatin and by Western (Fig. and of micrococcal nuclease, to of DNA in chromatin concentrations the released to but to DNA from the chromatin pellet (Fig. This is with studies, which have reported that a fraction of chromatin is to nuclease at concentrations G. Biol. PubMed Scopus Google Scholar, E. Knippers R. J. Mol. Biol. PubMed Scopus Google Scholar, A. Baack M. M. Knippers R. Biol. Chem. 1998; Google Scholar). of the DEK protein were released already at concentrations However, the of DEK associated with an nuclear at micrococcal nuclease concentrations and times not To the the of by and with the fraction of DNA as a function of the used The that a fraction of DEK was already released with micrococcal nuclease indicating that a fraction of DEK resides in chromatin that more to chromatin. To on the the of micrococcal nuclease digestion were separated on glycerol of the were and by gel to the position of chromatin in the gradient of the gradient were used to the DEK protein (Fig. We found that of the DEK protein released treatment of nuclei with micrococcal nuclease with chromatin of that DEK associated with chromatin nuclease We the that at DEK be to and the chromatin with RNase on glycerol a small portion of DEK was released from and RNase treatment isolated DEK (Fig. RNase However, the of DEK was clearly to chromatin DNase digestion of chromatin in and the release of the DEK protein (Fig. Our is that a major portion of DEK is associated with chromatin. and J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar) have shown that of micrococcal chromatin a of active and inactive chromatin. We have used to investigate the of DEK to different chromatin regions. nuclei were for and min with micrococcal nuclease and to the supernatant fraction This fraction has been described to of active chromatin that is in histone and in proteins J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar). The pellet was resuspended in an buffer and to supernatant fraction which has been shown to be of J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar). The fraction the nuclear with The to be to associated protein complexes as RNA J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar) or the which has also been in fraction C. M. J. Cell Biol. 1997; PubMed Scopus Google Scholar). shown by DNA on an the in nuclease accessibility of chromatin 5% and of the cellular is of whereas fraction of cellular showed a of DNA The with of cellular of DNA (Fig. 5 of and were to and the DEK protein was by with antibodies (Fig. 5 We found that the DEK protein is in three However, into that fraction the major part of the the DEK protein is in fraction with to fraction This that the DEK protein is more abundant in active chromatin. In order to investigate the localization of DEK during the cell HeLa cells were at the and S phase by a double thymidine block R.E. P. R. The Cell A Scholar). of as determined by the of DNA 1 and thymidine and for not cell were at the times thymidine release and as described (Fig. Proteins were separated by and the DEK protein was with antibodies. a Western were also with antibodies maintenance proteins to from chromatin during S phase T. Musahl C. Laskey R.A. Knippers R. J. Cell Sci. PubMed Google Scholar), but to chromatin at the of mitosis (12Ritzi M. Baack M. Musahl C. Romanowski P. Laskey R.A. Knippers R. J. Biol. Chem. 1998; 273: 24543-24549Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). shown in DEK on chromatin during of the cell cycle. Furthermore, DEK in could be eluted at 250 mm from that the of does not change during S phase chromatin is The is the MCM5 protein, which was found on phase chromatin (Fig. but not on chromatin from S phase and phase cells were release of HeLa cells from a no change in the amount and localization of the DEK protein during the phase not was used to investigate the of DEK with chromatin during HeLa cells were released from a thymidine block and later and with The localization of DEK was with antibodies and antibodies We found that DEK is associated with chromatin during of in to and not In addition, we chromosomes from cells (Fig. After treatment with we found a of the Thus, DEK to mitotic chromatin. Our recent experiments have shown that DEK changes the topology of DNA in chromatin in vitro (3Alexiadis V. Waldmann T. Andersen J. Mann M. Knippers R. Gruss C. Genes Dev. 2000; 14: 1308-1312PubMed Google Scholar). It was of to DEK is in with chromatin in vivo. In we that the DEK protein is associated with chromatin in vivo. We found that of the DEK protein be released from nuclei with micrococcal nuclease The fraction in the nucleus with of micrococcal of