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 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.
Highlights
DNA in the nucleus is organized into a hierarchy of structures with the nucleosome as the basic building block
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]
Our recent experiments have shown that DEK changes the topology of DNA in chromatin in vitro [3]
Summary
Cell Culture and Cell Cycle Synchronization—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 [12]. S phase was determined by cell counting and by pulselabeling with [3H]thymidine and mitosis by mitotic indexes exactly as described recently (Fig. 1) [12]. Immunoblotting and Antibodies—Immunoblotting was carried out according to standard procedures. Proteins were separated by SDSPAGE [13] 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
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