Histone modification pattern of the T-cellular Herpesvirus saimiri genome in latency

Abstract

Herpesvirus saimiri (HVS) subgroup C strains are able to growth transform human T lymphocytes in vitro. The stably persisting and nonintegrating HVS episome represents an optimal prerequisite for the investigation of the epigenetic state of latent herpesvirus genomes in vitro. Quantitative chromatin immunoprecipitation experiments using seven different histone acetylation- or methylation-specific antibodies revealed repressive marks at four lytic gene promoters and a variable pattern at the weakly transcribed LANA/orf73 promoter. The constitutive stpC/tip promoter regulating the viral oncoproteins and, more interestingly, the noncoding repetitive H-DNA elements flanking the coding region, showed a permissive chromatin structure. This study provides an appropriate model for the analysis of epigenetic herpesvirus genome modifications and their dynamics in T cells.

FIG. 1.

(A) Oligonucleotides used for PCR. (B) Genomic localization of the PCR amplicons in the promoter regions of the respective viral genes.

FIG. 2.

Histone acetylation profile of HVS episomes. Latent viral genomes from transformed human T cells were investigated by ChIP and quantitative SYBR green PCR. Briefly, 2 × 10⁶ cells per precipitating antibody were cross-linked for 10 min at 37°C by adding formaldehyde to a final concentration of 1%. Cells were washed repeatedly with ice-cold phosphate-buffered saline containing protease inhibitors (aprotinin, leupeptin, and AEBSF), lysed with buffer containing sodium dodecyl sulfate and protease inhibitors, and sonicated to shear the DNA to fragments of approximately 200 to 1,000 bp. The soluble fraction holding the chromatin was diluted in ChIP dilution buffer (Upstate), and two aliquots of input DNA were drawn. The samples were precleared with salmon sperm DNA/protein A agarose (Upstate) before the precipitating antibodies were added for overnight incubation at 4°C in a rotator. After recovery of the antibody-histone complex with salmon sperm DNA/protein A agarose, followed by several washing steps with rising stringency, the antibody-histone-DNA complex was eluted from the agarose, and the cross-links were reversed by the addition of sodium chloride (0.2 M final concentration) and incubation at 65°C for 4 h. Finally, the DNA was purified by proteinase K digestion, phenol extraction, and ethanol precipitation using glycogen as carrier. The samples were then subjected to quantitative SYBR green PCR using an Applied Biosystems 7500 sequence detection system. The PCR amplification reaction was carried out with the following conditions: 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 33 s at 60°C. To ensure the specificity of the PCR products, these were subsequently evaluated by dissociation curve analysis. The results from two independent experiments are shown. (A to C) Acetylation of histone H3 lysine residues 9 and 14 (K9, K14) (A), acetylation of histone H4 lysine residues (K4, K7, K11, K15) (B), and acetylation of histone H3 K9 (C). Cellular promoters of the housekeeping genes ADH5 and GAPDH served as euchromatic (EU) controls, and cellular juxtacentromeric and centromeric satellite regions served as controls for heterochromatin (HE). For a no-antibody control, see Fig. 3C.

FIG. 3.

Histone trimethylation profile of HVS episomes. Latent viral genomes from transformed human T cells were investigated by ChIP and quantitative SYBR green PCR. The results from two independent experiments are shown. (A and B) Trimethylation of histone H3 lysine residue 4 (K4) (A) and trimethylation of histone H3 lysine residue 9 (K9) (B). Cellular promoters of the housekeeping genes ADH5 and GAPDH served as euchromatic (EU) controls, and cellular juxtacentromeric and centromeric satellite regions served as controls for heterochromatin (HE). (C) No-antibody control.

FIG. 4.

Histone dimethylation profile of HVS episomes. Latent viral genomes from transformed human T cells were investigated by ChIP and quantitative SYBR green PCR. The results from two independent experiments are shown. (A and B) Dimethylation of histone H3 lysine residue 4 (K4) (A) and dimethylation of histone H3 lysine residue 9 (K9) (B). Cellular promoters of the housekeeping genes ADH5 and GAPDH served as euchromatic (EU) controls, and cellular juxtacentromeric and centromeric satellite regions served as controls for heterochromatin (HE). (C) No-antibody control.