Figure 2

HDAC3 but not HDAC2 interacts with the MIE locus of HCMV. A. ChIP on Chip assay. HFF cells were infected with the Toledo strain at MOI 5, and the DNA samples from the infected cells were prepared by using the EZ-ChIP commercial kit. Anti-HDAC2, anti-HDAC3, and normal IgG (as a negative control) antibodies were used to precipitate the fragmented Protein-DNA complexes. The DNA fragments pulled down with HDAC3 and IgG antibody were amplified and labeled with Alexa Flour 555 and Alexa Flour 647, respectively and vice-versa. The labeled DNA was applied to the HCMV genomic Microarray containing the entire HCMV (Toledo strain) genomic DNA divided into 593 small fragments. The array was scanned at 532 nm (for Cy3) and 635 nm (for Cy5). HDAC3 was detected only in the MIE locus as indicated with black arrows. The grey arrows indicate no-specific hybridizations. B. PCR verification of the ChIP-on-chip assay. A series of PCR reactions covering the MIE region was performed on the sample before ChIP (Input) and ChIP samples, as indicated on left. C. HDAC3-interacting map. The ORFs spanning the MIE locus are shown. HDAC3-interacting region is indicated as green. D. HDAC2/3 ChIP on hTERT promoter. Mrc-5 cells were cross-linked and ChIP samples were made using the EZ-ChIP commercial kit. Anti-HDAC2 (Anti-2), anti-HDAC3 (Anti-3), and normal IgG (as a negative control) antibodies were used to precipitate the fragmented Protein-DNA complexes. PCR was performed to detect the precipitated hTERT promoter DNA using the primers and PCR protocol as reported [27].