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Interaction of gamma-herpesvirus genome maintenance proteins with cellular chromatin.

Mughal N, Coppotelli G, Callegari S, Gastaldello S, Masucci MG - PLoS ONE (2013)

Bottom Line: We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line.While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs.Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.

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RHV-GMPs recruit BRD2 and BRD4 to the site of chromatin remodeling.Representative confocal images illustrating the nuclear fluorescence of A03-1 cells co-transfected with plasmids expressing mCherry-LacR-VP16 or mCherry-LacR-GMP fusion proteins and YFP-tagged BRD2 or BRD4. The recruitment of BRD2 and BRD4 to the site of chromatin decondensation, visualized by the accumulation of green fluorescence overlapping with the red fluorescent array, is indicated by arrows in cells expressing VP16 or the RHV-GMPs.
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pone-0062783-g005: RHV-GMPs recruit BRD2 and BRD4 to the site of chromatin remodeling.Representative confocal images illustrating the nuclear fluorescence of A03-1 cells co-transfected with plasmids expressing mCherry-LacR-VP16 or mCherry-LacR-GMP fusion proteins and YFP-tagged BRD2 or BRD4. The recruitment of BRD2 and BRD4 to the site of chromatin decondensation, visualized by the accumulation of green fluorescence overlapping with the red fluorescent array, is indicated by arrows in cells expressing VP16 or the RHV-GMPs.

Mentions: Powerful transcriptional transactivators, such as VP16, promote chromatin decondensation via recruitment of histone acetyltransferases and ATP-dependent chromatin remodelers whereas members of the HMG protein family initiate the remodeling process by competitive displacement of linker histones. We have previously shown that, similar to HMGAs, EBNA1 promotes chromatin remodeling with a slow kinetics and without recruitments of ATP-dependent remodeling complexes [9]. We now tested whether these properties are shared by other GMPs. To this end, we compared mCherry-LacR-tagged VP16 and GMPs for their ability to recruit a panel of GFP- or YFP-tagged ATPase subunits of known ATP-dependent remodeling complexes, including the BRG1 subunit of SWI/SNF complexes, the SNF2H subunit of ISWI complexes and the CHD4 subunit of NuRD complexes, histone acetyltransferases, such as GCN5, pCAF and p300, and two bromo and extra terminal domain (BET) proteins, BRD2 and BRD4, that bind to acetylated histones and are often hijacked by viruses to promote transcription [33]. As illustrated by the representative micrographs shown in Figure 5, Figure S1, Figure S2, and summarized in Table 2, all the tested proteins were recruited to the site of chromatin decondensation in cells expressing mCherry-LacR-VP16, as expected. In accordance with our previous findings, none of the proteins was recruited by EBNA1 and a similar behavior was observed with baEBNA1 that shares with EBNA1 a highly conserved AT-hook like chromatin-targeting module. In line with the presence of high affinity binding sites for BRD2 and BRD4 in the C-terminal domain of LANA1 [15], the two BET proteins were recruited to the site of chromatin remodeling in cells expressing mCherry-LacR-LANA1, and a similar recruitment was observed with all the RHV encoded GMPs (Figure 5 and Table 2). Recruitment of other components of ATP-dependent chromatin complexes and histone acetyltransferases was occasionally observed with different RHV GMPs (Figure S1, Figure S2 and Table 2). Thus, in some of the cells LANA1 recruited BRG1 and p300, and mnR1-LANA recruited pCAF, p300 and GCN5. These differences were highly reproducible in at least three independent experiments performed with each test combination, and could not be attributed to variations in the relative expression of the GMPs or the co-transfected cellular proteins.


Interaction of gamma-herpesvirus genome maintenance proteins with cellular chromatin.

Mughal N, Coppotelli G, Callegari S, Gastaldello S, Masucci MG - PLoS ONE (2013)

RHV-GMPs recruit BRD2 and BRD4 to the site of chromatin remodeling.Representative confocal images illustrating the nuclear fluorescence of A03-1 cells co-transfected with plasmids expressing mCherry-LacR-VP16 or mCherry-LacR-GMP fusion proteins and YFP-tagged BRD2 or BRD4. The recruitment of BRD2 and BRD4 to the site of chromatin decondensation, visualized by the accumulation of green fluorescence overlapping with the red fluorescent array, is indicated by arrows in cells expressing VP16 or the RHV-GMPs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646995&req=5

pone-0062783-g005: RHV-GMPs recruit BRD2 and BRD4 to the site of chromatin remodeling.Representative confocal images illustrating the nuclear fluorescence of A03-1 cells co-transfected with plasmids expressing mCherry-LacR-VP16 or mCherry-LacR-GMP fusion proteins and YFP-tagged BRD2 or BRD4. The recruitment of BRD2 and BRD4 to the site of chromatin decondensation, visualized by the accumulation of green fluorescence overlapping with the red fluorescent array, is indicated by arrows in cells expressing VP16 or the RHV-GMPs.
Mentions: Powerful transcriptional transactivators, such as VP16, promote chromatin decondensation via recruitment of histone acetyltransferases and ATP-dependent chromatin remodelers whereas members of the HMG protein family initiate the remodeling process by competitive displacement of linker histones. We have previously shown that, similar to HMGAs, EBNA1 promotes chromatin remodeling with a slow kinetics and without recruitments of ATP-dependent remodeling complexes [9]. We now tested whether these properties are shared by other GMPs. To this end, we compared mCherry-LacR-tagged VP16 and GMPs for their ability to recruit a panel of GFP- or YFP-tagged ATPase subunits of known ATP-dependent remodeling complexes, including the BRG1 subunit of SWI/SNF complexes, the SNF2H subunit of ISWI complexes and the CHD4 subunit of NuRD complexes, histone acetyltransferases, such as GCN5, pCAF and p300, and two bromo and extra terminal domain (BET) proteins, BRD2 and BRD4, that bind to acetylated histones and are often hijacked by viruses to promote transcription [33]. As illustrated by the representative micrographs shown in Figure 5, Figure S1, Figure S2, and summarized in Table 2, all the tested proteins were recruited to the site of chromatin decondensation in cells expressing mCherry-LacR-VP16, as expected. In accordance with our previous findings, none of the proteins was recruited by EBNA1 and a similar behavior was observed with baEBNA1 that shares with EBNA1 a highly conserved AT-hook like chromatin-targeting module. In line with the presence of high affinity binding sites for BRD2 and BRD4 in the C-terminal domain of LANA1 [15], the two BET proteins were recruited to the site of chromatin remodeling in cells expressing mCherry-LacR-LANA1, and a similar recruitment was observed with all the RHV encoded GMPs (Figure 5 and Table 2). Recruitment of other components of ATP-dependent chromatin complexes and histone acetyltransferases was occasionally observed with different RHV GMPs (Figure S1, Figure S2 and Table 2). Thus, in some of the cells LANA1 recruited BRG1 and p300, and mnR1-LANA recruited pCAF, p300 and GCN5. These differences were highly reproducible in at least three independent experiments performed with each test combination, and could not be attributed to variations in the relative expression of the GMPs or the co-transfected cellular proteins.

Bottom Line: We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line.While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs.Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.

Show MeSH
Related in: MedlinePlus