Geminin is Targeted for Repression by the Retinoblastoma Tumor Suppressor Pathway through Intragenic E2F Sites

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The geminin protein is a critical regulator of DNA replication. It functions to control replication fidelity by blocking the assembly of prereplication complexes in the S and G2 phases of the cell cycle. Geminin protein levels, which are low in G0/G1 and increase at the G1/S transition, are controlled through coordinate transcriptional and proteolytic regulation. Here we show that geminin is regulated transcriptionally by the retinoblastoma tumor suppressor (RB)/E2F pathway. Initially, we observed that the activation of RB led to the repression of geminin transcription. Conversely, Rb-null mouse embryonic fibroblasts have enhanced the expression of geminin relative to wild type mouse embryonic fibroblasts. Similarly, an acute loss of Rb in mouse adult fibroblasts deregulated geminin RNA and protein levels. To delineate the responsible regulatory motifs, luciferase reporter constructs containing fragments of the geminin promoter were generated. An analysis of the critical regulatory cis-acting elements in the geminin promoter indicated that intragenic E2F sites down-stream of the first exon were responsible for RB-mediated repression of geminin. The direct analysis of the endogenous geminin promoter revealed that these intragenic E2F sites are occupied by E2F proteins, and the mutation of these sites eliminates responsiveness to RB. Together, these data link the expression of geminin to the RB/E2F pathway and represent the first promoter analysis of this important regulator of DNA replication.

Geminin was identified originally as a protein that is degraded by mitoticXenopus egg extracts, but not by interphase extracts (1), and concurrently during a screen to identify proteins that affect Xenopus development (2). Geminin is a small (25 kDa) protein expressed during the S and G2 phases of the cell cycle but degraded in M phase at the metaphase/anaphase transition via the anaphase-promoting complex-mediated ubiquitination (3, 4). Functional analyses demonstrated that geminin acts to prevent the relicensing of replication origins after they have fired once. This is accomplished by binding to CDT1 (5), a requisite factor for loading MCMs1 into the prereplication complex. Beginning at the G1/S transition, geminin protein levels accumulate and become sufficient to inhibit CDT1 activity. As soon as the origins of replication have fired once in the S phase, this inhibition of CDT1 prevents the reloading of MCMs onto chromatin until the completion of mitosis when geminin is degraded. Although the levels of geminin mRNA have been shown to increase 2–3-fold at the G1/S transition (6), the mechanism of this transcriptional regulation remains to be elucidated.

RB has several emerging roles in the control of diverse processes outside of the G1/S transition. These include DNA repair, cell death, and DNA replication (7–10). In the case of DNA replication, the activation of the RB pathway results in the repression of numerous target genes. Classically, this repression is achieved through assembling repressor complexes at the E2F family of transcription factor binding sites (11–13). For example, the known targets of RB-mediated repression include several genes that encode components of the MCM complex, which are required for the initiation of DNA synthesis (14–16). DNA polymerase α and PCNA, both required for DNA replication to proceed, also are repressed via the activation of RB (14–16). As the noted replication factors all stimulate DNA replication, their negative regulation is consistent with the action of RB in the inhibition of the S phase. However, in a recent microarray screen, we identified geminin as a putative target for RB-mediated repression. This finding is difficult to reconcile with the classical cell cycle inhibitory role of RB. If geminin in fact is repressed via RB, this would represent a novel function because it would indicate that RB plays an important role not only in inhibiting replication but also in enabling replication to commence. In this study, we observed the repression of endogenous geminin by active RB as well as deregulation of geminin expression with a loss of RB signaling. A comparison of the geminin genes in mouse and rat revealed conserved E2F sites. We cloned the geminin promoter and identified critical regulatory elements. An analysis of the cis-acting elements by reporter assays showed that the geminin promoter is repressed via active RB functioning through intragenic E2F sites.