THAP1 binds to the RRM1 promoter in vitro and in vivo. (A) Sequence of the RRM1 promoter indicating the positions of THAP1 binding sites (THABS), E2F cell-cycle regulatory elements, and NF-Y/CCAAT box. The mRNA region is indicated in uppercase letters. The initiating methionine is shown in bold. (B) Dnase I footprinting analysis of RRM1 promoter region. Lane 0, partial DnaseI cleavage of DNA fragment without incubation with protein. Lanes 0.1 and 1, partial DnaseI cleavage after incubation with 0.1 μM or 1 μM THAP domain of human THAP1, respectively. Lanes C+T and A+G, Maxam-Gilbert chemical sequencing references (cleavage after pyrimidine C+T and purine A+G). (C) In vitro EMSA using the first THABS motif of RRM1 promoter and 0.1 μM THAP domain of human THAP1. A 50-fold molar excess of wild-type (THABS) and mutant (mutTHABS) cold competitor oligonucleotides were used to show specificity of binding. (D) Identification of endogenous THAP1 on the RRM1 promoter in vivo using ChIP assays. Cross-linked chromatin from proliferating HUVECs was subjected to immunoprecipitation with antibodies against THAP1, NF-YB (positive control), and Flag epitope (negative control). The NF-YA promoter was used as a positive control for the NF-YB transcription factor and a negative control promoter for THAP1. (E) ChIP-qPCR assays were used to quantify the amount of RRM1 or NF-YA promoter DNA precipitated by anti-THAP1 or anti-Flag antibodies. Immunoprecipitated DNA was quantified in triplicate by qPCR using the percent of input method (see “Materials and methods”). A representative experiment out of 3 is shown. (F) Fold enrichment of THAP1 on the RRM1 promoter was calculated by dividing the amount of RRM1 promoter DNA precipitated by anti-THAP1 antibodies to the amount of DNA precipitated from the NF-YA negative control promoter. No enrichment was observed with anti-Flag negative control antibodies. Error bars in panels E and F indicate SD.