Mammalian cells contain differentially functional compartments called organelles that are separated from cytoplasm by a lipid bilayer membrane. The nucleus is an extremely dynamic organelle and highly organized compartment with multiple functions [reviewed in
]]. When analyzed by indirect immunofluorescence microscopy, many nuclear proteins are seen to localize in distinct structures with punctate staining patterns
]. Nuclear structures, such as speckles, paraspeckles, nucleoli, Cajal bodies, polycomb bodies, and nuclear domain 10 (ND10, a.k.a. promyelocytic leukemia— PML body) are formed primarily by protein-protein, protein-RNA, or protein-DNA interactions
]. ND10 is a subnuclear structure that gathers many different SUMOylated nuclear proteins (such as Daxx and SP100). The formation of ND10 depends on PML protein. Past observations confirm that PML knockout cells lack ND10 and that transfecting exogenous PML into PML knockout cells results in the restoration of ND10. Most DNA viruses replicate DNA and transcribe genes in the nucleus after their genomic DNA enters the nucleus by facilitated transport through the nuclear pore complex
]. Once inside the nucleus, viral genomes distribute randomly, but it appears that only those at ND10 replicate and transcribe predominantly
], suggesting specifically that the environment at ND10 is particularly advantageous for the virus. However, the ND10 proteins (such as PML, Sp100, and Daxx) are interferon-upregulated and have repressive effects on viral replication
]. Moreover, most DNA viruses encode an immediate-early protein that induces the dispersion of ND10
], and in the absence of these viral proteins, replication is severely retarded
]. These findings have led to the hypothesis that ND10 sites are also a part of a nuclear defense mechanism
]. Therefore, the effects of ND10 on viral replication remain to be settled.
What is known definitively is that RNA transcripts distribute throughout the nucleus in either a diffuse or speckled pattern
]. As determined for both SV40 and HSV-1, the origin DNA was necessary but not sufficient for the virus to transcribe RNA and replicate DNA at ND10
]. The origin-binding proteins (large T-antigens of SV40 or ICP4 of HSV-1) were also required, suggesting that a DNA-protein complex precedes virus-ND10 association
]. The Everett group has observed that the HSV-1 genome is associated with ND10 proteins after infection and demonstrated that this association was formed by a new aggregation of ND10 components rather than by the migration of preexisting intact ND10 structures
]. Furthermore, Everett and colleagues recently discovered that the SUMO pathway is important for the recruitment of ND10-associated proteins to the HSV-1 genome
]. The studies of both SV40 and HSV-1 led to a new concept, that DNA-protein complexes might be sensed by ND10 or ND10 proteins. In in vivo
studies observing chromatin structure, the Belmont group introduced repetitive lac operator (lacO) sequences and a tightly binding lac repressor protein that was fused with GFP (GFP-lac repressor) into the nucleus and found that the GFP-lac repressor/Operator complexes localize at ND10
]. Later, the Spector group observed a dynamic interaction between PML and GFP-lac repressor/Operator complexes
]. Those observations led to our conceptual hypothesis that ND10 might be a “sensor” for recognizing DNA/protein complexes
]. We wondered whether the GFP-lac repressor/Operator system can be used to determine the effects of ND10 on gene expression.
In the present study, we first showed that the lac operator alone is not associated with ND10, although ND10 recognizes GFP-lac repressor/Operator complexes at a rate of 100%. We next inserted repetitive DNA into HSV-1 amplicons to make them visible and useful in the analysis of viral DNA sequences that, in the presence of DNA binding protein, are deposited at ND10. Our findings with respect to infectious DNA and transfected DNA suggest that instead of having sensors for proteins, cells possess mechanisms for recognizing deposits of DNA/protein complexes and segregating such complexes into loci containing several ND10-associated proteins. In addition, we found that HPV origin DNA/Origin-binding protein (E2) complexes can also be recognized by ND10. However, endogenous DNA/protein complexes were not associated with ND10. Therefore, our observations suggest that foreign DNA/protein complexes might be able to recruit or be recognized by ND10 proteins. Most importantly, the gene expression at ND10 was detected with less intensity than that was not associated with ND10, which demonstrated that ND10 is a restrictive site for gene expression for the tested DNA/protein complexes.