ABSTRACT
Upon virus infection of a cell, the uncoated DNA is usually blocked by the host intrinsic immune system inside the nucleus. Although it is crucial for the virus to counteract the host intrinsic immune system and access its genome, little is known about how viruses can knock down host restriction and identify their blocked genomes for later viral gene activation and replication. We found that upon baculovirus transduction into Vero E6 cells, the invading viral DNA is trapped by the cellular death domain-associated protein (Daxx) and histone H3.3 in the nucleus, resulting in gene inactivation. IE2, a baculovirus transactivator, targets host Daxx through IE2 SUMO-interacting motifs (SIMs) to indirectly access viral DNA and forms unique nuclear body structures, which we term clathrate cage-like apparatus (CCLAs), at the early transduction stage. At the later transduction stage, CCLAs gradually enlarge, and IE2 continues to closely interact with viral DNA but no longer associates with Daxx. The association with Daxx is essential for IE2 CCLA formation, and the enlarged CCLAs are capable of transactivating viral but not chromosomal DNA of Vero E6 cells. Our study reveals that baculovirus IE2 counteracts the cellular intrinsic immune system by specifically targeting Daxx and H3.3 to associate with viral DNA indirectly and efficiently. IE2 then utilizes this association with viral DNA to establish a unique CCLA cellular nanomachinery, which is visible under light microscopy as an enclosed environment for proper viral gene expression.
IMPORTANCE The major breakthrough of this work is that viral protein IE2 localizes and transactivates its own viral DNA through a most unlikely route, i.e., host proteins Daxx and H3.3, which are designed to efficiently restrict viral DNA from expression. By interacting with these host intrinsic immune factors, IE2 can thus target the viral DNA and then form a unique spherical nuclear body, which we name the CCLA, to enclose the viral DNA and necessary factors to assist in high-level transactivation. Our study represents one of the most complete investigations of nuclear body formation. In addition, so far only RNA or protein molecules have been reported as potential nucleators for initiating nuclear body formation; our study may represent the first example showing that DNA can be a nucleator for a new class of nuclear body formation.
INTRODUCTION
Viruses are efficient entities at invading many cell types and are capable of exploiting host cellular machineries, known or unknown, in many ways for their own benefit. Upon entering the cell nucleus, invading viral DNA usually encounters host intrinsic immune mechanisms, including death domain-associated protein (Daxx), which is known to be an important factor in the cellular control of viral gene expression (1). Daxx is usually associated with promyelocytic leukemia nuclear bodies (PML-NBs), the transcription repressor Sp100, and alpha-thalassemia X-linked mental retardation protein (ATRX) that together form an effective intrinsic immune response machinery to inhibit the gene expression of invading DNA (2). Further studies have shown that Daxx acts as a chaperone for the histone H3.3 variant for chromatin deposition (3).
Viruses have developed mechanisms to counteract this strong host machinery that blocks expression of invading DNA. The immediate early ICP0 protein of herpes simplex virus 1 (HSV-1) is a RING finger ubiquitin ligase that can disrupt PML-NB, overcoming the restriction mediated by these host proteins (4, 5). BNRF1 of Epstein-Barr virus (EBV) disrupts Daxx-ATRX-mediated H3.3 loading on viral chromatin to activate viral early gene transcription (6). Although there have been extensive studies on mammal-specific viruses and substantial knowledge has been gained about how they interact with their hosts and how their viral products disrupt or degrade Daxx for viral propagation (1), insect viruses are much more diverse and have a much longer evolutionary history than mammalian viruses (7, 8), so markedly unique but unknown insect host cellular machineries could have been exploited by viruses long before the divergence of vertebrates and invertebrates, which persist in the insect lineage.
Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the archetype alphabaculovirus, is an enveloped, rod-shaped insect virus that possesses a large double-stranded DNA genome (133 kbp) and contains around 155 open reading frames (9, 10). It is one of the most widely used tools for protein expression in its natural host, insect cells (11). In nonpermissive mammalian systems, most of the transduced baculovirus genes remain silent (12–15), but foreign genes inserted into the viral genome can be turned on by mammalian promoters, such as cytomegalovirus immediate early promoter (CMV) or simian virus 40 early promoters (SV40) for efficient gene delivery, tissue engineering, and gene therapy, thus making baculovirus a very useful vector system broadly used in mammalian cells and organisms (9, 11, 16–19).
Baculovirus immediate early gene 2 (ie2) encodes the transactivator IE2, originally called IEN, for baculovirus gene expression in insect cells (20). It is one of the earliest baculovirus gene products produced immediately upon viral infection in cells of lepidopteran insects (21, 22). This highly structured protein contains two nuclear localization sequences (NLS) at its N-terminal end, a C3HC4-type RING finger domain, and a coiled-coil domain important for IE2 protein oligomerization at its C terminus (23) (Fig. 1A). IE2 protein has been shown to cause S-phase cell cycle arrest in Sf21 cells, to exhibit ubiquitin ligase activity in vitro, and to form distinct nuclear foci (21, 23). Previously, we found that IE2 can also strongly stimulate the activity of the CMV promoter in mammalian cells, which could increase baculovirus transgene expression in mammalian systems (9, 23). These nonhost mammalian cells provide a unique opportunity to study the intriguing mechanism by which the “naive” viral gene ie2 can function so well in mammalian cells.
IE2 transactivates expression of foreign genes driven by the TriEx promoter in Vero E6 cells. (A) Schematic showing the known domains of IE2. NLS, nuclear localization sequences; RING, RING finger domain; coiled-coil, coiled-coil domain. Numbers indicate amino acid boundaries. (B) Plasmid constructs in this study. All the expression constructs are driven by a composite of p10, CMV, and T7 promoters (TriEx) that allows target gene expression in insect, mammalian, and bacterial cells. pAcIE2, wt IE2; pAcSIM-66, pAcSIM-108, and pAcSIM-DM, IE2 constructs harboring mutations that disrupt the putative SIMs at positions 66 (SIM-66), 108 (SIM-108), or both (SIM double mutant [SIM-DM]), respectively; pAcC230S, IE2 RING domain mutant; pAcEGFP, green fluorescence reporter gene; pAcLuc, luciferase reporter gene. (C) Transactivation of IE2 in Vero E6 cells. The green fluorescence images of Vero E6 cells transduced with different recombinant viruses were captured at the indicated times. hpt, hours posttransduction. (D) Western blot (WB) analysis for the Vero E6 cell samples in panel C using anti-His antibody. GAPDH was used as a loading control. N, cell-only control; EG, cells transduced with vAcEGFP; EG+IE2, cells transduced with both vAcEGFP and vAcIE2.
