Coordination of a Transcriptional Switch by HMGI(Y) Acetylation Nikhil Munshi, Theodora Agalioti, Stavros Lomvardas, Menie Merika, Guoying Chen, and Dimitris Thanos

Supplemental Figure 1. Mutant HMGI(Y) expression vectors (K71R and K65R) were generated by PCR mutagenesis, and the presence of the mutation was verified by sequencing. Other expression vectors (PCAF and PCAF HAT^-) have been described elsewhere, and transfections were carried out as previously described (8). HeLa cells were cotransfected with the -110 IFN Luc reporter (1 g), along with expression vectors (3 g) encoding HMGI(Y), HMGI(Y) K65R, or HMGI(Y) K71R in the absence (A) or the presence of PCAF (B) or PCAF HAT^- (C). The cells were virus infected for the indicated amount of time, and the luciferase activity was determined and plotted as relative activity. Shown is the average of three independent experiments, and the variability was less than 25%. Note the differences in scale among (A), (B), and (C). (D) DNAseI footprinting experiments using PCAF- or mock-acetylated HMGI(Y) K71R (100 ng) and recombinant IFN- activators (30 ng NF-B, 400 ng ATF-2/c-JUN, and 30, 100, 200, 400, or 800 ng IRF-1). (E) Liquid HAT assays were performed as previously described (8). Peptides (21mer, Ac K65, and Ac K71) were obtained from Research Genetics, Inc., and the indicated amounts were used in HAT assays for one hour at 30°C. Reactions were stopped by spotting on P-81 filter paper (Whatman), and incorporated radioactivity was determined in a scintillation counter. Liquid HAT assays were done using recombinant CBP HAT (30 ng) or PCAF HAT (10 ng) domain proteins and the indicated concentrations of peptide. Data was fitted using the Prizm software package, and approximate Km and Vmax values are shown.

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