FIG. 4.

Bromodomain of ANCCA binds preferentially to H3K14ac. (A) Amino acid sequence alignment of bromodomains from ANCCA and other human proteins. Arrows indicate amino acid sequences changed in the mutant form of the ANCCA bromodomain.(B) Histones either acid extracted from butyrate-treated HeLa S3 cells (top) or purified from E. coli and in vitro acetylated by PCAF (bottom) were subjected to Western analysis for acetylation with the indicated antibodies. (C) GST-ANCCA bromodomain proteins, wild type or mutant, were incubated with histones from HeLa cells. (D) GST-ANCCA bromodomain proteins were incubated with histones in vitro acetylated by PCAF. Histones retained on the glutathione beads were detected by Western analysis with the indicated antibodies. (E, F) GST-ANCCA bromodomain proteins (E) or nuclear extract from HeLa cells expressing HA-tagged full-length ANCCA (F) was incubated with biotinylated histone tail peptides with the indicated acetylation. After being washed extensively, ANCCA proteins retained on the beads were analyzed by Western analysis with anti-GST (E) or anti-HA (F) antibodies, respectively.