Subcellular Localization of the Human Proto-oncogene Protein DEK

Abstract

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 cosedimentates 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.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 (1, 2).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 (3). 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 (4). DEK was later identified as an autoimmune antigen in patients with pauciarticular onset juvenile rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune diseases (5-7). 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 (8).In a recent study, it was demonstrated that DEK associates with splicing complexes through interactions promoted by SR 1 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 (9). In addition DEK was found in a ϳ335-kDa five-component complex at a conserved position 20 -24 nucleotides upstream of exon-exon junctions (10).Our recent experiments have identified DEK as a protein that induces alterations in the superhelical density of DNA in chromatin (3). The change in topology was only observed with chromatin but not with naked DNA and depends on...

DEK over-expression promotes mitotic defects and micronucleus formation

“…This finding is in contrast to a previous report 51 showing DEK bound to metaphase chromosomes. This discrepancy may be due to previous results being obtained from cells being treated with nocodazole, compared to our studies carried out in untreated cells.…”

“…42,43,51 However, we find that DEK is mostly absent Figure 4. A DEK-GFP fusion protein is retained on mitotic chromosomes and associates with lagging chromosomes.…”

“…2A), with the proportion of cells in G1, S and G2/M quantified in Figure 2B. Interestingly, while DEK protein levels were relatively constant upon G1 and S arrest as previously reported, 51 DEK protein levels decreased dramatically in mitotically enriched cells following mitotic shake off (Fig. 2C).…”

“…38 Previous studies have suggested that DEK associates with mitotic chromatin, potentially in a stage-dependent manner. For instance, using immunofluorescence and metaphase spreads, Kappes et al 2001 observed that DEK is detectable on HeLa cell chromatin during metaphase and anaphase but appears to be absent in prometaphase. Two other reports confirm DEK association with chromatin in telophase cells, 42,43 with one showing DEK absence from metaphase cells.…”

Defining a 0.5-Mb Region of Genomic Gain on Chromosome 6p22 in Bladder Cancer by Quantitative-Multiplex Polymerase Chain Reaction

“…While its physiological function is not known, DEK may influence the topology of DNA in chromatin 29,30 and regulate specific post-splicing steps in gene expression. 31 The role of DEK in the pathogenesis of disease is also unknown.…”