Chromatin Immunoprecipitation (ChIP).

ChIP was performed as described previously (19, 39, 42, 44, 64, 65) using thymi from DOX -injected mice. See http://genomecenter.ucdavis.edu/farnham/farnham/protocols/tissues.html for tissue ChIP. The antibodies were recovered by using Protein G-sepharose, and washed extensively (64). The immunoprecipitated DNA was recovered by digestion of the samples with proteinase K and RNase. ~250 base pair fragments covering each p53-consensus sequences on the mouse p21^Cip1 (site #1) and Bbc3 (site #1) promoters, and mouse Thbs1 intron 7 (Fig. 1A) were amplified by PCR using 1 µCi of [α−³²P] dATP (GE Healthcare), separated in a 10 % non-denaturing polyacrylamide gel.

The sequences of primers used in ChIP PCR at Fig. 4A are as follows: p21^Cip1_1580SE 5’CCCTGT CCTTTTCTGGAAGTG-3’, p21^Cip1_1975AS 5’-CTGGGGTCTCTGTCTCCATTC-3’; Bbc3_1201SE 5’-GGACCAAAATCATGGCTTCA-3’; Bbc3_1377AS 5’-TGGGGAGACC ACAGTTCAAA-3’. Thbs1_310SE 5’-GAAAGCCCTACTGGTCCATCC-3’, Thbs1_560AS 5’-TGCACCATCACCACATTTCTC-3’.

A. p53-dependent binding of Dmp1 to the Bbc, p21^Cip1, and Thbs1 genomic loci in mouse thymus in response to DNA damage. Wild type and p53-null mice (6-week-old) were tail-injected with 0.6 mg DOX/30 g of a mouse, thymi were harvested at 0 and 4 hrs, and binding of Dmp1 to p53 target gene promoters were studied by chromatin immunoprecipitation. Significant binding of Dmp1 to the Bbc, p21^Cip1, and Thbs1 promoters were found in wild type, but not in p53-null thymus, suggesting that Dmp1 binding to these p53 target genes was indirectly mediated by p53. The Dmp1 protein did not bind to the genomic DNA without Dmp1 or p53 consensus sequences (data not shown).

B. Schematic representation of the findings obtained in this study and previously published data[47]. The Dmp1 promoter is activated by oncogenic Ras and overexpression of HER2, which subsequently activates the Arf-p53 pathway to induce cell cycle arrest or apoptosis to prevent the emergence of transformed cells. The promoter is also activated by TNFα and dsDNA breaks caused by DOX or etoposide. In Arf-deficient cells, Dmp1α (amino acids 87–392) directly binds to the p53 C-terminus (amino acids 290–360) and neutralizes all the known functions for Mdm2 on p53 (47) published data. This effect is mutually exclusive of DNA-binding of Dmp1α and thus is independent of Arf. In addition, Dmp1α-p53 binding accelerates DNA-binding of p53 to the target genes, which has been shown by EMSA with supershift assays with six different antibodies to Dmp1. See Materials and Methods about the epitope of each antibody.