8042 Nucleic Acids Research, 2010, Vol. 38, No. 22 Figure 1. Widespread translational changes are associated with each 4E-BP deletion. (A) A254 traces showing polysome proﬁles from exponential phase cultures for the strains used. RNA fractions collected were pooled into monosomal (M) or polysomal (P) fractions from wild-type (w) caf20D (c) and eap1D (e) cells for translational-proﬁling array experiments. (downward arrow) ‘‘halfmer’’ peaks. (B) Graphical representation of global changes in translation accompanying deletion of CAF20 or (C) EAP1. Mean polysome-to-monosome ratio intensities for each probeset from mutant (y-axis) is plotted against the wild-type (x-axis). Points above or below a log2 = 0.9 cut-off are considered signiﬁcant changes (coloured red and blue, respectively). Plots (D) and (E) as B and C except spots representing translationally regulated genes (red and blue) from the eap1D plot (C) are shown on the caf20D plot (D), and regulated genes from the caf20D plot (B) on the eap1D plot (E). (F) Venn-style diagram showing the number of genes altered (in parenthesis) and the overlap between data sets. Numbers in black ﬁlled circles represent ‘potentiated’ genes (see text). (G) Immunoblots of Caf20p-Myc and Eap1-Myc levels in strains indicated. eIF2g (GCD11) loading control from Caf20-Myc tagged cells is also shown. intensities. A ‘change in translational state’ was determined by comparing the ratios from mutant cells (m) to that from wild-type cells (w). These analyses are represented as scatter plots in Figure 1B and C and nu- merically (Supplementary Tables S5–S8). The plots reveal that the majority of mRNA probe sets fall on or around the diagonal, indicating that the translation of most genes is not affected by deletion of either gene. In line with previous studies using similar techniques, we applied a signiﬁcance cut off of log2 = ±0.9 (i.e. >1.86-fold) change (21,29). This revealed that caf20D altered the translation of 784 mRNAs (471 up- and 313 down-regulated), while eap1D affected 329 mRNAs (176 up and 153 down) (Figure 1F). The fact that more mRNAs are Caf20p targets (784/1028 = 76%) than Eap1p agrees with our observations that the relative abun- dance of Myc tagged Caf20p is 2.8 ± 0.63-fold greater than Eap1 (data not shown). These data are consistent with Caf20p regulating translation of a greater number of mRNAs than Eap1p. Eap1p acts as a translational repressor for mRNAs that are poorly associated with polysomes under normal growth conditions. This observation is consistent with the idea that Eap1p plays a role repressing translation of a subset of genes during normal growth. This is illustrated graphically in Figure 1D, where the spot location is derived from the caf20D experiment, but spots are coloured according to the eap1D data. It is evident that most eap1D up-regulated genes (red spots) are to the left half of the plot, while the down-regulated ones (blue) are on the right. Therefore the majority of genes which are up-regulated in eap1D cells (red spots in Figure 1C and D) are among those transcripts which are poorly associated with polysomes in wild-type cells (81% or 142/176 log2[Pw/Mw]<0). A reciprocal arrangement holds for down-regulated genes (14% or 21/153 log2[Pw/Mw]<0). This is much less evident for Caf20p regulated transcripts. Figure 1E illustrates where the caf20D regulated genes are highlighted on the eap1D plot. Although >50% of the genes up-regulated by caf20D are