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CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.

In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.

Proteinase-activated receptors (PARs) are G-protein-coupled receptors that have been linked to an array of cellular processes, including inflammation, migration, and proliferation. Although signal transduction downstream of PARs has been actively investigated, little is known about the mechanisms that lead to changes in transcriptional programs. Here we show that the CUX1 homeodomain protein is a downstream effector of PAR2. Treatment of epithelial and fibroblastic cells with trypsin or the PAR2-activating peptide (PAR2-AP) caused a rapid increase in CUX1 DNA binding activity. The stimulation of CUX1 was specific to PAR2 because no effect was observed with thrombin or the PAR1-AP. Using a panel of recombinant CUX1 proteins, the regulation was found to involve the cut repeat 3 (CR3) and the cut homeodomain, two DNA binding domains that are present in all CUX1 isoforms. Expression analysis in cux1(-/-) mouse embryo fibroblasts led to the identification of three genes that are regulated downstream of both PAR2 and CUX1 as follows: interleukin-1alpha, matrix metalloproteinase-10, and cyclo-oxygenase-2. p110 CUX1 was able to activate each of these genes, both in reporter assays and following the infection of cells. Moreover, the treatment of Hs578T breast tumor cells with trypsin led to a rapid recruitment of p110 CUX1 to the promoter of these genes and to a concomitant increase in their mRNA steady-state levels. Altogether, these results suggest a model whereby activation of PAR2 triggers a signaling cascade that culminates with the stimulation of p110 CUX1 DNA binding and the transcriptional activation of target genes.

The transcriptional regulator Cux1 (CDP, Cutl1) is aberrantly expressed in mouse models for polycystic kidney disease. Here we show that p75-Cux1, the shortest isoform of Cux1, transcribed from an alternative promoter within intron 20, is also deregulated in polycystic kidneys derived from Pkd1 mutant embryos. To determine the role of the p75-Cux1 isoform in cystogenesis, we generated transgenic mice expressing p75-CUX1 in the kidneys and other tissues. Strikingly, these animals developed polycystic kidneys at variable penetrance and severity, correlating with transgene expression levels. Histological and marker analysis of p75-CUX1-derived polycystic kidneys revealed renal cysts derived from the tubular nephron, supporting a model of autosomal dominant polycystic kidney disease. Transgenic p75-CUX1 kidneys additionally showed an up-regulation of the protooncogene c-myc and a down-regulation of the cyclin-dependent kinase inhibitor p27. Chromatin affinity purification experiments confirmed the direct interaction of Cux1 with the c-myc and p27 promoters. These molecular alterations were accompanied by an increase in cilia length and in the proliferative index of epithelial cells lining the cysts. Together, these results identify an important role for the short isoform of CUX1 in polycystic kidney disease development.

Proteolytic processing at the end of the G(1) phase generates a CUX1 isoform, p110, which functions either as a transcriptional activator or repressor and can accelerate entry into S phase. Here we describe a second proteolytic event that generates an isoform lacking two active repression domains in the COOH terminus. This processing event was inhibited by treatment of cells with synthetic and natural caspase inhibitors. In vitro, several caspases generated a processed isoform that co-migrated with the in vivo generated product. In cells, recombinant CUX1 proteins in which the region of cleavage was deleted or in which Asp residues were mutated to Ala, were not proteolytically processed. Importantly, this processing event was not associated with apoptosis, as assessed by terminal dUTP nick end labeling assay, cytochrome c localization, poly(ADP-ribose) polymerase cleavage, and fluorescence-activated cell sorting. Moreover, processing was observed in S phase but not in early G(1), suggesting that it is regulated through the cell cycle. The functional importance of this processing event was revealed in reporter and cell cycle assays. A recombinant, processed, CUX1 protein was a more potent transcriptional activator of several cell cycle-related genes and was able to accelerate entry into S phase, whereas mutants that could not be processed were inactive in either assay. Conversely, cells treated with the quinoline-Val Asp-2,6-difluorophenoxymethylketone caspase inhibitor proliferated more slowly and exhibited delayed S phase entry following exit from quiescence. Together, our results identify a substrate of caspases in proliferating cells and suggest a mechanism by which caspases can accelerate cell cycle progression.

