FIG 10.

ChIP experiments reveal specific SPOC1 binding within the proximal enhancer region of the major immediate early promoter. (A) Primary HFF cells were infected with TB40/E at an MOI of 0.5, proteins were cross-linked at 8 hpi, and subsequent ChIP was performed with anti-SPOC1 antibody CR56 or anti-HA as a control. After washing and elution, the samples were subjected to proteinase K digestion and purified with the QIAquick MinElute PCR purification kit (Qiagen, Hilden, Germany). Subsequently, samples were subjected to SYBR green qRT-PCR with primers targeting different regions up- and downstream of the transcription start site of the IE1 and IE2 genes (regions shown relative to position +1 of the transcription start site). (B) HFF/SPOC1 (Tet-On) cells were induced with doxycycline (500 ng/ml) 1 day prior to infection with TB40/E (MOI of 0.5). At 8 h postinfection, proteins were cross-linked, and ChIP was performed with anti-SPOC1 antibody CR56. SPOC1 binding sites within the HCMV genome were mapped onto the reference sequence under GenBank accession number EF999921.1. Reads were deduplicated with SAMtools 1.3 and subjected to peak calling using the MACS2 IDR pipeline. (C) Inset of the peak from panel B depicting open reading frames, the proximal and distal enhancers of the MIEP, and the regions amplified by the primer pairs from panel A.