Figure 1.

Both p19^Arf and p16^Ink4a promoters are activated by cyclin D1 and the cyclin D1T286A mutant. A: The Arf (−281) or Ink4a (−400) promoter luciferase construct was cotransfected with indicated amounts of cyclin D1 [wild-type (wt)], D1T286A (D1TA), or D1Δ142–253 (D1Δ)²¹ expression vectors in NIH 3T3 cells. The numbers show the fold activation of the luciferase reporter normalized by internal controls of SEAP. B: The Arf (−281) or Ink4a (−400) promoter luciferase construct was cotransfected with increasing amounts of cyclin D1 expression vector in Dmp1^+/+ or Dmp1^−/− MEFs. The numbers show the fold activation of the luciferase reporter normalized by internal controls of SEAP. C: Luciferase reporter construct encoding Arf (−281) promoter or Ink4a (−400) promoter was cotransfected with 1 μg of pFLEX1-Dmp1 together with either 3 or 5 μg of pFLEX1-cyclin D1 or pFLEX1-D1Δ142–253 into Dmp1-null MEFs. The numbers show the fold activation of the luciferase reporter normalized by internal controls of SEAP. D: Primary Dmp1^+/+ (black bars) or Dmp1^−/− (white bars) MEFs were infected with retrovirus carrying an empty vector or vector expressing cyclin D1 or D1T286A protein. After neomycin selection, cells were harvested for the analysis of Arf and Ink4a mRNA levels by real-time PCR using β-actin as a control. E: ChIP analysis of cyclin D1 binding to the Arf and Ink4a promoters in Dmp1^+/+ and Dmp1^−/− MEFs infected with lentivirus expressing HA-cyclin D1. Lysates derived from Dmp1^+/+ and Dmp1^−/− MEFs were immunoblotted for Dmp1 and HA. β-Actin was used as a loading control. The asterisk indicates a nonspecific band. F: ChIP analysis for the binding of endogenous cyclin D1 to the Arf and Ink4a promoters in MMTV-neu tumors. Tissue ChIP was conducted with formalin-fixed tumors from the MMTV-neu mice with wild-type Dmp1. The cyclin D1 protein was precipitated by two different antibodies to cyclin D1 (SP4 and H295). Data are means ± SD from n = 3 individual experiments (A–D). Ab, antibody; pro, promoter.