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. 2012 Sep 28:9:222.
doi: 10.1186/1743-422X-9-222.

Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression

Yisel A Rivera-Molina  1 , Bruno R Rojas, Qiyi Tang
Affiliations

Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression

Yisel A Rivera-Molina et al. Virol J. .

Abstract

Background: DNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription.

Results: Our recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML-/-, Daxx-/-, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10.

Conclusion: Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.

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Figures

Figure 1
Figure 1
Localization of integrated and transfected operator repeats containing no transcription units. Components labeled are indicated in the upper corners of the panels with their respective colors. (A) HEp-2-Op (derived from HEp-2 cells integrated with 10 kb = 256 operator repeats) probed by in situ hybridization for the location of the operator repeat insert; the operator DNA does not colocalize with PML. (B) HEp-2-Op (256 repeats) cells fused with HEp-2gfplacI cells. The single integrated operator repeat insert from 3 HEp-2-Op cell nuclei is found within PML-stained regions (yellow). The GFP-repressor is diffusely distributed in the three HEp-2gfplacI cell nuclei. (C) HEp-2-Op cells with 128 operator repeats were transfected with the GFP-repressor. Integrated operator repeats appear green (arrow). Insets show the color-separated small operator repeats at higher magnification. (D) HF cells probed by in situ hybridization for the location of the U2 gene locus relative to PML. The respective loci do not colocalize. (E) The same as (D), but showing a U2 locus localized adjacent to PML (arrow). (F) HEp-2 cells transfected with operator repeats and probed by in situ hybridization for operator location relative to PML. Operator plasmid aggregates do not colocalize with PML. (G) HEp-2 cells cotransfected with operator repeats and the GFP-repressor expression vector. The “green” operator/repressor complexes in the nucleus colocalize with PML. (H) MEF PML−/− cotransfected with operator repeats and the GFP-lac repressor expression vector and labeled for Daxx. Daxx colocalizes with the operator/repressor complex. The inset shows the area framed in blue at a higher magnification and with the colors separated. (I) MEF PML−/− cotransfected with operator repeats and the GFP-repressor expression vector and labeled for SUMO, which colocalizes with the operator/repressor complex. The inset shows the area framed in blue at a higher magnification and with the colors separated. (J) Mouse Daxx−/− cells cotransfected with operator repeats and the GFP-repressor expression vector and labeled for PML, which colocalizes with the operator/repressor complex. The inset shows the area framed in blue at a higher magnification and with the colors separated. (K) HEp-2 cells transfected with a plasmid-containing cellular heat shock promoter. In situ hybridization shows plasmid aggregates not colocalizing with PML. The inset shows the area framed in blue at a higher magnification and with the colors separated.
Figure 2
Figure 2
Localization of HPV origin DNA repeats in the nucleus after cotransfection with GFP-E2–expressing plasmid. Components labeled are indicated in the upper corners of the panels with their respective colors. (A)-(C): MEF cells cotransfected with pHPVori128 and pgfpE2; the oriDNA-GFPE2 complex is shown in green (B) and PML is shown in red (C); the merged picture is in (A). (D-F): The same as in (A-C), but cells are MEF PML−/− cells and show Daxx in red (F). (G-I), the same as in (A-C), but the cells are MEF Daxx−/− cells.
Figure 3
Figure 3
Localization of integrated operator repeats containing no transcription units after cotransfection with gfplacI- and CMV IE1-expressing plasmids. Components labeled are indicated in the upper corners of the panels with their respective colors. HEp-2-Op cells were cotransfected with pgfplacI and pIE1 for 24 hours, and then the cells were stained blue for IE1 (C) and red for PML (D). The lac Operator/GFP-lac repressor complexes are shown in green (B). All colors are merged in (A).
Figure 4
Figure 4
Gene transcription, ND10, and splicing compartments. (A) The plasmids used for making HSV-1 amplicons. (B) HEp-2 cells were transfected with pgfplacI; 6 hours later, the cells were super-infected with the amplicon ASK/E-Op for 2 hours. Then the cells were stained for ND10 using PML antibody and Texas Red-conjugated secondary antibody. Pictures were taken to show the relationship of ND10 and input amplicon DNA. (C) The same as (B), but the infection with the amplicon was at 12 hours, and the in situ hybridization experiment examined RNA (in blue), showing the relationship between lacZ gene transcription and ND10. (D) HEp-2 cells were infected with the amplicon (ASK/E-Op) for 2 hours; in situ hybridization was performed to show amplicon DNA (green). Immunofluorescence shows ND10 (red). (E) The same as (D), but the infection was for 12 hours; in situ hybridization was performed to show diffused β-gal RNA (DNA was degraded by DNAse treatment). (F) The same as (E), but the picture was taken to show the putative RNA. (G) HEp-2 cells were infected with the amplicon (ASK/E) and ICP0-deleted HSV-1 (as helper virus) for 12 hours; FISH was performed to show the distribution of lacZ RNA (green), ND10 (red), and splicing compartments (blue). (H) The same as (G) to show the relationship of RNA and splicing compartments. (I) The same as (G), to show the relationship of ND10 and RNA.
Figure 5
Figure 5
Beta-galactosidase assay. Detection of the activity of β-galactosidase was carried out using the β-Galactosidase Reporter Gene Staining Kit from Sigma-Aldrich, following the manufacturer’s protocol. Twenty-four hours after transfection of the plasmids (as indicated in Figure 5) in 293-T cells, the cells were washed three times with PBS and lysed with lysis buffer. Mock-infected 293T cells were used as controls. The samples were normalized to the sample amount of total protein. The β-Galactosidase activity in the sample was calculated taking into consideration the OD, final reaction volume, the absorbance of 1mM for an optical path of 1cm, and the incubation period (tmin). For the assays involving isopropyl-thio-b-D-galactopyranoside (IPTG), a final concentration of 0.4mM of IPTG was used. IPTG was added to the supplemented medium and left overnight. Each experiment was performed in triplicate and an average value obtained.
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