A Transcriptional Switch Mediated by Cofactor Methylation Wei Xu, Hongwu Chen, Keyong Du, Hiroshi Asahara, Marc Tini, Beverly M. Emerson, Marc Montminy, Ronald M. Evans

Supplementary Material

Supplemental Figure 1. Direct interaction between CBP/p300 and CARM1. (A) Immuno-detection of p300 in GST pull-down assays with bacterially-expressed GST (lane 2), wild type GST-CARM (lane 3), and GST-CARM (189VLD-AAA191) mutant (lane 4) proteins. (B) Mammalian two-hybrid transfection assay. 15 ng of CMX-GAL-CBP and CMX-VP16-CARM1 were co-transfected with 35 ng of MH100-TK-Luc reporter into CV1 cells in 48-well plates. Luciferase activity was normalized with internal -gal activity. (C) Coimmunoprecipitation of CBP and CARM1 by anti-CBP antibody in mock (vector), CMX-Flag CBP transfected or CMX-CARM1 and CMX-Flag CBP co-transfected 293 cells. CARM1 was detected by western blotting in immunoprecipitates by anti-CBP antibodies from the transfected cell extracts (WB). CBP or CARM1 levels in whole cell extract were detected with anti-CBP (A22, Santa Cruz Biotech.) or anti-CARM1 (T16, Santa Cruz Biotech.) antibodies (Input).

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Supplemental Figure 2. Coomassie staining of proteins used in the in vitro methylation experiment.

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Supplemental Figure 3. Methylation-dependent partitioning of transcriptional coactivator CBP/p300 in inducible gene activation. CBP/p300 and CARM1 are recruited to nuclear receptor by p160 coactivator in response to hormone, where HAT and HMT activity stimulate transcription by remodeling chromatin through subsequent histone acetylation and methylation in the vicinity of hormone response element (HRE). In contrast, methylated CBP/p300 fails to mediate CREB-dependent transcription because methylation blocks CBP/p300 and CREB interaction.

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Methods for the DNA fragmentation assay in Fig. 4B. NGF-treated PC12 cells were transfected with wild type (lane 2), mutant CARM1 (lane 3), CARM1 and VP16-CREB (lane 4) constructs. Cells treated with 0.1 mM staurosporine were used as a positive control (lane 5). Cells were grown in 10 ng/ml NGF in low serum media (DMEM supplemented with 2% BSA). Genomic DNA was purified after 2 days and the fragments were amplified using LM-PCR kit (Clontech). Equal amount of en-2 DNA were amplified from each sample as control (data not shown).