Distinctions in the specificity of E2F function revealed by gene expression signatures

Abstract

The E2F family of transcription factors provides essential activities for coordinating the control of cellular proliferation and cell fate. Both E2F1 and E2F3 proteins have been shown to be particularly important for cell proliferation, whereas the E2F1 protein has the capacity to promote apoptosis. To explore the basis for this specificity of function, we used DNA microarray analysis to probe for the distinctions in the two E2F activities. Gene expression profiles that distinguish either E2F1- or E2F3-expressing cells from quiescent cells are enriched in genes encoding cell cycle and DNA replication activities, consistent with many past studies. E2F1 profile is also enriched in genes known to function in apoptosis. We also identified patterns of gene expression that specifically differentiate the activity of E2F1 and E2F3; this profile is enriched in genes known to function in mitosis. The specificity of E2F function has been attributed to protein interactions mediated by the marked box domain, and we now show that chimeric E2F proteins generate expression signatures that reflect the origin of the marked box, thus linking the biochemical mechanism for specificity of function with specificity of gene activation.

Fig. 1.

Classification of samples based on E2F1 or E2F3 expression. (A) An E2F1 signature. (Left) Expression patterns of MEFs infected with AdCMV (control) or Ad-HAE2F1. Columns represent independent infections, and rows are ordered vertically by the estimated regression weights. (Center) Two-factor analysis of Ad-CMV vs. Ad-HAE2F1. Individual samples depicted in the scatter plot on two dominant factors underlying 100 genes selected in discrimination of the training cases. Each independent infection is indicated by a number and is color-coded, indicating control infections in blue and the Ad-HAE2F1 infections in red. (Right) Crossvalidation analysis defines an E2F gene expression signature. One-at-a-time crossvalidation predictions of classification probabilities for the training cases from the factor regression analysis. The values on the horizontal axis are estimates of the overall factor score in the regression analysis. The corresponding values on the vertical axis are estimated classification probabilities with the corresponding 95% probability intervals marked as dashed lines to indicate uncertainty about these estimated values. Control samples are red squares, Ad-HAE2F1 are blue triangles, and Ad-HAE2F3 are green triangles. (B) An E2F3 signature. Details are the same as in A, except for the use of Ad-HAE2F3.

Fig. 2.

Distinguishing E2F1 and E2F3 samples. (A) Expression patterns of MEFs infected with Ad-HAE2F1 or Ad-HAE2F3. Columns represent independent infections, and rows are ordered vertically by the estimated regression weights of the 100 predictive genes. (B) Two-factor analysis of Ad-HAE2F1 vs. Ad-HAE2F3. Individual samples depicted in the scatter plot on two dominant factors underlying 100 genes selected in discrimination of the training cases. Ad-HAE2F1 samples are colored in red and numbered by independent infection. Ad-HAE2F3 infections are colored in blue and numbered independently. (C) Crossvalidation analysis defines an E2F3 gene expression signature. One-at-a-time crossvalidation analysis predicts classification probabilities for the training cases from the factor regression analysis. Values on the horizontal axis are estimates of the overall factor score from the regression analysis, and the corresponding values on the vertical axis are the estimated classification probability of E2F3 activity, with 95% probability intervals marked as dashed lines to indicate uncertainty about the estimated values. Control infections are labeled as red squares, Ad-HAE2F1 as blue triangles, and Ad-HAE2F3 as green triangles.

Fig. 3.

Classification of E2F chimeras. (A) Schematic of protein domain structure of E2F1 and E2F3. Domain structures of Ad-HAE2F1, Ad-HAE2F3, Ad-HA111331, and Ad-HA333113. (B) Expression patterns of MEFs infected with Ad-HAE2F1, Ad-HAE2F3, Ad-HA333113, and Ad-HA111331. Columns represent independent infections, and rows are ordered vertically by estimated regression weights of the 100 predictive genes for E2F3 expression. (C) Prediction of E2F chimeras based on E2F3 gene expression signature. One-at-a-time crossvalidation predictions of classification probabilities of chimeric protein expression based on the trained pattern derived from the E2F3-vs. E2F1-expressing fibroblasts. Ad-HA111331 is designated as a green triangle and labeled E2F3 by origin of the marked box, and Ad-HA333113 is designated as a blue triangle and labeled E2F1 by origin of the marked box.