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Small GTP-binding proteins of the Ras superfamily regulate a wide array of cellular functions and play critical roles in signal transduction, gene regulation, intracellular trafficking of vesicles, cell proliferation, and survival (37). Proteins in the Rho subfamily (Rho, Rac, and Cdc42) are best characterized for their effects on actin polymerization and cytoskeleton organization (15). Through their effects on the cytoskeleton, Rho GTPases are essential in cell morphology, cell migration and chemotaxis, and establishment of cell polarity (6, 18, 44). They also participate in several aspects of cell cycle progression. During mitosis, Cdc42 regulates spindle microtubule attachment to kinetochores in metaphase, whereas RhoA, which localizes at the cleavage furrow during telophase and ends up at the midbody at the abcission step, is involved in cytokinesis (25, 43, 59). Indeed, interfering with RhoA activity, by treating cells with Clostridium botulinum C3 exoenzyme, or with RhoA expression, by small interfering RNA (siRNA) silencing, prevents contraction of the actomyosin ring and leads to failure of cytokinesis and multinucleation in various cell types (25, 60). During these steps, RhoA activity is regulated by the exchange factor Ect2 and the GTPase activating protein MgcRacGAP (hereafter, RacGAP) which colocalize with a kinesin known as MKLP1, Kif23, or kinesin-like protein 5 (35, 52, 61). MKLP1 associates with RacGAP in a complex known as centralspindlin and is thought to mediate recruitment of RacGAP to the mitotic spindle. Protein interactions between Ect2 and RacGAP lead to conformational changes that activate the exchange activity of Ect2 (20, 26). During mitosis progression, dynamic protein interactions between Ect2, RacGAP, and MKLP1 are regulated by phosphorylation/dephosphorylation events mediated by the Cdk1 and Aurora B kinases (34, 36, 53). The dynamics and functions of this complex are evolutionarily conserved as the Drosophila orthologs (Pebble/RacGAP50/Pavarotti) and the Caenorhabditis elegans orthologs (LET-21/CYK4/ZEN4) also control cytokinesis with similar regulations. Genes whose protein products play specific roles in cell cycling and exert their functions at defined steps of cycle progression are often regulated in a cell cycle-dependent manner (7, 14). These genes include not only the well-characterized mitotic cyclins and their associated kinases or inhibitors but also additional kinases (e.g., Plk-1 and Aurora) and components of the anaphase promoting complex/cyclosome (3, 56, 58). Promoters for cell cycle-regulated genes contain binding sites for specific transcription factors (14). The E2F family contains eight identified members acting as transcriptional activators or repressors (11). E2F1 was initially characterized as an activator of genes expressed at the G1/S transition to regulate cell cycle progression but was also shown to regulate G2/M genes as well as the apoptosis pathway (19, 62). Besides its coactivator DP, E2F may interact with other transcription factors such as B-Myb, itself an E2F target in G1/S (24, 45, 62). CUX1 (Cut homeobox 1) belongs to the family of Cut homeodomain-containing transcription factors that is present in all metazoans. In vertebrates, the protein was first identified as the CCAAT-displacement protein and has been variously called CDP, CUX, or CUTL1 (2, 41, 57). Whereas the CCAAT-displacement activity, which depends on rapid but transient interactions with DNA, appears to be constant throughout the cell cycle, a more stable interaction with DNA arises at the end of the G1 phase as the result of dephosphorylation by Cdc25A and proteolytic processing by a nuclear isoform of cathepsin L (8, 17, 38). The N-terminally truncated isoform thus generated, p110 CUX1, was shown to accelerate entry into S phase (46). The regulatory effect of p110 CUX1 was found to be promoter dependent: it repressed the p21cip and cyclin H genes but activated most genes including those coding for cyclin A2, DNA polymerase (Pol) α, and the Cdc25 proteins (8, 21, 46, 55). Another signature of cell cycle-regulated genes is the frequent occurrence of cell cycle-dependent element/cycle homology region (CDE/CHR) sequences close to the transcription start site. Although factors binding to such elements have not yet been unambiguously characterized, their functionality has clearly been established by mutation or deletion analysis in several cell cycle genes such as cdc25C, Cyclin A, or aurora B (22, 27, 29, 30). We report here that the mitotic complex genes Ect2, RacGAP, and MKLP1 are coordinately induced in S phase in proliferating T lymphocytes as well as in epithelial cells, depending upon activity of the CUX1 and E2F1 transcription factors. We have also identified CHR elements that mediate repression of these promoters in the G1 phase of the cell cycle.

The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase alpha gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase alpha gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene.