Limits...
Identification and characterization of the Spodoptera Su(var) 3-9 histone H3K9 trimethyltransferase and its effect in AcMNPV infection.

Li B, Li S, Yin J, Zhong J - PLoS ONE (2013)

Bottom Line: Su(var) 3-9 protein was found to be localized in the nucleus in Sf9 cells, and interact with histone H3, and the heterochromatin protein 1a (HP1a) and HP1b.A dose-dependent enzymatic activity was found at both 27 °C and 37 °C in vitro, with higher activity at 27 °C.Addition of specific inhibitor chaetocin resulted in decreased histone methylation level and host chromatin relaxation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, People's Republic of China.

ABSTRACT
Histone H3-lysine(9) (H3K9) trimethyltransferase gene Su(var) 3-9 was cloned and identified in three Spodoptera insects, Spodopterafrugiperda (S. frugiperda), S. exigua and S. litura. Sequence analysis showed that Spodoptera Su(var) 3-9 is highly conserved evolutionarily. Su(var) 3-9 protein was found to be localized in the nucleus in Sf9 cells, and interact with histone H3, and the heterochromatin protein 1a (HP1a) and HP1b. A dose-dependent enzymatic activity was found at both 27 °C and 37 °C in vitro, with higher activity at 27 °C. Addition of specific inhibitor chaetocin resulted in decreased histone methylation level and host chromatin relaxation. In contrast, overexpression of Su(var) 3-9 caused increased histone methylation level and cellular genome compaction. In AcMNV-infected Sf9 cells, the transcription of Su(var) 3-9 increased at late time of infection, although the mRNA levels of most cellular genes decreased. Pre-treatment of Sf9 cells with chaetocin speeded up viral DNA replication, and increased the transcription level of a variety of virus genes, whereas in Sf9 cells pre-transformed with Su(var) 3-9 expression vector, viral DNA replication slow down slightly. These findings suggest that Su(var) 3-9 might participate in the viral genes expression an genome replication repression during AcMNPV infection. It provided a new insight for the understanding virus-host interaction mechanism.

Show MeSH

Related in: MedlinePlus

Activity of Su(var) 3-9 in Sf9 cells.Sf9 cells were harvested 3 d post treatments with Chaetocin at appropriate concentration determined by MTT assay (A) and transient overexpression of S. frugiperda Su(var) 3-9. Nuclear proteins were extracted and the global methylation level was evaluated (B). Cellular DNA was isolated and digested with DNase I. The percentage of DNA resistant to the nuclease was determined with qPCR with primers for S. frugiperda β-Actin and β-Tubulin (C, D). The error bars indicate the standard deviation calculated from at least three independent parallel experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3722159&req=5

pone-0069442-g005: Activity of Su(var) 3-9 in Sf9 cells.Sf9 cells were harvested 3 d post treatments with Chaetocin at appropriate concentration determined by MTT assay (A) and transient overexpression of S. frugiperda Su(var) 3-9. Nuclear proteins were extracted and the global methylation level was evaluated (B). Cellular DNA was isolated and digested with DNase I. The percentage of DNA resistant to the nuclease was determined with qPCR with primers for S. frugiperda β-Actin and β-Tubulin (C, D). The error bars indicate the standard deviation calculated from at least three independent parallel experiments.

Mentions: Cell viability assay showed that Sf9 cells could tolerate up to 500 nM chaetocin in the medium (Figure 5A). When the cells were treated with increased dosage of chaetocin, general histone methylation level was gradually inhibited, indicating the presence of Su(var) 3-9 activity in the cell (Figure 5B). In addition, the cellular DNA became more sensitive to DNase I at β-Actin and β-Tubulin loci, which reflected the decreased chromatin compaction (Figure 5C). In contrast, the opposite trends of results were observed when the cells were transfected with a plasmid overexpressing Su(var) 3-9 (Figure 5D).


Identification and characterization of the Spodoptera Su(var) 3-9 histone H3K9 trimethyltransferase and its effect in AcMNPV infection.

Li B, Li S, Yin J, Zhong J - PLoS ONE (2013)

Activity of Su(var) 3-9 in Sf9 cells.Sf9 cells were harvested 3 d post treatments with Chaetocin at appropriate concentration determined by MTT assay (A) and transient overexpression of S. frugiperda Su(var) 3-9. Nuclear proteins were extracted and the global methylation level was evaluated (B). Cellular DNA was isolated and digested with DNase I. The percentage of DNA resistant to the nuclease was determined with qPCR with primers for S. frugiperda β-Actin and β-Tubulin (C, D). The error bars indicate the standard deviation calculated from at least three independent parallel experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722159&req=5

pone-0069442-g005: Activity of Su(var) 3-9 in Sf9 cells.Sf9 cells were harvested 3 d post treatments with Chaetocin at appropriate concentration determined by MTT assay (A) and transient overexpression of S. frugiperda Su(var) 3-9. Nuclear proteins were extracted and the global methylation level was evaluated (B). Cellular DNA was isolated and digested with DNase I. The percentage of DNA resistant to the nuclease was determined with qPCR with primers for S. frugiperda β-Actin and β-Tubulin (C, D). The error bars indicate the standard deviation calculated from at least three independent parallel experiments.
Mentions: Cell viability assay showed that Sf9 cells could tolerate up to 500 nM chaetocin in the medium (Figure 5A). When the cells were treated with increased dosage of chaetocin, general histone methylation level was gradually inhibited, indicating the presence of Su(var) 3-9 activity in the cell (Figure 5B). In addition, the cellular DNA became more sensitive to DNase I at β-Actin and β-Tubulin loci, which reflected the decreased chromatin compaction (Figure 5C). In contrast, the opposite trends of results were observed when the cells were transfected with a plasmid overexpressing Su(var) 3-9 (Figure 5D).

Bottom Line: Su(var) 3-9 protein was found to be localized in the nucleus in Sf9 cells, and interact with histone H3, and the heterochromatin protein 1a (HP1a) and HP1b.A dose-dependent enzymatic activity was found at both 27 °C and 37 °C in vitro, with higher activity at 27 °C.Addition of specific inhibitor chaetocin resulted in decreased histone methylation level and host chromatin relaxation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, People's Republic of China.

ABSTRACT
Histone H3-lysine(9) (H3K9) trimethyltransferase gene Su(var) 3-9 was cloned and identified in three Spodoptera insects, Spodopterafrugiperda (S. frugiperda), S. exigua and S. litura. Sequence analysis showed that Spodoptera Su(var) 3-9 is highly conserved evolutionarily. Su(var) 3-9 protein was found to be localized in the nucleus in Sf9 cells, and interact with histone H3, and the heterochromatin protein 1a (HP1a) and HP1b. A dose-dependent enzymatic activity was found at both 27 °C and 37 °C in vitro, with higher activity at 27 °C. Addition of specific inhibitor chaetocin resulted in decreased histone methylation level and host chromatin relaxation. In contrast, overexpression of Su(var) 3-9 caused increased histone methylation level and cellular genome compaction. In AcMNV-infected Sf9 cells, the transcription of Su(var) 3-9 increased at late time of infection, although the mRNA levels of most cellular genes decreased. Pre-treatment of Sf9 cells with chaetocin speeded up viral DNA replication, and increased the transcription level of a variety of virus genes, whereas in Sf9 cells pre-transformed with Su(var) 3-9 expression vector, viral DNA replication slow down slightly. These findings suggest that Su(var) 3-9 might participate in the viral genes expression an genome replication repression during AcMNPV infection. It provided a new insight for the understanding virus-host interaction mechanism.

Show MeSH
Related in: MedlinePlus