released chromatin on glycerol revealed that DEK with DNA After treatment of these with a small fraction of DEK was released and at the position of free DEK This that a fraction of DEK is associated with complexes in as has been shown (9McGarvey T. Rosonina E. McCracken S. Li Q. Arnaout R. Mientjes E. Nickerson J.A. Awrey D. Greenblatt J. Grosveld G. Blencowe B.J. J. Cell Biol. 2000; 150: 309-320Crossref PubMed Scopus (110) Google Scholar). A of a fraction of DEK protein with splicing complexes was also by with anti-SR antibodies. In addition to the of DEK, which by treatment with micrococcal nuclease and and DEK, a fraction which could not be released from nuclei by micrococcal nuclease or DNase treatment but is eluted with 250 mm and might be associated with regions. the of DEK in the nucleus different of the protein has to be In DEK be (11Fornerod M. Boer J. van Baal S. Jaegle M. von Lindern M. Murti K.G. Davis D. Bonten J. Buijs A. Grosveld G. Oncogene. 1995; 10: 1739-1748PubMed Google Scholar), but or other the protein to different chromatin or to is not DEK is not nuclear proteins that to chromatin and to RNA of factors with dual RNA and DNA binding the DEK protein, of these factors also in human To the protein which to DNA but also splicing factor in the nucleus D. M. A. van V. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, C. E. Genes Dev. 1998; 12: PubMed Scopus Google Scholar). In addition the to DNA and also with protein splicing factors S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the factor a specific factor has been shown to splicing activity M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google M. A. S. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). is the fusion of the DEK protein with expressed in acute (4von Lindern M. Fornerod M. van Baal S. Jaegle M. de Wit T. Buijs A. Grosveld G. Mol. Cell. Biol. 1992; 12: 1687-1697Crossref PubMed Scopus (332) Google Scholar), the of DEK on chromatin and to the of the protein or alterations in RNA for the DEK is an abundant protein with more a A of the DEK acid with in data identified of and expressed but not a significant with of the DEK protein show to the P. M. EMBO J. 1997; PubMed Scopus Google Scholar, M. T. van van R. M. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar), also and Biochem. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), a in chromosomal This acid a of conserved and separated by a that an M. T. van van R. M. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). The in might with the DNA have been found in different proteins for the and the protein, in DNA Furthermore, the is associated with different proteins in the of complexes Biochem. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and might a of in to active chromatin. It has also been shown that the chromosomes by binding to and release by the chromosomes to during cell and chromatin P. M. EMBO J. 1997; PubMed Scopus Google Scholar, M. M. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the a of proteins to specific chromosomal In the of the DEK protein, the with chromatin might be mediated by the binding of the to DNA and by interactions with histone (3Alexiadis V. Waldmann T. Andersen J. Mann M. Knippers R. Gruss C. Genes Dev. 2000; 14: 1308-1312PubMed Google Scholar). In with experiments (11Fornerod M. Boer J. van Baal S. Jaegle M. von Lindern M. Murti K.G. Davis D. Bonten J. Buijs A. Grosveld G. Oncogene. 1995; 10: 1739-1748PubMed Google Scholar), data show a of the DEK protein with chromatin during In addition we could demonstrate that is no change in the amount and localization of the DEK protein during the cell indicating that DEK is associated with chromatin during the cell cycle. This that the DEK protein might be in of chromatin. Recent experiments have demonstrated that different proteins as the binding protein the splicing factor and the protein which in nuclear the nucleus T. Nature. 2000; PubMed Scopus Google Scholar). In addition it was found that the of histone protein is to chromatin at but is chromatin T. A. R. M. Nature. 2000; PubMed Scopus Google Scholar, M.A. X. M.J. Nature. 2000; PubMed Scopus Google Scholar). Thus, protein on chromatin in cells for and to an binding T. A. R. M. Nature. 2000; PubMed Scopus Google Scholar). Our studies on the of the DEK protein with nuclear structures have analyzed the of the DEK In of the data that most proteins in the it that the DEK protein also chromatin or chromatin and We to and for and of the The expression and the DEK antibodies were a gift from Gerald The mAb NM4 antibodies were kindly provided by Benjamin

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