Previously, we observed that IE2 forms nuclear bodies that colocalize with actin and RNA polymerase II (Pol-II) in Vero E6 cells (23). The underlying mechanisms of how nuclear bodies are formed and their role in high level gene expression are intriguing but largely unknown. In this study, we have elucidated a novel mechanism by which a viral factor copes with the host intrinsic immune response to release the viral genome from host suppression and stimulates strong gene expression through generation of a unique functional structure, which we call a clathrate cage-like apparatus (CCLA). “Clathrate” is a term conventionally used for a chemical structure consisting of a lattice that traps or contains molecules (24, 25). This newly discovered IE2 CCLA structure, visible under light microscopy, is a novel transactivation unit. CCLAs were found to build on viral DNA, actively recruit the factors necessary for transactivation of viral genomic DNA, generate abundant mRNA, and eventually result in high-level protein expression. We provide new insights into important virological subjects concerning how viruses initiate their infections and activate gene transactivation against general host restriction mechanisms. Furthermore, baculoviruses are now broadly used in mammalian systems for efficient gene delivery (11, 17, 18, 26–29), so our study may also provide a way for enhanced baculovirus applications in these systems.
RESULTS
The transactivation function of IE2 and colocalization of IE2 CCLAs with viral DNA.Previously, we reported that transduction of IE2 into Vero E6 cells could significantly upregulate the expression of a CMV promoter (23). Intriguingly, evidence shows that IE2 may not directly bind to the specific DNA sequence during its transactivation (23, 30–32). In order to study the mechanisms underlying the functions of IE2, we constructed recombinant baculoviruses carrying ie2 genes with or without mutations of functional IE2 motifs (Fig. 1B). Whereas vAcEGFP mediated enhanced green fluorescent protein (EGFP) expression in Vero E6 cells, cotransduction of vAcIE2 (whereby IE2 is driven by the CMV promoter within the composite TriEx promoter) resulted in a pronounced increase of EGFP expression from the early transduction stage (24 h posttransduction [hpt]). This transactivation was further enhanced at the later transduction stage (48 hpt), as revealed by both the fluorescence signal (Fig. 1C) and our Western blotting (Fig. 1D). We previously observed that IE2 colocalized with the transcription-related factors actin and RNA Pol-II in the nuclei of vAcIE2-transduced Vero E6 cells (23). We termed this IE2 nuclear body structure together with its enclosed cellular components as IE2 CCLA or just CCLA. We wondered if this colocalization is important for the transactivation function of IE2. Through immunofluorescence (IF) and DNA fluorescence in situ hybridization (FISH), we found that IE2 CCLAs strictly colocalized with the transduced viral DNAs at 48 hpt (Fig. 2). Since IE2 does not bind directly to any known specific DNA sequence, we speculated that the formation of CCLA structures may be crucial for IE2 to interact with the DNA to facilitate transactivation.
IE2 tightly colocalizes with viral DNA inside the nucleus. The association between IE2 and viral DNA was detected by DNA FISH. After transduction of vAcIE2 virus into Vero E6 cells (48 hpt), IE2 protein was labeled by anti-IE2 antibody (red), and a purified baculovirus genomic DNA was used as the probe for detecting the location of viral DNA (green). IE2 (red) was found to closely associate with viral DNA (green). Individual channels of the cell delimited by the white box are shown at the right. Bars: 10 μm.
Daxx and H3.3 interact with IE2 CCLAs in the early transduction stage.It has become increasingly clear in recent years that Daxx and H3.3 can suppress foreign DNA and viral gene transcription through epigenetic chromatin silencing (33–35). We investigated if IE2 CCLAs interact with Daxx and H3.3, thereby indirectly targeting viral DNA. Our IF analysis of AcIE2-transduced Vero E6 cells showed that IE2 colocalized well with Daxx and H3.3 by 24 hpt (Fig. 3A), manifested as tiny nuclear foci. By 48 hpt, some of the IE2 CCLAs began to enlarge (Fig. 3B; see inset for an example), but by this stage colocalization of IE2 CCLAs with both Daxx and H3.3 decreased significantly. This decrease was especially evident when we compared line scans of fluorescent signals between the early (24 hpt) and late (48 hpt) stages; the fluorescence peak of IE2 (red) remained high at 48 hpt, but those of Daxx (green) and H3.3 (blue) did not (Fig. 3A and B, bottom rows), as corroborated by the colocalization coefficient evaluated from multiple cells (Fig. 3C). As a mock control, we determined the localization of Daxx and H3.3 in Vero E6 cells in the absence of virus transduction, and we found that Daxx and H3.3 protein were diffusely distributed but colocalized with each other in the nuclei of cells at both 24 and 48 hpt (Fig. 3D to F).
IE2 associations with Daxx and H3.3 at the early transduction stage and escapes Daxx suppression of gene expression at the late transduction stage. Associations between IE2 (red), Daxx (green), and histone H3.3 (blue) were detected by IF analysis at 24 (A) and 48 (B) hpt. Insets are magnifications of regions marked by white boxes. Bottom rows show the intensity line plots along yellow arrows. (C) The colocalization coefficients were calculated for IE2, Daxx, and H3.3 from 38 randomly selected cells separately at 24 hpt and 48 hpt. Cells without virus transduction (mock transduction) were also examined at 24 hpt (D) and 48 hpt (E) as negative controls. (F) Colocalization coefficients calculated for IE2, Daxx, and H3.3 in mock-transduced cells. Results are shown as means and SDs (error bars). *, significant difference (P < 0.05) by unpaired t test. ns, not significant. Bars: 5 μm.
Although we studied proteins with and without colocalization by IF in these experiments, further studies are necessary to fully establish their associations. We also performed coimmunoprecipitation (co-IP) experiments to study the association between IE2 and Daxx. As shown in Fig. 4, lysates from cells transduced with mock control, vAcEGFP, vAcIE2, and vAcSIM-DM (SIM-DM) were individually immunoprecipitated using Daxx antibody and then subjected to Western blot analysis using antibody against IE2, H3.3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The recombinant virus vAcSIM-DM expresses IE2 with double mutations on SUMO-interacting motifs (SIMs) (Fig. 1A and B), allowing us to study the role played by SUMO in the associations between IE2 and Daxx. We found that the wild-type (wt) IE2 from vAcIE2 associated with Daxx at 24 hpt but not at 48 hpt (Fig. 4). This result reveals that the association between IE2 and Daxx happens only at the early transduction stage (24 hpt) and not at the later stage (48 hpt). The experiments involving vAcSIM-DM transduction also showed that IE2 does not associate with Daxx hosting SIM double mutations at both transduction stages, and the function of these SIMs is described further below.
