Figure 9.

Recruitment of p52 to p53-regulated promoters is independent of p52 DNA binding. (A) Schematic diagram showing the R52A/R54A p52 DNA-binding mutation. Information shown about base contacts was obtained from (Cramer et al, 1997). (B) EMSA analysis of HA-tagged wild-type p52 and p52-DBM. Nuclear protein lysates prepared from U-2 OS cells transfected with either HA control plasmid, HA-tagged p52 or HA-tagged p52 DBM were analysed by EMSA using a ³²P labeled H2 κB element probe (upper panel). Western blot analysis of nuclear protein extracts demonstrates equivalent expression of p52 and p52-DBM (lower panel). Nonspecific bands recognized by the HA antibody indicate equivalent loading. (C, D) The p52 DNA-binding mutant does not bind the Cyclin D1 promoter but is still recruited to p53 target genes. ChIP analysis of the Cyclin D1 (C) or p21, DR5 and PUMA (D) promoters in U-2 OS cells transfected with either HA control plasmid, HA-tagged p52 or HA-tagged p52 DBM. The antibodies used are indicated.