Ep400

The preparation of chromatinized G5E4T, normalization of chromatin amounts, in vitro transcription reactions on chromatin templates, immunodepletion and IT procedures have been described in our previous publications (Black et al., 2006 (link); Lin et al., 2011 (link)). Briefly, transcription or IT assays were carried out on 50 ng of chromatin templates that were bound with saturating levels of GAL4-VP16. Where indicated, immobilized chromatin was acetylated with 20 ng of Esa1, 20 ng Gcn5 and 1 mM Acetyl-CoA for 45 min. Transcription was measured by ³²P-primer extension and visualized on an Amersham Biosciences Typhoon 9400. Immunoblots were processed on a LI-COR Odyssey and quantitated by Image Studio 2. Antibodies included pan-acetyl Histone H3 (Active Motif), MED23 (BD Pharmingen), Pol II (QED Bioscience), TFIIB (Tantin et al., 1996), EP400, Tip60 (Abcam), ASH2L, CHD1 (Bethyl Laboratories) and all others from Santa Cruz Biotechnology.

Stable U2OS Tet-On Luciferase cells were obtained from Clontech Laboratories Inc., Mountain View, CA, and cultured in DMEM with 10% Tet-system approved FBS (Clontech). After addition of 1 μg/ml Doxycycline, transcription and ChIP assays were as previously described (Black et al., 2006 (link)). Antibodies used in ChIP included MED26 (Santa Cruz Biotechnology, sc-48776), Pol II (QED Bioscience), H3.3 (Abnova, H00003021-M01), EP400 (Abcam, ab70301), H2AZ (Abcam, ab4174), and H3K4me1 (Abcam, ab8895). Antibody against H3K18ac was in house (Ferrari et al., 2008 (link)). Immunoprecipitates were washed and DNA was quantitated by real-time semi-quantitative PCR. Alternately, mRNA was isolated and quantified by Reverse Transcriptase-PCR.