Analysis of IE2 and Daxx associations by the co-IP assays. A co-IP assay was performed to assess associations between Daxx and IE2. Vero E6 cells were transduced with recombinant baculoviruses. The transduced cells were collected, and the input cellular lysates (Input) were analyzed by antibodies against Daxx, IE2, H3.3, and GAPDH. Then the lysates were immunoprecipitated with anti-Daxx antibody and analyzed for the presence of Daxx, IE2, H3.3, and GAPDH by individual antibodies against these proteins. N, mock control; EG, cells transduced with vAcEGFP for EGFP expression; IE2, cells transduced with vAcIE2 for wt IE2 expression; SIM-DM, cells transduced with vAcSIM-DM for the expression of an IE2 double SIM mutants at both residues 66 to 68 and 108 to 110.
Daxx and H3.3 dissociate from viral DNAs in the late transduction stage in the presence of IE2.Since Daxx and H3.3 are known to target invading DNA and we showed that Daxx and H3.3 did not interact with IE2 in the late stage (48 hpt) (Fig. 3B and C and Fig. 4), we wondered if Daxx and H3.3 still associates with viral DNA at this stage. The colocalization of Daxx (Fig. 5) and H3.3 (Fig. 6) with IE2 and viral DNA were analyzed by IF and DNA FISH. We found that while IE2 CCLAs tightly associated with viral DNA from early to late stages, Daxx and H3.3 colocalized with viral DNA and IE2 CCLAs only at 24 hpt (Fig. 5A and Fig. 6A) and largely dissociated from them at the late 48 hpt stage (Fig. 5B and C and Fig. 6B and C).
Colocalization study of IE2, Daxx, and DNA at different transduction stages. The colocalization of IE2, Daxx, and DNA was determined by DNA FISH assay at 24 hpt (A) and 48 hpt (B). IE2 CCLAs (blue) were found to colocalize with viral DNA (green) at both 24 hpt and 48 hpt but only with Daxx (red) at 24 hpt. Regions marked with a white box are magnified as insets. Fluorescence intensity along arrows is plotted at the bottom. Bars: 5 μm. (C) Colocalization coefficients were calculated from 40 randomly selected cells separately at 24 hpt and 48 hpt. Results are shown as means and SDs (error bars).
Colocalization study of IE2, H3.3, and DNA at different transduction stages. DNA FISH assay determined the colocalization of IE2, H3.3, and DNA at 24 hpt (A) and 48 hpt (B). IE2 CCLAs (blue) colocalized with viral DNA (green) at both 24 hpt and 48 hpt but only with H3.3 (red) at 24 hpt. Regions marked with a white box are magnified as insets. Fluorescence intensity along arrows is plotted at the bottom. Bars: 5 μm. (C) Colocalization coefficients were calculated from 40 randomly selected cells separately at 24 hpt and 48 hpt. Results are shown as means and SDs (error bars).
In order to confirm this late dissociation of these factors from viral DNA, chromatin immunoprecipitation (ChIP) using Daxx-specific antibody was performed to isolate the Daxx-associated chromatins from cells transduced with vAcEGFP, vAcIE2, vAcC230S (C230S, an IE2 RING domain mutant virus), and vAcSIM-DM at both 24 and 48 hpt. We then analyzed by quantitative PCR (qPCR) the sequence contents of the CMV, ie1, and gp64 promoters from the baculovirus genome, as well as the gapdh and hsp70 promoters from the host genome, in the precipitated chromatins versus the input chromatins (Fig. 7A and B). A rabbit IgG antibody was used in parallel to immunoprecipitate the same chromatins as in Fig. 7A and B to evaluate the nonspecific background signal (Fig. 7C and D). Our results show that Daxx associated with viral DNA at the early transduction stage (24 hpt) in both the presence and absence of functional IE2, which was not the case for host DNA (Fig. 7A, gapdh and hsp70). Although Daxx associated much more strongly with viral DNA at 48 hpt for most of the recombinant viruses we tested, Daxx was found to significantly dissociate from the viral DNA at this stage upon introduction of functional IE2 by vAcIE2 transduction (Fig. 7B).
Chromatin immunoprecipitation (ChIP) analysis using Daxx-specific antibody to analyze Daxx-associating DNA sequences. Vero E6 cells were transduced with vAcEGFP (EG), vAcIE2 (IE2), vAcC230S (C230S), and vAcSIM-DM (SIM-DM) viruses. Cell lysates were harvested at 24 hpt (A) and 48 hpt (B) and the extracted chromatins were precipitated by an anti-Daxx antibody to analyze the specific DNA fragments bound by Daxx. Chromatins extracted from the identical cell lysates precipitated by a rabbit IgG antibody were used to detect the background signal (C and D), and the chromatins without any antibody precipitation served as the input. Both the precipitated and input chromatins were analyzed by qPCR using primers for specific promoter regions. Data are expressed as the percentage of precipitated DNA relative to input DNA (mean values ± SDs). †, significant difference (P < 0.05) versus the respective IgG ChIP signal; *, significant difference (P < 0.05) versus IE2 by unpaired t test.
Requirement for Daxx in IE2 CCLA growth and transactivation.To determine the role of Daxx in the formation and function of IE2 CCLAs, we knocked down Daxx by small interfering RNA (siRNA) (Fig. 8). Compared to cells treated with the negative-control siRNA (siControl), cells treated with siRNA targeting Daxx (siDaxx) showed weak or almost no IF Daxx signal in the nuclei (Fig. 8A to C). Western blotting also showed a decrease of Daxx protein expression in siDaxx-treated cells (Fig. 8D). In the absence of Daxx, IE2 could only form small dots in the nuclei of siDaxx-treated cells at 24 hpt (Fig. 8A), and it no longer formed enlarged CCLAs at 48 hpt (Fig. 8B). A luciferase activity assay, in which the virus-introduced luciferase coding region was driven by the CMV promoter (vAcLuc), was also performed (Fig. 9). The transactivation of IE2 to CMV promoter could thus be quantified by measuring the activity of luciferase using a luminometer. We found that Daxx knockdown (siDaxx) resulted in a general increase of luciferase expression compared to the control (siControl). Interestingly, IE2 could enhance viral gene expression only upon proper Daxx expression (Fig. 9, columns 4 and 14), but IE2 could not stimulate better CMV promoter activation under Daxx knockdown (Fig. 9, columns 9 and 19). Consistent with our IF analyses, these results suggest that IE2 CCLAs may specifically target the Daxx/H3.3 complex to antagonize its repression of viral DNA transcription.
