CCAAT enhancer-binding protein ␤ (C͞EBP␤), a basic-leucine zipper transcription factor, is an important effector of signals in physiologic growth and cancer. The identification of direct C͞EBP␤ targets in vivo has been limited by functional compensation by other C͞EBP family proteins and the low stringency of the consensus sequence. Here we use the combined power of expression profiling and high-throughput chromatin immunoprecipitation to identify direct and biologically relevant targets of C͞EBP␤. We identified 25 potential C͞EBP␤ targets, of which 88% of those tested were confirmed as in vivo C͞EBP␤-binding sites. Six of these genes also displayed differential expression in C͞EBP␤ ؊/؊ livers. Computational analysis revealed that bona fide C͞EBP␤ target genes can be distinguished by the presence of binding motifs for specific additional transcription factors in the vicinity of the C͞EBP␤ site. This approach is generally applicable to the discovery of direct, biologically relevant targets of mammalian transcription factors.C CAAT enhancer-binding proteins (C͞EBPs) constitute a family of basic-leucine zipper (bZIP) transcription factors that are critical for the regulation of numerous biological processes, including differentiation, metabolic homeostasis, proliferation, tumorigenesis, inflammation, and apoptosis (1-9). C͞EBP proteins are regulated at multiple levels, including gene transcription, translation, and phosphorylation, in response to a variety of stimuli including hormonal, cytokine and growth factor-signaling pathways (1). C͞EBP proteins are able to form hetero-and homodimeric complexes with other C͞EBP family members, thereby creating additional diversity in target sequence recognition.C͞EBP␤ is an important effector of growth signals in experimental models of physiologic and neoplastic growth, the acutephase response, and metabolic homeostasis (1-11). Livers from C͞EBP␤ Ϫ/Ϫ mice exhibit a blunted regenerative response associated with prolonged hypoglycemia and altered expression of several cell-cycle-associated genes (10). In addition, a recent microarray analysis of human tumors has implicated C͞EBP␤ as a downstream mediator of cyclin D (12). Although these studies provide strong support for the role of C͞EBP␤ as a regulator of cell growth, at present, neither the mechanism by which C͞EBP␤ modulates the growth effects of cyclin D1 nor the targets of C͞EBP␤ in this pathway have been elucidated.A variety of approaches has been used to identify C͞EBP␤-binding sites, including cell culture systems, C͞EBP␤ Ϫ/Ϫ mice, and analyses of promoter sequences. However, several obstacles have limited the identification of direct C͞EBP␤-dependent transcriptional targets in vivo. All C͞EBP family members with the exception of C͞EBP possess identical in vitro DNA-binding affinity for C͞EBP consensus sequences, suggesting that other C͞EBP family members may be able to compensate for the loss of C͞EBP␤ (13). Second, the application of computational sequence analysis to identify C͞EBP promoter sequences has been impede...

The E2F transcription factors play an essential role in regulating the G(1)- to S-phase transition of the cell cycle. Previous studies have identified the importance of interactions between E2Fs and other transcription factors as a mechanism for transcriptional control of a subset of E2F regulated target genes. However, the mechanisms responsible for E2F target gene specificity remain incompletely understood. Here we report that in a mammalian in vivo model of synchronized proliferation, C/EBPbeta occupancy on the promoters of E2F-regulated growth-related genes increases as a function of cell cycle progression. C/EPBbeta binding to these promoters is associated with recruitment of the coactivator CBP/p300, histone H4 acetylation, and maximal activation of E2F target genes. Moreover, binding of CBP/p300 to E2F targets is markedly reduced in C/EBPbeta null mice, resulting in reduced expression of E2F regulated genes. These findings identify C/EBPbeta as a direct activator of E2F target genes in mammalian cell cycle progression through a mechanism that involves recruitment of CBP/p300. The demonstration of a functional link between C/EBPbeta and CBP/p300 for E2F target gene activation provides a potential mechanism for how coactivators such as CBP/p300 can be selectively recruited to E2F target genes in response to tissue-specific growth stimuli.

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