Snf2h

Cells were lysed in RIPA buffer (50 mM Tris‐HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % NP‐40, 0.5 % sodium deoxycholate, 0.1 % SDS) supplemented with protease inhibitors (cOmplete cocktail, Roche, Basel, Switzerland). For denaturation, protein extracts were incubated in Laemmli buffer at 95 °C for 10 min, separated by SDS–PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA) by semidry blotting (Biorad, Hercules, CA, USA). Primary antibodies and dilutions used were NM1 (1:1000), β-actin (Sigma, 1:50000), WSTF (Abcam, 1:2000), SNF2h (Abcam, 1:500), PCAF (Abcam, 1:500), Set1/Ash2 (Abcam, 1:500). Immunoreactive bands were visualized by chemiluminescence (Amersham, GE Healthcare Life Sciences, Pittsburgh, USA).

ChIP was performed as previously described (34 (link)). Immunoprecipitated DNA was quantified by polymerase chain reaction (PCR) using the Syber Green mix (Kapa) and all precipitations, except for those performed on histones and histone modifications, were compared to signals at the H27 site in the 45S rDNA (35 (link)) or to the actin promoter. The primer pairs used are given in the Supplementary Information (Supplementary Tables S2 and S3). Antibodies used were WSTF (Abcam), SNF2h (Abcam), RNA pol III (kind gift from Dr. Henry and from Abcam), TFIIIA (Abcam), TFIIIC220C (Santa Cruz and Abcam), Brf1 (Abnova and Abcam), GCN5 (Abcam), PCAF (Abcam), p300 (Abnova), TRRAP (Santa Cruz), c-Myc (Santa Cruz and Abcam), Max (Santa Cruz and Abcam), RNA pol II CTD (Abcam), H3-Ac, H3K9-Ac, H3K14-Ac, H3K27-Ac, H3K4-me3, H3K9-me3, H3K27-me3, H4, H4K8-Ac and H4K16-Ac (Abcam).