CXXC5 (RINF) is a direct target of retinoic acid. (A) RINF mRNA expression levels were measured by quantitative RT-PCR after 4 hours of 1 μM ATRA treatment. Inhibition of translation with cycloheximide (CHX) used at 10 μg/mL did not block ATRA-induced increase in RINF mRNA level, demonstrating that this process does not require de novo protein synthesis, and strongly suggests that RINF is a primary target of retinoic acid. The histogram represents means from 2 independent cell-culture treatments (± SEM). (B) In vivo binding of RARα and PML-RARα to the RINF promoter in NB4 cells. Cross-linked chromatin was prepared from NB4 cells treated or not with 1 μM ATRA for 3 hours and immunoprecipitated with anti-RARα or anti-PML antibodies. The precipitates were subjected to PCR analysis using primer pairs spanning the human RINF or RARβ2 gene promoters. The primers were designed to encompass the putative retinoid-responsive element found in the RINF promoter (ggagttcatgaggtgagc) or the well-established RARE (ggttcaccgaaagttca) in RARβ2 promoter (here used as a positive control); bold letters in the sequences represent the direct repeats. Input, PCRs performed on total chromatin from NB4 cells before IP; No Ab (no antibody control), PCRs performed on sample obtained after IP with an irrelevant antibody or without any antibody.