Daxx knockdown blocks the enlargement of IE2 CCLAs. Vero E6 cells were transfected by specific Daxx-targeting siRNAs (siDaxx) or siRNA control (siControl), and IF analysis was used to detect the presence of IE2 (vAcIE2 transduction [red]) and Daxx (green) at 24 hpt (A) and 48 hpt (B) after Daxx knockdown by siRNA. Cells treated with siControl were tested as a negative control. Bars: 5 μm. (C) Colocalization coefficients were calculated from 51 randomly selected cells separately at 24 hpt and 48 hpt. Results are shown as means and SDs (error bars). (D) Western blot analysis showing Daxx knockdown efficiency for the cell samples used in panels A and B. The levels of Daxx were analyzed by antibody against Daxx, and the levels of cellular GAPDH were employed as loading controls. N, cell-only control.
IE2-induced transactivation requires a functional Daxx. A luciferase assay was performed to determine transactivation activity in the presence and absence of Daxx. siControl or siDaxx was transfected into Vero E6 cells, followed by vAcEGFP (as control), vAcIE2, or vAcSIM-DM transduction. Luciferase virus (vAcLuc), in which the luc gene was driven by the CMV promoter, was cotransduced as a reporter. The cell extracts were separately harvested at 24 hpt (A) and 48 hpt (B) for luciferase assay. Results are shown as means and SDs (error bars) of at least four replicates. *, significant difference from value for vAcIE2 (P < 0.05).
IE2 interacts with Daxx through SIMs.Although we have established that formation of IE2 CCLAs and their role in DNA transactivation require an association with Daxx, it is still unclear how the IE2 and Daxx proteins interact with each other. Previously, Daxx was shown to interact with other proteins via SUMO-SIM interactions (36). Since our previous studies also demonstrated that IE2 interacts specifically with SUMO1 proteins (9, 23), we investigated the possible mechanism of IE2/Daxx association through SUMO. By analyzing the domain features of IE2 using the protein GPS-SBM SUMO software (37), we found that IE2 contains two putative SIMs at the positions 66 to 69 and 108 to 111 near the N-terminal region (Fig. 10A). Thus, it is possible that SUMO protein bridges the IE2/Daxx association. In order to investigate this possibility, we constructed three IE2 mutants by PCR mutagenesis according to a previously established method for characterizing putative SIMs (36, 38–40) in which we mutated the first three amino acid residues in the putative SIM 66 to 69 (SIM-66) or 108 to 111 (SIM-108) or both (IE2 SIM double mutant [SIM-DM]) (Fig. 1B). We examined colocalization of IE2 and SUMO2/3 at 48 hpt by IF (Fig. 11). Whereas wt IE2 displayed normal CCLAs that colocalized with SUMO2/3, both single SIM mutants (SIM-66 and SIM-108) exhibited diminished colocalization, and the SIM-DM even formed irregular CCLAs that showed no colocalization with SUMO2/3 (Fig. 11). Since our results showed that the putative SIMs are involved in the association of SUMO2/3 with IE2 and likewise affected CCLA formation, we performed further experiments to determine transactivation of the CMV promoter by these IE2 variants by cotransducing vAcIE2, vAcSIM-66, vAcSIM-108, and vAcSIM-DM with the luciferase reporter virus vAcLuc. The activity of luciferase was assayed using a luminometer (Fig. 10B), and luciferase protein amounts were confirmed by Western blotting (Fig. 10C and D). In this analysis, luciferase assays showed that baculovirus carrying wt IE2 (vAcIE2) and the SIM-66 mutant could both activate the CMV promoter, although the level of SIM-66 stimulation was lower than that of wt IE2, but no obvious stimulation was seen by the SIM-108 or SIM-DM mutants (Fig. 10B). These results suggest that SIMs play an important role in IE2-induced transactivation.
The SIMs are crucial for IE2-induced transactivation. (A) IE2 map indicating the position and sequence of SIMs. (B) vAcEGFP, vAcIE2, vAcSIM-66, vAcSIM-108, and vAcSIM-DM were cotransduced with vAcLuc, and luciferase activities were assayed at both 24 and 48 hpt. Results are shown as means and SDs (error bars) of at least four replicates. *, significant difference from value of vAcIE2 in the same time interval (P < 0.05) by unpaired t test. (C and D) Western blot analysis using anti-luciferase antibody (WB: Luc) of the same cell samples at both 24 hpt (C) and 48 hpt (D).
Colocalization of IE2 variants with SUMO2/3. Vero E6 cells were transduced with vAcIE2, vAcSIM-66, vAcSIM-108, or vAcSIM-DM, and their colocalizations with SUMO2/3 were analyzed by IF at 48 hpt. All IE2 SIM mutants (SIM-66, SIM-108, and SIM-DM) exhibited reduced colocalization with SUMO2/3, and the SIM-DM mutant showed the most severe morphological disruption of CCLA formation. Bars: 5 μm.
We then performed IF experiments to study the possible colocalization of the IE2 SIM-DM protein with Daxx. Unlike our previous results showing that wt IE2 and Daxx colocalized at 24 hpt but dissociated at 48 hpt (Fig. 3), we found little or no evidence of colocalization between IE2 SIM-DM and Daxx at either early (24 hpt [Fig. 12A]) or late (48 hpt [Fig. 12B]) stages, which was confirmed by our co-IP data (Fig. 4, SIM-DM). These results indicate that the mechanism of IE2-Daxx association is mediated by SUMO-SIM.
Colocalization studies of wt and SIM-DM IE2 with Daxx. Vero E6 cells were transduced with vAcIE2 or vAcSIM-DM. The colocalization of wt (green) and SIM-DM (red) IE2 with Daxx was analyzed by IF at 24 hpt (A) and 48 hpt (B). Bars: 5 μm. (C) Colocalization coefficients were calculated from 30 randomly selected cells separately at 24 hpt and 48 hpt. Results are shown as means and SDs (error bars).
To further confirm that bridging of IE2 and Daxx via SUMO-SIM interaction is important for both CCLA formation and IE2 transactivation, we used a SUMO E2 enzyme UBC9 inhibitor, 2-D08, to block the SUMOylation pathway in vAcIE2-transduced Vero E6 cells. In the presence of 2-D08, IE2 could not form enlarged CCLAs at 48 hpt (Fig. 13). Protein colocalization patterns were also significantly altered in response to 2-D08 treatment. IE2 CCLAs in dimethyl sulfoxide (DMSO)-treated cells were colocalized with Daxx and H3.3 at 24 hpt (Fig. 13A) and dissociated from Daxx and H3.3 at 48 hpt (Fig. 13C), as also revealed in Fig. 3A and B. In contrast, for cells treated with 2-D08, IE2 CCLAs showed weak or almost no colocalization with Daxx or H3.3 at both 24 and 48 hpt (Fig. 13B and D).
