E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node–derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. A comparison of ChIP-chip data with expression profiling data on resting E2f2−/− T lymphocytes identified a subset of 51 E2F2-specific target genes, most of which are upregulated on E2F2 loss. Luciferase reporter assays showed a retinoblastoma-independent role for E2F2 in the negative regulation of these target genes. Importantly, we show that the DNA binding activity of the transcription factor CREB contributes to E2F2-mediated repression of Mcm5 and Chk1 promoters. siRNA-mediated CREB knockdown, expression of a dominant negative KCREB mutant or disruption of CREB binding by mutating a CRE motif on Mcm5 promoter, relieved E2F2-mediated transcriptional repression. Taken together, our data uncover a new regulatory mechanism for E2F-mediated transcriptional control, whereby E2F2 and CREB cooperate in the transcriptional repression of a subset of E2F2 target genes.

E2F transcription factors control the expression of genes involved in a variety of essential cellular processes and consequently their activity needs to be tightly regulated. Protein-protein interactions are thought to be key modulators of E2F activity. To gain insight into the mechanisms that regulate the activity of E2F2, we searched for novel proteins that associate with this transcription factor. We show that the nuclear protein ALY (THO complex 4), originally described as a transcriptional co-activator, associates with DNA-bound E2F2 and represses its transcriptional activity. The capacity of ALY to modulate gene expression was analyzed with expression microarrays by characterizing the transcriptome of E2F2 expressing HEK293T cells in which ALY was either overexpressed or silenced. We show that ALY influences the expression of more than 400 genes, including 98 genes bearing consensus E2F motifs. Thus, ALY emerges as a novel E2F2-interacting protein and a relevant modulator of E2F-responsive gene expression. Molecular & Cellular