File S1 - Histone Deacetylase 1 and p300 Can Directly Associate with Chromatin and Compete for Binding in a Mutually Exclusive Manner

Figure S1 Direct recruitment of HDACs by MMTV promoter. (A) Recombinant Flag-HDAC1 was expressed from baculovirus infected insect cells and purified through affinity purification. The puified Flag-HDAC1 was subjected to SDS-PAGE and coomassie blue staining. * indicates Flag-HDAC1. The bovine serum albumin (BSA) is served as loading control. (B) Purified Flag-HDAC1 from insect cells and HDAC1 complex (HDAC1 com.) from 3134 cells were subjected to Western blot with HDAC1 and RbAp 46/48 antibodies. #1 and #2 are two biological repeats of purification. (C) Recruitment of HDAC1 by MMTV recon. The purified Flag-tagged HDAC1, CoREST, or LSD1 was incubated with the reconstituted MMTV mononucleosome. After extensive washes, proteins bound to the Dynabeads were separated in SDS-PAGE and detected by Western blotting with the anti-Flag antibody. (D) Recruitment of HDACs by MMTV promoter sequence. The purified Flag-tagged HDAC1, HDAC2, HDAC3, or LSD1 was incubated with MMTV promoter DNA fragments. Proteins bound to DNA were separated in SDS-PAGE and detected by Western blotting. Flag-tagged HDAC1, HDAC2, and HDAC3 were incubated with unbound beads (beads) as a negative control. Figure S2 HDAC1 interacts with various DNA sequences. (A) Schematic representation of biotin labelled DNA fragments. (B) DNA associated Flag tagged HDAC1 and HDAC2 were detected by Western blotting with the anti-Flag antibody. The experiment was repeated three times. (C) Bacterial expressed GST-HDAC1 was purified and eluted by 50 mM Glutathione and incubated with various biotin labelled DNA fragments. DNA associated GST HDAC1 was detected by Western blotting with the anti-HDAC1 antibody. The experiment was repeated three times. (D) Coomassie blue staining of purified GST tagged HDAC1. * indicates GST-HDAC1. Figure S3 Histone H3 tail interacts with HDACs. The purified GST-histone H3 tail 1-57 was incubated with Flag-tagged HDAC1, 2, 3, 4, 5 and 6. Proteins bound to GST-H3 1-57 were separated in SDS-PAGE and detected by Western blotting. The experiment was repeated at least three times. Figure S4 Treatment of TSA does not affect binding of histone H3 with HDAC1. The glutathione sepharose immobilized GST-histone H3 protein was incubated with recombinant flag tagged HDAC1 with or without the presence of 100 nM or 300 nM TSA in a pull down assay. Histone H3 associated HDAC1 was detected by Western blotting with an anti-Flag antibody. The experiment was repeated at least three times. Figure S5 HDAC2 and p300 are directly recruited to histone H3 in a competitive manner. The glutathione sepharose immobilized GST-histone H3 protein was first incubated with HDAC2 and subsequently incubated with various concentrations of p300 or vice versa. The histone H3 bound p300 and HDAC2 were detected by Western blotting with anti-Flag and anti-p300 antibodies. The experiment was repeated three times. Figure S6 HDAC1 protein level is decreased in p300 knockdown cells. The protein levels in p300 knockdown cells (p300 KD) and control cells (NC) were examined by Western blotting with anti-p300, anti-HDAC1, and anti-β-actin antibodies. The experiment was repeated three times. Table S1 Primers used in CHIP analysis.
(PPT)