Regulation of cell cycle progression by forkhead transcription factor FOXO3 through its binding partner DNA replication factor Cdt1

Abstract

To ensure genome stability, DNA must be replicated once and only once during each cell cycle. Cdt1 is tightly regulated to make sure that cells do not rereplicate their DNA. Multiple regulatory mechanisms operate to ensure degradation of Cdt1 in S phase. However, little is known about the positive regulators of Cdt1 under physiological conditions. Here we identify FOXO3 as a binding partner of Cdt1. FOXO3 forms a protein complex with Cdt1, which in turn blocks its interaction with DDB1 and PCNA. Conversely, FOXO3 depletion facilitated the proteolysis of Cdt1 in unperturbed cells. Intriguingly, FOXO3 deficiency resulted in impaired S-phase entry and reduced cell proliferation. We provide data that FOXO3 knockdown mimics Cdt1 down-regulation and affects G1/S transitions. Our results demonstrate a unique role of FOXO3 in binding to Cdt1 and maintaining its level required for cell cycle progression.I n eukaryotic cells, DNA replication initiates from thousands of replication origins. Each origin acquires replication competence through the assembly of a prereplication complex (pre-RC) occurring in late mitosis and early G1 (1-3). Pre-RCs are assembled at the origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1, and MCM2-7 (4). In S phase, pre-RCs are sequentially acted on by two protein kinases, Cdc7 and Cdk2, which promote recruitment of proteins required for helicase activation and replisome assembly, leading to origin unwinding and DNA synthesis. To ensure that no replication origin fires more than once, the assembly of the replication apparatus at origins is tightly regulated by the cell cycle machinery. Among the most important of these regulatory mechanisms are the degradation of Cdt1 during S phase and the sequestration of Cdt1 by the geminin protein (5-7). Phosphorylation of Cdt1 by Cdk2 promotes its binding to SCF-Skp2 E3 ubiquitin ligase (8-10), which results in its degradation in S phase. In addition to the Skp2 pathway, PCNA/ DDB1/Cul4-dependent signaling was found to degrade Cdt1 during S phase via the interaction of Cdt1 with PCNA (11-15). Recently, APC/C Cdh1 was proposed as a third ubiquitin ligase regulating Cdt1 degradation (16). Cdt1 is also targeted for degradation after DNA damage to stop licensing of new origins until after DNA repair. Both the SCF-Skp2 complex and the Cul4-DDB1 complex have been reported to induce degradation of Cdt1 after UV irradiation (17,18).FOXO transcription factors are critical for the regulation of cell cycle arrest, cell death, and DNA damage repair. Ample evidence has suggested that FOXO exerts a negative effect on cell cycle progression. In dividing cells, overexpression of the active form of FOXO family members promotes cell cycle arrest at the G1/S boundary. Target genes that mediate FOXO-induced cell cycle arrest are the Cdk inhibitors p27KIP1 and p21 (in the presence of TGF-β), the Rb family member p130, and cyclin D1 and D2. The ectopically expressed active form of FOXO factors can cause G1 arrest both by up-regulat...

Geminin facilitates FoxO3 deacetylation to promote breast cancer cell metastasis

“…Previously, we reported that depletion of FoxO3 in MCF-7 cells led to impaired G 1 /S transition due to its failure to stabilize CDT1 (15). However, in MDA-MB-231 and LM2 cells, FoxO3 silencing had no detectable effect on the protein levels of CDT1 Figure 7G).…”

“…We previously found that FoxO3 has a unique role in regulating G 1 /S transition via stabilizing the chromatin licensing and DNA replication factor 1 (CDT1) protein, a binding partner of geminin (15). Geminin is a negative factor involved in DNA replication by blocking CDT1, thereby maintaining chromosomal integrity and preventing aneuploidy (16,17).…”

GSK3 Protein Positively Regulates Type I Insulin-like Growth Factor Receptor through Forkhead Transcription Factors FOXO1/3/4

“…Previous studies also demonstrate that FOXO contributes to Akt or mTOR inhibitor resistance in cancer cells through up-regulating receptor tyrosine kinases (54). In addition, a recent study reveals that FOXO3 directly binds to DNA replication factor Cdt1 and promotes cellular proliferation (27). This demonstrates that FOXO1/3/4 are positive regulators of hepatoma cell proliferation.…”

“…Although many studies indicate that FOXOs are bona fide tumor suppressors, recent studies demonstrate that they have roles in promoting tumorigenesis and drug resistance. Depletion of FOXO3 reduced G 1 /S transition and cell proliferation in MCF7 breast cancer cells and H1299 lung cancer cells (27). Elevated FOXO3 expression is associated with poor prognosis in patients with acute myeloid leukemia exhibiting normal cytogenetics, and genetic ablation of FOXO3 reduces disease burden in a murine model of chronic myeloid leukemia (28,29).…”

Chromatin Structure and Replication Origins: Determinants of Chromosome Replication and Nuclear Organization

“…Transcription factors and other nuclear proteins can contribute to the replication profile by binding to specific genomic loci and helping to recruit replication factors 73; 74 or by facilitating the formation of structural features such as local chromatin loops. For example, the transcription factor Myc forms a functional chromatin domain 75 in Drosophila, and Myc binding sites which retain Myc through mitosis colocalize with insulator proteins, implying a structural function.…”