Visualizing Dynamic E2F-Mediated Repression In Vivo

Classic E2F target genes include those that regulate cell cycle progression (e.g., CcnE, Cdc6, Cdc25A, Mcm2-7, Orc, CcnA, and Cdc2) or the maintenance of nucleotide pools (e.g., Dhfr, Rnr, Tk, and Ts) (reviewed in references 5, 6, and 56). Typically, these have been identified by mutation of the E2F site [consensus sequence TTT(C/G)(C/G)CGC] in reporter constructs, leading to the deregulated expression of the putative target gene across the cell cycle. A number of E2F target genes have been identified whose products stimulate apoptosis (e.g., p73, Apaf1, Arf, and caspases). Most of the genes encoding E2F family members (E2f1-E2f3a and E2f6-E2f8) are themselves E2F targets, many of which are thought to contribute to a feed-forward amplification loop to generate sufficient E2F activity to stimulate cell cycle progression following pRB phosphorylation (1, 9, 11, 14, 22, 28, 35, 48, 66). More recently, gene expression profiling with inducible E2F expression and chromatin immunoprecipitation (ChIP-on-chip) analysis in cultured cells have greatly expanded the sheer number (estimated to be in the hundreds) and classes (e.g., DNA repair, cell cycle checkpoints, and chromatin dynamics) of E2F target genes substantially (24, 36, 39, 43, 44, 57). However, little is still known about the significance of any single E2F site in the normal regulation of an E2F target gene in vivo. Interestingly, two Rb family members (Rb and p107) are E2F target genes (20, 65), which suggests that substantial complexity may exist in the transcriptional circuitry connecting the Rb and E2f family members and that E2F may lie upstream and downstream of pRB in a genetic sense. Apart from the well-documented ability of cyclin/cyclin-dependent kinase (CDK)-mediated phosphorylation to regulate pRB function (50), transcription of the human RB gene or mouse Rb gene plays a role in regulating pRB function. Notably, point mutations and deletions in the human RB promoter have been identified in low-penetrance retinoblastomas, emphasizing the importance of the proper levels of RB transcription for tumor suppression (4, 10, 45, 63). Additionally, Rb transcription increases as cells undergo differentiation (e.g., P19 cells with retinoic acid) (41, 52, 62), which is consistent with the role of Rb in promoting differentiation of numerous cell types, particularly the neuronal lineage (18, 32, 37). The presence of elevated levels of mutant RB mRNA in many retinoblastomas has prompted speculation that pRB autoregulates its own promoter, and mutation of the RB gene leads to its increased transcription (15, 20). In light of the recently demonstrated dispensability of G1 cyclins and CDKs during most of development, an exploration of alternative routes to regulating pRB function seems warranted (42, 51). Indeed, transcriptional control of Rb levels during development could provide an alternative mechanism that would bypass the need for G1 cyclin/CDK-mediated phosphorylation in many tissues. A well-conserved 26-bp cluster of binding sites lying 180 bp upstream of the translational start site accounts for much of the human RB and the mouse Rb promoter activity in vitro (19, 62). Binding sites for Sp1, Ets, ATF, and E2F are present, the first two of which are partially overlapping and are referred to hereafter as Sp1/Ets (see Fig. ​Fig.1A).1A). A subset of the aforementioned point mutations in low-penetrance retinoblastomas maps into this Sp1/Ets site or into the adjacent ATF site of the RB promoter (45, 63), which is consistent with these being activator sites. In vitro studies have shown that mutation of the E2F site in this cluster activates RB gene expression in cell lines and that overexpression of pRB can repress Rb promoter expression of this putative repressor site (20, 40, 49, 62). FIG. 1. Generation of wild-type and mutant Rb promoter reporter lines. To generate the RbP(WT)-LacZ transgene construct, a fragment (4.3 kb) of the wild-type Rb promoter containing a cluster (green box) of transcription factor binding sites (Sp1, Ets sites, ATF ... To evaluate the importance of a single E2F site in vivo and to understand how E2F-mediated repression of a critical target, such as the Rb tumor suppressor, is regulated during development, we constructed a novel series of wild-type and mutant Rb promoter-LacZ transgenic lines, which allowed us to visualize Rb promoter activity in every tissue throughout development. These novel RbP-LacZ reporter mice express in a tissue- and temporal-specific manner, giving new insight into the role of E2F in vivo and the complex and dynamic balance between transcriptional activation and repression ongoing in the whole animal.