Treatment of the SUMO E2 inhibitor 2-D08 blocks the formation of IE2 CCLAs. Vero E6 cells were transduced with vAcIE2, and then either 30 μM 2-D08 (B and D) or an equal volume of DMSO (A and C) was added at 12 hpt. The cells were fixed at both 24 (A and B) and 48 (C and D) hpt, and colocalization of IE2 with H3.3 and Daxx was assessed by IF. Bars: 5 μm.
High levels of mRNA was generated in IE2 CCLAs.Previously, we showed that IE2 CCLAs enclose high levels of nuclear actin and colocalize with concentrated RNA Pol-II (9, 23). Nuclear actin is known to form a complex with RNA Pol-II that is important for its functioning (41) and also binds to heterogeneous ribonucleoproteins (hnRNPs) during transcriptional elongation (42–44).
To further determine the localization of transcribed mRNAs, we performed an RNA FISH assay with cells transduced with vAcIE2. In situ mRNA hybridization using an antisense RNA probe against ie2 transcript (driven by the CMV promoter) identified abundant ie2 mRNA accumulation in the IE2 CCLAs at 48 hpt (Fig. 14A), whereas the control sense RNA probe did not (Fig. 14B). These mRNA signals colocalized well with actin (Fig. 14A, RNA+Actin) and they were enclosed by IE2 in the CCLAs (Fig. 14A, IE2+Actin and IE2+RNA) where the viral DNA resides (Fig. 5B and Fig. 6B).
IE2 CCLAs actively accumulate mRNA. Results for RNA FISH show that IE2 CCLAs actively accumulate mRNA at 48 hpt, as revealed by an antisense RNA probe against the ie2 transcripts (A). The sense RNA probe acted as a negative control (B). The color codes for different targets are as follows: blue, IE2; green, G-actin; and red, mRNA. Insets are magnified regions marked by white boxes. Bottom rows show the intensity of line plots of IE2, actin, and RNA signals along arrows. Bars: 5 μm. Colocalization coefficients for IE2, actin, and RNA were calculated from 30 randomly selected cells separately at 24 hpt (C) and 48 hpt (D). Results are shown as means and SDs (error bars).
DISCUSSION
Baculovirus is known to enter numerous mammalian cells with high efficiency, but it neither replicates nor results in significant gene expression for most viral gene in mammalian cells (12–15). Thus, baculovirus has been routinely used for proper gene transduction and expression in mammalian cells using mammalian promoters for broad applications, including gene delivery, protein expression, and tissue engineering (9, 11, 16, 17). Previously, we found that baculovirus IE2 can strongly activate foreign gene expression in mammalian cells. In this study, we investigated the mechanism of IE2 transactivation. IE2 can transactivate several different mammalian promoters, including CMV and SV40, in transduced mammalian cells. With no specific DNA sequence binding capability (23), IE2 must apply a special strategy to approach the transduced viral DNA to initiate gene transactivation. We illustrate that the formation of IE2 CCLAs is mediated by a SUMO-SIM interaction with the host intrinsic immune response proteins Daxx and H3.3. The association with IE2 abrogates the binding of Daxx and H3.3 to viral DNA in the late transduction stage, which is consistent with the observed upregulated foreign gene expression. IE2 likely builds CCLAs using viral DNA, because the line scans of fluorescent patterns of both DNA and IE2 overlap at 48 hpt (Fig. 5 and 6).
One of the highlights of this study is the specific association of IE2 with the host Daxx and H3.3 proteins. Daxx works as a transcriptional coregulator, and it is known to interact with H3.3 to target and suppress viral gene transcription (1, 6, 33, 35, 45–48), so how does IE2 instead manage to activate gene expression after interacting with these intrinsic immune proteins? Our DNA FISH analysis reveals that Daxx (Fig. 5) and H3.3 (Fig. 6) strongly colocalize with IE2 and viral DNA at 24 hpt but dissociate from Daxx and H3.3 at 48 hpt. The observation that Daxx interacts strongly with viral DNA and dissociates from it only in the presence of wt IE2 at the late stage, is proven by our ChIP analysis (Fig. 7), whereas in the absence of IE2 (i.e., EG for EGFP) or upon transduction of IE2 mutants (i.e., C230S and SIM-DM), viral DNA sequences were consistently captured by Daxx at both early 24 hpt and late 48 hpt stages (Fig. 7). These results indicate that IE2 is necessary for the release of Daxx from viral DNA. Furthermore, when we knocked down Daxx by siRNA, IE2 failed to form enlarged CCLAs (Fig. 8), which resulted in loss of IE2 transactivation activity (Fig. 9). These results suggest that the formation of IE2 CCLAs requires Daxx. The Daxx/H3.3 antivirus complex is a host machinery well designed for trapping and inactivating invading DNA (49–51). By means of this unexpected mechanism, IE2 can properly and efficiently target viral DNA, but not the host chromosomal DNA, through the host defensive Daxx/H3.3 complex.
An additional highlight of our study is the importance of SUMO/SIM for both CCLA formation and IE2 transactivation activity. Daxx was reported to be sumoylated by UBC9 as a transcriptional corepressor (36, 52, 53), and we found that IE2 contains two putative SIMs, so we hypothesized that IE2 may associate with Daxx via SUMO-SIM interaction. In our study, there is no physical association between IE2 SIM-DM and Daxx in our co-IP assay (Fig. 4), there was no evidence of IE2 SIM-DM colocalization with Daxx (Fig. 12), and IE2 SIM-DM formed distorted CCLA structures at the late stage (48 hpt) (Fig. 12B). Our experiments also show that the colocalization of IE2 and SUMO was disrupted by the IE2 SIM-DM mutations (Fig. 11). The assumption of IE2-SUMO association is further strengthened by our observation that 2-D08 treatment prevented formation of enlarged IE2 CCLA structures (Fig. 13), indicating that SUMO plays an important role in the IE2-Daxx association.
Nuclear bodies are highly organized structures frequently observed in the cell nucleus. The structures of nuclear bodies have been identified as distinct nuclear foci by light and electron microscopy. Numerous nuclear bodies have been characterized thus far, including nucleoli, PML-NBs, Cajal bodies (CBs), nuclear speckles, and Polycomb bodies (54). Recent studies show that during virus infection, the invading virus expresses many viral proteins that interact with host nuclear bodies (4, 5, 55). Currently, RNA or protein molecules have been reported as potential nucleators for initiating nuclear body formation (54), but our study represents a unique example of a viral factor (IE2) assembling as a nuclear body, the CCLA, using DNA. This raises the possibility that DNA nucleators may be recognized as a new class of nuclear body in the future.
