Methyltransferase Recruitment and DNA Hypermethylation of Target Promoters by an Oncogenic Transcription Factor Luciano Di Croce, Veronica A. Raker, Massimo Corsaro, Francesco Fazi, Mirco Fanelli, Mario Faretta, Francois Fuks, Francesco Lo Coco, Tony Kouzarides, Clara Nervi, Saverio Minucci, and Pier Giuseppe Pelicci

Supplementary Material

Supplemental Figure 1. (A) Methylation status of the RAR2 promoter. Genomic DNA from APL blasts was subjected to sodium bisulphite modification and the RAR2 promoter amplified as described (Fig. 1E). (B) Methylation-specific PCR of normal (CD34⁺) and APL blasts. Cells were left untreated or incubated with RA, TSA, 5-Aza-dC, as indicated. Genomic DNA was subjected to sodium bisulphite modification, and amplified by use of primers specific for methylated (M) and unmethylated (U) DNA. (C) Immunofluorescent localization of PML, PML-RAR and Dnmts. Representative confocal laser scanning microscopy images from 293T cells (expressing Myc-tagged Dnmt1, Myc-tagged Dnmt3a, or PML-RAR) and PR9 cells, are shown. Following incubation with primary antibody, coverslips were probed with the secondary antibody conjugated with either Alexa (green) or Cy3 (red). Merge (right) of left and middle panels indicates areas of co-localization (yellow). (D) APL blasts were treated or not for 48 h with RA, 5-Aza-dC, or TSA, as indicated. RT-PCR of RAR2 expression was performed as described in Fig. 1C. (E) Bisulphite genomic sequencing was performed on RAR2 promoter from APL blasts. RA or 5-Aza-dC was added as indicated for 48h.

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