The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase ␣ gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase ␣ gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.CUX1 (cut homeobox) belongs to a family of transcription factors involved in the control of proliferation and differentiation (reviewed in reference 52). Several CUX1 isoforms can be expressed as a result of proteolytic processing or transcription initiation at alternative sites (21,23,45,49,70). Cux1 gene ablation in mice resulted in high perinatal lethality. Surviving mice exhibited a number of mutant phenotypes, including growth retardation, male infertility, curly whiskers, abnormal hair follicle morphogenesis, and a shortage of T and B cells (13,43,63,73). Transgenic mice expressing p200 CUX1 exhibited multiorgan hyperplasia and organomegaly (36), whereas those expressing p75 or p110 CUX1 displayed enhanced susceptibility to malignancies in various tissues and cell types (4; C. Cadieux and A. Nepveu, unpublished data).Initial studies of mammalian CUX1 revealed its role as a transcriptional repressor that is expressed in differentiating precursor cells and serves to down-regulate the expression of genes expressed only in terminally differentiated cells (41,55,64,66,67). CUX1 was also reported to regulate the expression of cell cycle-regulated genes such as those encoding p21 WAF1 (7), histones H1, H2A, H2B, H3, and H4 (12, 27, 74, 76), and DNA polymerase (Pol) ␣ (71). The full-length protein, p200 CUX1, interacts transiently with DNA and is expressed througho...