IE2 CCLAs represent a novel cellular transcription factory that actively recruits abundant factors for efficient transcription. IE2 CCLAs form a spherical barrier to enclose viral DNA (this study) and then dynamically recruit actin and RNA Pol-II (23) after eliminating transcription restriction by Daxx and H3.3 (this study). IE2 CCLAs represent a highly efficient machinery for gathering cellular resources for targeted gene activation. It is quite striking that baculovirus IE2 can employ such a complex strategy to commandeer the mammalian immune and transcriptional machineries to stimulate strong gene expression. Since insects, the host of baculovirus, evolutionarily diverged from mammals approximately 900 million years ago (56), IE2 CCLA-mediated transactivation may represent a very primitive but long-conserved machinery heretofore unknown in mammalian systems.
As mentioned in the introduction, baculovirus has become a frequently used tool for gene delivery in mammalian cells, so the main purpose of our experiments was to study the mechanism regarding how an exogenous protein (IE2) can strongly activate gene expression in these cells. Nevertheless, we still studied possible IE2 associations with Daxx and H3.3 in an insect system (Fig. 15). We used wt baculovirus to infect Sf21 cells, i.e., the conventional cell line used for baculovirus infection, and found that colocalizations between IE2 and Daxx and between IE2 and H3.3 did occur (Fig. 15A). Since IE2 nuclear bodies have been well reported to diminish after 8 h after virus infection (hpi) in insect cells (22, 57–59), we cannot study later association and disassociation events among these proteins as we have done for the mammalian system. We reason that the reduction of IE2 nuclear bodies in insect cells may arise from the effects of other viral genes, e.g., IE1 (59, 60) or other gene products, during baculovirus infection. We verified this supposition by conducting plasmid transfection into insect cells. An IE2 expression construct (Fig. 15B) driven by a heat shock promoter and fused with an enhancer (the homologous repeat [hr] [66]) was used to transfect insect Sf21 cells. Interestingly, we found that IE2 nuclear bodies lasted longer, grew, and colocalized well with Daxx and H3.3 (Fig. 15C), i.e., very similar to the IE2 CCLA observed in Vero E6 cells. In order to study whether SUMO is also involved in IE2-Daxx associations in insect host cells, we further performed experiments using 2-D08 treatment to confirm the important role of SUMOylation in the development of IE2 CCLAs and the colocalization with other proteins in Sf21 cells (Fig. 16). Again, the results matched our observations in Vero E6 cells (Fig. 13), indicating that SUMO also plays a role in the IE2-Daxx association in insect host cells. Since specific antibodies against Daxx and H3.3 in insect cells are not currently available, we had to use mammalian antibodies against these two protein homologs in insect cells to perform these experiments. Thus, it is difficult to make the firm conclusion that IE2 actually operates via the same mechanism in mammalian and insect cells. Nevertheless, these results may suggest that upon baculovirus infection in insect cells, IE2 associates with Daxx and H3.3 at the early virus infection stage to localize the loci of the infecting viral genome and that IE2 is then either inhibited or degraded by another viral factor(s) to expose the viral genome for better gene activation and DNA replication.
Study of IE2 CCLAs in insect cells during baculovirus infection. Sf21 cells were infected by wt AcMNPV. (A) IF studies were performed at 6 h postinfection (hpi). IE2 formed the CCLA structures in the nucleus, and the colocalization with Daxx and H3.3 can be seen in the merged images. Bars: 5 μm. (B) Plasmid construction for IE2 expression in insect cells. Schematic showing the construction of phr-hsp-IE2, in which the ie2 coding region is driven by an hr-heat shock protein fusion promoter. (C) Colocalization studies of the plasmid-expressed IE2 with Daxx and H3.3 in insect cells. Upon transfection of phr-hsp-IE2, IE2 CCLAs (red) could be observed in insect cells at 24 hpt. Colocalization of IE2 CCLAs with Daxx (green) could be observed, whereas colocalization with H3.3 (green, bottom) was also found at 24 hpt. Bars: 5 μm.
Treatment with the SUMO E2 inhibitor 2-D08 also blocks the formation of IE2 CCLAs in insect cells. Sf21 cells were transfected with phr-hsp-IE2 and either 30 μM 2-D08 (B and D) or an equal volume of DMSO (A and C) was added at 12 hpt. The cells were fixed at both 24 (A and B) and 48 (C and D) hpt, and colocalization of IE2 with H3.3 and Daxx was assessed by IF. Bars: 2 μm.
Taken together, our findings have revealed a unique CCLA nanomachinery organized by baculovirus IE2 protein in cell nuclei. Although IE2 CCLAs look like tightly sealed nanoparticles, they can actively and selectively recruit factors like RNA Pol-II and actin (9, 23) as well as viral DNA to induce high-level viral gene activation (Fig. 17). Nuclear bodies or foci generated by cellular or viral proteins are commonly observed in many cell systems (54, 61). The complex yet efficient system operated by IE2 CCLAs reported here represents one of the most unique nuclear body machineries, using DNA as a new class of nucleator, responsible for high-level gene expression.
Model of IE2 CCLA formation and gene transactivation. During baculovirus transduction, the exposed viral DNA is targeted by Daxx and H3.3 to restrict DNA expression (1). Then IE2 targets Daxx to specifically associate with viral DNA (2) and then segregates Daxx to allow CCLA formation (3). Mature IE2 CCLAs are capable of recruiting factors like RNA Pol-II and actin (23), necessary for abundant viral mRNA production (4).
MATERIALS AND METHODS
Cell lines and cell culture.Vero E6 (ATCC CRL1586) derived from vervet monkey (Cercopithecus aethiops) kidney cells and Spodoptera frugiperda IPLB-Sf21 (Sf21) cells were cultured as previously described (23, 57). Briefly, Vero E6 cells were cultured with MEM-alpha medium (Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and maintained in a humidified incubator with 5% CO2 at 37°C. Sf21 cells were cultured at 26°C in TC100 insect medium (Gibco, Thermo Fisher Scientific) with 10% FBS. Recombinant AcMNPV was generated and propagated in Sf21 cells as previously described (23). Transfection was performed using PolyJet (SignaGen), following the manufacturer’s protocol. For baculovirus transduction, Vero E6 cells were seeded to reach an optimal 70 to 80% confluency prior to transduction, and then recombinant baculovirus was added directly into culture medium at various multiplicities of infection (MOIs). After gentle mixing, the transduced cells were incubated at 37°C with 5% CO2 for 24 or 48 h, depending on the experiment.
