Figure 5

REST/NRSF recruited corepressor CoREST to HSV-1 RE-1/NRSE and induced histone H4 deacetylation. A. Over-expression of REST/NRSF in 293HEK cells by transfection of pFLAG-REST. Lane 1: 293HEK cells transfected with pICP4. Lane 2: 293HEK cells transfected with pICP4 and pFLAG-REST. Immunoblot was performed using anti-REST antibody. B. EMSA using transfected cell extract. Lane 1. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with control plasmid. Lane 2. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 3: Labeled mutant oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 4: Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with pCMVp73. Lane 5: Labeled wild-type ds oligo containing 10× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 6: Labeled wild-type ds oligo containing 25× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Noted that endogenous REST/NRSF produced a shifted band (Lane 1). C. Analysis of REST/NRSF binding, histone H4 acetylation alteration, and CoREST recruitment by ChIP. 293HEK-pICP4 cell line was transfected with pFLAG-REST and subjected to ChIP assay. Samples prior to the immuno-precipitation are used for input control. The samples were amplified by PCR and subjected to 1.2% agarose electrophoresis staining with ethidium bromide. 1. pICP4 + Control. 2. pICP4 + pFLAG-REST. 3. pICP4 + pCMVp73.