Supplementary Figure Legends s4

Supplementary figure legends

Figure S1. Inactivation of p53/p21 inhibits p21 expression and SAHF in H2A.Z-depleted WI-38. DNA staining by DAPI (A to D –1), p21 staining by a monoclonal antibody (A to D –2, and p21 staining by a polyclonal antibody (A to D –3) and prolonged exposure (C/D-4). The cell indicated in A is control WI-38, B is H2A.Z-depleted WI-38, C are p53 and H2A.Z-depleted WI-38, and D are p21 and H2A.Z-depleted WI-38. C-4 and D-4 are prolonged microscopic exposures of C-2 and D-2 respectively.

Figure S2. Localization of H2A.Z and H2A at the p21 promoter region. (A) ChIP assay in U2OS cells with H2A.Z and H2A represented as % input. (B) Same experiment as (A) but in SaOS2 cells.

Figure S3. Localization of H2A.Z at the p21 promoter in HCT116 p53+/+ and p53-/- isogenic cells.

(A) Diagram of the p21 promoter region and the segments analyzed in these experiments. (B) H2A.Z enrichment at the p21 promoter in HCT116 p53+/+ and p53-/- cells.

Figure S4. H2A.Z and p400 colocalize at p53 binding sites in multiple p53 target genes. (A) ChIP experiments demonstrating the localization of H2A.Z and p400 in the intronic region of GADD45 that contains a p53-binding site. (B) Localization of p400 and H2A.Z at the BAX, and (C) the MDM2 promoter region. Schematic diagrams of GADD45, BAX, and MDM2 demonstrate the p53 binding sites and the amplicons used for Q-PCR. Relative binding of H2A.Z is expressed as fold enrichment over H2A and p400 binding is percent input multiplied by 100.

Figure S5. p400 incorporates H2A.Z into chromatin around the p53 binding sites at p21.

(A) Expression levels of p21 in U2OS cells before and after p400 and SRCAP knockdown (left panel). The level of SRCAP RNA knock down by the shRNA is shown in the right panel. The mRNA levels were normalized against 36B4, as measured by Q-PCR. (B) Diagram of the p21 promoter region and the segments analyzed in these experiments. (C) ChIP assays of H2A.Z enrichment at the p21 promoter demonstrating the effect of p400 and SRCAP knockdowns on the incorporation of H2A.Z into chromatin.

Figure S6. p400 is evicted from the p53-binding sites at the p21 gene following induction with daunorubicin. (A) p21 regions analyzed by ChIP. (B) ChIP experiments showing the relative binding of p400 at the p21 promoter before and after induction with daunorubicin.

Figure S7. Effect of TPA on the induction of p21 in U2OS cells. Expression levels of p21, before and after treatment with TPA for 4hrs, was measured as described in the Experimental procedures section.