Plasmid and virus construction.The ie2 gene was amplified from the AcMNPV genome and cloned into pTriEx-3 plasmid (Novagen), which contains p10, CMV, and T7 promoters for expression in insect, mammalian, and bacterial cells (23, 57). Recombinant baculoviruses vAcIE2, vAcC230S, and vAcEGFP, which express wt IE2, RING domain mutant IE2, and EGFP, respectively, by the TriEx promoter (Novagen) were generated as previously described (23). IE2 SIM single and double mutants were generated by site-directed mutagenesis using pairs of complementary primers (Table 1) to replace the amino acid residues VQII at positions 66 to 69 (SIM-66) and VILI at positions 108 to 111 (SIM-108) with residues AAAI. All IE2 mutants contained His tags at their C-terminal regions and were cloned into the pTriEx-3 vector to generate pAcSIM-66, pAcSIM-108, and pAcSIM-DM, respectively. The luciferase gene was obtained by PCR from pTre-luc plasmid as previously described (23, 57) and inserted into pTriEx-3 to generate the pAcLuc vector. The pAcSIM-66, pAcSIM-108, pAcSIM-DM, and pAcLuc vectors were cotransfected with FlashBAC (a modified AcMNPV baculovirus genome; Mirus) into Sf21 cells by Cellfectin (Thermo Fisher; 10362100). The resulting recombinant baculoviruses—vAcSIM-66, vAcSIM-108, vAcSIM-DM, and vAcLuc—were isolated through endpoint dilutions as described previously (23, 57). For the insect experiment, the baculovirus enhancer sequence hr1 (homologous region 1) was obtained by PCR using 2× Ultra Hi-Fi PCR Master Mix (TTC-PE15, TOOLS) as previously described (23, 57) and inserted into the pAB-6×His vector (Invitrogen). The PCR-amplified fragments of the Drosophila hsp70 promoter and baculovirus ie2 gene sequence were obtained as described previously (23, 62) and subcloned into the pAB-6×His vector containing the hr sequence to obtain the phr-hsp-IE2 plasmid. The plasmid was transfected into Sf21 cells by using Cellfectin.
Primers used in this study
IF experiments.Vero E6 cells (4 × 104) were seeded into 8-well Millicell EZ slides (Millipore) and transduced with recombinant baculovirus using an MOI of 100. The slides were centrifuged at 2,000 rpm for 30 min at room temperature (RT) and then incubated at 37°C for various time frames. For the insect IE2 CCLA formation assay, Sf21 cells were plated at a density of 1 × 105 per well in 8-well Millicell EZ chamber slides 1 day before transfection. Plasmid was transfected at 500 ng per well. The slides were centrifuged at 2,000 rpm for 30 min at RT and then incubated at 26°C for various time frames. At each desired time point, the cells were fixed with 4% paraformaldehyde, permeabilized with 100% acetone at −20°C, and blocked with 3% bovine serum albumin (BSA) for 1 h. The cells were then incubated with primary antibody overnight at 4°C. Antibodies used included mouse anti-His-tagged antibody (1:5,000; GeneTex; GTX628914), rabbit anti-Daxx antibody (1:200; Santa Cruz; SC-7152), rabbit anti-histone H3.3 antibody (1:500; GeneTex; GTX112955), rabbit anti-SUMO1 antibody (1:500; Santa Cruz; SC-9060), and rabbit anti-SUMO2/3 antibody (1:500; GeneTex; GTX62763). After overnight incubation, the cells were washed three times with DPBST (Dulbecco’s phosphate-buffered saline [DPBS] plus 0.1% Tween 20) and incubated with 1:200 dilutions of Alexa Fluor 555 goat anti-mouse IgG secondary antibody or Alexa Fluor 594 goat anti-mouse IgG secondary antibody (Invitrogen) and Alexa Fluor 488 goat anti-rabbit IgG secondary antibody. G-actin was specifically stained with Alexa Fluor 488 conjugated with DNase I (1:500; Invitrogen D12371) as previously described (23). Images were obtained with a Zeiss laser confocal microscope (LSM780) using a Fluor 63×/1.40 numerical aperture (NA) oil immersion objective. All images were acquired using 1,024 by 1,024 diameter pixels, and fluorescence intensity was analyzed by ZEN 2010 software (Zeiss).
DNA FISH.DNA hybridization was conducted according to the protocol described in the FISH Tag DNA kit (Invitrogen; F32947), with slight modifications. Vero E6 cells were transduced with virus using an MOI of 100. Cells were fixed at 48 hpt by 4% paraformaldehyde, and the target proteins were stained using specific antibodies. After the last washing step, the slides were refixed with 4% paraformaldehyde. The baculovirus genome (purified from Escherichia coli containing BacMid bMON14272) conjugated with Alexa Fluor 488 was used as a probe to detect DNA signal. Slides were dried at room temperature and then incubated in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 2 min at 73°C. Hybridization buffer (70% formamide, 2× SSC) was then added to the slides. Denatured probes were added to the slides and incubated overnight at 37°C. Samples were then washed three times with hybridization buffer with 0.1% NP-40 at 37°C. These washing steps were followed by two 5-min washes with 50% DPBST–50% hybridization buffer at room temperature. Samples were then washed three times with DPBS before being sealed with ProLong Diamond antifade mounting reagent (Thermo Fisher; P36965). Images were obtained with a Zeiss laser confocal microscope (LSM780) by using a Fluor 63×/1.40 NA oil immersion objective. All images were acquired using 1,024 by 1,024 diameter pixels, and fluorescence intensity was analyzed by ZEN 2010 software (Zeiss).
Co-IP assay.Vero E6 cells transduced with vAcEGFP, vAcIE2, and vAcSIM-DM with MOIs of 100 were collected at 24 and 48 hpt and then washed twice with DPBS buffer. Cell fractions were prepared in EMBK buffer (25 mM HEPES [pH 7.6], 5 mM MgCl2, 1.5 mM KCl, 75 mM NaCl, 175 mM sucrose) with 0.1% NP-40 containing proteinase inhibitor. To detect the association of IE2 with Daxx proteins, 20 mM freshly dissolved N-ethyleimide (NEM) and 5 mM iodoacetic acid (IAA) were added to the EMBK buffer. After 30 min of incubation, samples were pelleted down by centrifugation and suspended in immunoprecipitation buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.4], 1 mM ZnSO4, 0.2 mM phenylmethylsulfonlyl fluoride [PMSF], 1% NP-40 with 20 mM NEM and 5 mM IAA). The samples were sonicated to ensure complete dissipation of the nucleus. After sonication, nuclear extracts were incubated with magnetic protein A/G beads (Pierce) conjugated with mouse anti-His-tagged antibody (1:5,000; GeneTex; GTX628914), rabbit anti-Daxx antibody (1:200, Santa Cruz; SC-7152), or rabbit anti-GAPDH (1:5,000; GeneTex; GTX100118) overnight at 4°C. The samples were then pelleted down using a magnetic separator. The supernatant was then carefully removed by pipette, and samples were washed with immunoprecipitation buffer at least seven times before being lysed in Laemmli sample buffer (TAAR-TB2; TOOLS, Taiwan), separated by SDS-PAGE (TFU-GG420; TOOLS, Taiwan), and examined by Western blotting.
