The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

The ␣-fetoprotein gene (AFP) is tightly regulated at the tissue-specific level, with expression confined to endoderm-derived cells. We have reconstituted AFP transcription on chromatin-assembled DNA templates in vitro. Our studies show that chromatin assembly is essential for hepatic-specific expression of the AFP gene. While nucleosome-free AFP DNA is robustly transcribed in vitro by both cervical (HeLa) and hepatocellular (HepG2) carcinoma extracts, the general transcription factors and transactivators present in HeLa extract cannot relieve chromatin-mediated repression of AFP. In contrast, preincubation with either HepG2 extract or HeLa extract supplemented with recombinant hepatocyte nuclear factor 3 ␣ (HNF3␣), a hepatic-enriched factor expressed very early during liver development, is sufficient to confer transcriptional activation on a chromatin-repressed AFP template. Transient transfection studies illustrate that HNF3␣ can activate AFP expression in a non-liver cellular environment, confirming a pivotal role for HNF3␣ in establishing hepatic-specific gene expression. Restriction enzyme accessibility assays reveal that HNF3␣ promotes the assembly of an open chromatin structure at the AFP promoter. Combined, these functional and structural data suggest that chromatin assembly establishes a barrier to block inappropriate expression of AFP in non-hepatic tissues and that tissue-specific factors, such as HNF3␣, are required to alleviate the chromatin-mediated repression.

Aberrant expression of developmentally silenced genes, characteristic of tumor cells and regenerating tissue, is highly correlated with increased cell proliferation. By modeling this process in vitro in synthetic nuclei, we find that DNA replication leads to deregulation of established developmental expression patterns. Chromatin assembly in the presence of adult mouse liver nuclear extract mediates developmental stage-specific silencing of the tumor marker gene alpha-fetoprotein (AFP). Replication of silenced AFP chromatin in synthetic nuclei depletes sequence-specific transcription repressors, thereby disrupting developmentally regulated repression. Hepatoma-derived factors can target partial derepression of AFP, but full transcription activation requires DNA replication. Thus, unscheduled entry into S phase directly mediates activation of a developmentally silenced gene by (i) depleting developmental stage-specific transcription repressors and (ii) facilitating binding of transactivators.Cellular commitment, differentiation, and specificity are determined primarily by the interaction of protein complexes with chromatin DNA. This gene-regulatory programming is challenged during each cell cycle by passage of the replisome during DNA synthesis. DNA replication may facilitate competition between transiently disrupted histones and transacting factors at the replication fork, potentially changing established chromatin structure and gene expression patterns (2,7,20,29,59). During tumorigenesis, terminally differentiated cells that have withdrawn from the cell cycle are induced to resume cycling, often through functional inactivation of tumor suppressor genes such as Rb and INK4a (30,33). This cyclic disruption of chromatin structure provides a fertile environment, similar to that which exists during fetal development, for altering gene expression patterns. Aberrant adult activation of genes normally expressed only in the fetus is characteristic of many tumors (reviewed in references 31 and 39). For example, a hepatoma marker gene, alpha-fetoprotein (AFP), is transcriptionally repressed shortly after birth (reviewed in reference 52) and is reexpressed in mature hepatocytes only when they leave G 0 quiescence and begin cycling following partial hepatectomy or during hepatocellular carcinoma (HCC) (42; reviewed in reference 52). Expression of AFP is therefore closely linked to cell cycle progression; the renewal of DNA replication following transition from quiescence to S phase may play an epigenetic role in modulating gene expression.The relative local concentration of transacting factors at the time of replication can determine whether a given chromatin structure is maintained or converted to an alternate conformation (17, 58). Modulating the balance of repressors and activators present during replication could be achieved through a variety of mechanisms, including functional inactivation of repressors and activation of transcription factors. Alternatively, the availability of transactivators could be controlled by s...