Fig. 4.

TR4 prevents prostate tumorigenesis by regulating DNA repair gene ATM transcription.

(A) Scheme of putative TR4 binding element on ATM promoter. The sequence was determined by Multi-genome Analysis of Positions and Patterns of Elements of Regulation search engine (MAPPER). The sequence matching model M00966 is marked in bold with underline. (B and C) ATM promoter was cloned into luciferase reporter vector pGL3 and was transfected along with internal control pRL-tk into RWPE1 cells. The results show that knocking down TR4 reduced ATM-Luc activity in RWPE1 cell, whereas overexpression increased ATM-Luc activity. Y-axis FL/RL indicates firefly luciferase activity normalized by internal control renilla luciferase activity. The results were normalized by internal control and were repeated three times. P values are 0.0068 and <0.0001 in TR4 knockdown and TR4 overexpression groups, respectively, via Student’s t-test, respectively. (D) Chromatin immunoprecipitation assay shows TR4 can physically bind to ATM promoter region shown in Figure 6A. 1×10⁶ cells per 200 μl of SDS Lysis Buffer were sonicated and DNA was pulled down by either TR4 antibody or normal mouse IgG. DNA from each group was washed, eluted and amplified by specific primers flanking the sequences shown in A. Input sample (1% of total sample) was gathered before immunoprecipitation was performed.