The CCAAT displacement protein/Cut homeobox (CDP/Cux) transcription factor is expressed as multiple isoforms that may contain up to four DNA-binding domains: Cut repeats 1, 2, and 3 (CR1, CR2, CR3) and the Cut homeodomain (HD). The full-length protein, which contains all four DNA-binding domains, is surprisingly less efficient than the shorter isoforms in DNA binding. Using a panel of recombinant proteins expressed in mammalian or bacterial cells, we have identified a domain at the extreme N terminus of the protein that can inhibit DNA binding. This domain was able to inhibit the activity of full-length CDP/Cux and of proteins containing various combinations of DNA-binding domains: CR1CR2, CR3HD, or CR2CR3HD. Since inhibition of DNA binding was also observed with purified proteins obtained from bacteria, we conclude that autoinhibition does not require post-translational modification or interaction with an interacting protein but instead functions through an intramolecular mechanism. Antibodies directed against the N-terminal region were able to partially relieve inhibition. In vivo, the transition between the inactive and active states for DNA binding is likely to be governed by posttranslational modifications and/or interaction with one or more protein partners. In addition, we show that the relief of autoinhibition can be accomplished via the proteolytic processing of CDP/Cux. Altogether, these results reveal a novel mode of regulation that serves to modulate the DNA binding activity of CDP/Cux.

The CCAAT displacement protein, the homolog of the Drosophila melanogaster CUT protein, contains four DNA-binding domains: three CUT repeats (CR1, CR2, and CR3) and the CUT homeodomain (HD). Using a panel of fusion proteins, we found that a CUT repeat cannot bind to DNA as a monomer, but that certain combinations of domains exhibit high DNA-binding affinity: CR1+2, CR3HD, CR1HD, and CR2HD. One combination (CR1+2) exhibited strikingly different DNA-binding kinetics and specificities. CR1+2 displayed rapid on and off rates and bound preferably to two C(A/G)AT sites, organized as direct or inverted repeats. Accordingly, only CR1+2 was able to bind to the CCAAT sequence, and its affinity was increased by the presence of a C(A/G)AT site at close proximity. A purified CCAAT displacement protein/CUT protein exhibited DNA-binding properties similar to those of CR1+2; and in nuclear extracts, the CCAAT displacement activity also required the simultaneous presence of a C(A/G)AT site. Moreover, CR1+2, but not CR3HD, was able to displace nuclear factor Y. Thus, the CCAAT displacement activity requires the presence of an additional sequence (CAAT or CGAT) and involves CR1 and CR2, but not the CUT homeodomain.

The Drosophila and mammalian Cut homeodomain proteins contain, in addition to the homeodomain, three other DNA binding regions called Cut repeats. Cut-related proteins thus belong to a distinct class of homeodomain proteins with multiple DNA binding domains. Using nuclear extracts from mammalian cells, Cut-specific DNA binding was increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo. Sequence analysis of Cut repeats revealed the presence of sequences that match the consensus phosphorylation site for protein kinase C (PKC). Therefore, we investigated whether PKC can modulate the activity of mammalian Cut proteins. In vitro, a purified preparation of PKC efficiently phosphorylated Cut repeats, which inhibited DNA binding. In vivo, a brief treatment of cells with calphostin C, a specific inhibitor of PKC, led to an increase in Cut-specific DNA binding, whereas phorbol 12-myristate 13-acetate, a specific activator of PKC, caused a decrease in DNA binding. The PKC phosphorylation sites within the murine Cut (mCut) protein were identified by in vitro mutagenesis as residues Thr415, Thr804, and Ser987 within Cut repeats 1-3, respectively. Cut homeodomain proteins were previously shown to function as transcriptional repressors. Activation of PKC by phorbol 12-myristate 13-acetate reduced transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites was not affected. Altogether, our results indicate that the transcriptional activity of Cut proteins is modulated by PKC.

Homeodomain-containing proteins are believed to function as sequence-specific DNA binding proteins, regulating gene expression. Specificity of sequence recognition is conferred by the homeodomain acting either alone or in conjunction with other conserved DNA binding domains as is the case for Pou domain and Paired domain proteins. The recent isolation of cDNAs encoding mammalian homologues of the Drosophila Cut homeodomain protein has revealed that the 72-amino acid Cut Repeats are conserved in evolution. We have investigated the biochemical activity of human Cut Repeats by expressing fusion proteins containing glutathione S-transferase linked to various combinations of Cut Repeats and Cut homeodomain. We show by gel retardation and DNase footprinting assays that Cut Repeats can function as DNA binding domains, either independently or in cooperation with the homeodomain. The binding affinity (KD) to a specific recognition site was estimated to be 8 x 10(-9) M for Cut Repeat 3 and 4 x 10(-10) M for Cut Repeat 1. When both Cut Repeat 3 and the Cut homeodomain were present in the fusion protein, the binding affinity was increased to 4 x 10(-11) M. These results define a novel class of proteins that contain in addition to the homeodomain a second conserved protein domain, the Cut Repeats, that also function as a DNA binding domain.