ChIP assays.Chromatin was prepared using Magna ChIPT A 1-day chromatin immunoprecipitation kits (Millipore; 17-10085) following the manufacturer’s instructions. Briefly, 1 × 107 Vero E6 cells were seeded into T150 flasks for each sample. Vero E6 cells were transduced with vAcEGFP, vAcIE2, vAcC230S, and vAcSIM-DM viruses. Cell lysates were harvested at 24 and 48 hpt. The transduced cells were then cross-linked by adding 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by adding 10× glycine, followed by a PBS wash and resuspension in lysis buffer. After the lysis step, DNA was fragmented (200 ∼ 500 bp) by sonicator. The extracted chromatins were precipitated by rabbit anti-Daxx antibody (Santa Cruz; SC-7152) to analyze the specific DNA fragments bound by Daxx. Chromatins extracted from the identical cell lysates precipitated by normal rabbit IgG (Millipore; PP64B) were used to detect the background signal, and the chromatins without any antibody precipitation served as the input. Both the precipitated and input chromatin DNAs were washed and purified following the manufacturer’s protocol and then analyzed by real-time PCR using primers for specific promoter regions listed in Table 1.
Daxx knockdown.Daxx knockdown was achieved using Daxx siRNA (Santa Cruz; SC-35178) and control siRNA (Santa Cruz; SC-37007). Transfection was performed using JetPRIME transfection reagent following the manufacturer’s instructions (Polyplus; 114-01). After 24 h of incubation, fresh medium containing recombinant baculovirus was used to replace the siRNA-containing medium. The cells were incubated for an additional 48 h before Western blotting and luciferase assays.
SUMO inhibitor 2-D08 (2’,3′,4’-trihydroxyflavone) drug treatment.Mammalian Vero E6 and insect Sf21 cells were treated with DMSO or 2-D08 (Millipore; 505156). To eliminate the possibility that 2-D08 might interfere with virus entry into cells, cells were first incubated with virus for 5 h to ensure virus entry and then the original medium was replaced with medium containing 2-D08. A working concentration of 30 μM 2-D08 in DMSO was used to treat the transduced and infected cells based on a previous study (63). After 24 and 48 hpt, the cells were fixed with 4% paraformaldehyde before being subjected to the IF procedure.
Luciferase activity assay.Luciferase assays were conducted as described previously (23, 57). Briefly, 96-well plates were seeded with 1 × 104 Vero E6 cells and transduced with vAcLuc with or without wt IE2 or mutant IE2 baculoviruses. After 48 h of incubation, the cells were washed twice with DPBS and lysed with 100 µl of cell culture lysis reagent (CCLR) for 10 min. The cell lysates were centrifuged at 3,500 rpm for 30 min at 4°C. After cell lysis, 20 µl of supernatant was transferred into 180 µl of luciferase assay reagent and placed into the wells of a black 96-well microplate. Luciferase activity was measured with a luminometer (EnSpire; PerkinElmer) by injecting 50 µl of 0.2 mM luciferin (Promega) into each well.
RNA FISH.RNA hybridization was conducted as described in the FISH Tag RNA kit (Invitrogen; F32954), with slight modifications. For in vitro transcription, the T3 and T7 promoter sequences needed to be near the 5′ and 3′ ends of the DNA strand, respectively, of the DNA construct. To generate the construct, the ie2 gene was amplified from the pAcIE2 plasmid and the T7 and T3 promoter sequences were generated by PCR using the IE2-T7-BHI-F and IE2-T3-BHI-R primers for the sense and antisense strands, respectively. The RNA probes were amplified in vitro by PCR using either T3 or T7 RNA Pol. The transcripts were then labeled with Alexa Fluor 594 dye, purified, and used for RNA hybridization. RNA FISH was performed with IF, and then the slides were refixed with 4% paraformaldehyde and washed three times with DPBS. The samples were incubated in the hybridization buffer (containing 100 μg/ml of fragmented salmon DNA, 50% formamide, 5× SSC, 50 μg/ml of heparin, and 0.1% Tween 20) and then heated at 55°C for 5 min. Probes were diluted in hybridization buffer (1 μg/ml), denatured by heating at 80°C for 2 min, and then cooled on ice. These denatured probes were added to the slides and incubated overnight at 37°C. After the incubation, the probes were removed and samples were washed with hybridization buffer three times at 42°C. The washing steps were followed by two 5-min washes with 50% DPBST combined with 50% hybridization buffer at room temperature. After the washing step, the samples were further washed three times with DPBS and then sealed with ProLong Diamond antifade mounting reagent (Thermo Fisher P36965). Images were obtained by a Zeiss laser confocal microscope (LSM780) using a Fluor 63×/1.40 NA oil immersion objective. All images were acquired using 1,024 by 1,024 diameter pixels, and fluorescence intensity was analyzed by ZEN 2010 software (Zeiss).
Statistical analysis.For all the experiments in which we determined colocalization coefficients, quantification was based on images captured from more than 30 cells out of at least three independent experiments for the quantification. The numbers of cells used and the experiments performed are detailed in the individual figure legends. For the ChIP assay, three independent cell cultures were used for the IP process and each qPCR was done with three replicates. For the luciferase activity assays, all conditions were analyzed with eight repeats in three independent experiments. All quantitative data are shown as means and standard deviations (shown by error bars). Statistical analysis was performed using an unpaired t test (Excel 2016 software; Microsoft) for two group comparisons, and P values of <0.05 were considered significant.
ACKNOWLEDGMENTS
This research was funded by grants MOST 106-2321-B-001-012, MOST 106-2321-B-001-027, MOST 107-2321-B-033-002, and MOST 107-2311-B-001-030 from the Ministry of Science and Technology, NHRI-106A1-MRCO-0217171 from The National Health Research Institutes, and 022361 from Academia Sinica, Taiwan, Republic of China.
We thank Su-Ping Li of the Institute of Molecular Biology (IMB), Academia Sinica, for technical assistance in confocal studies and John O’Brien of the Academia Sinica IMB Editing Core for editing the manuscript.
We have no competing interests to declare.
FOOTNOTES
- Received 29 January 2019.
- Accepted 31 January 2019.
- Accepted manuscript posted online 6 February 2019.
- Copyright © 2019 American Society for Microbiology.
