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ChIP-chip and expression analyses indicated that CUX1 transcription factors regulate a large number of genes and microRNAs involved in multiple cellular processes. Indeed, in proliferating cells CUX1 was shown to regulate several genes involved in DNA replication, progression into S phase and later, the spindle assembly checkpoint that controls progression through mitosis. siRNA-mediated knockdown established that CUX1 is required for cell motility. Moreover, higher expression of short CUX1 isoforms, as observed in many cancers, was shown to stimulate cell migration and invasion. In parallel, elevated expression particularly in higher grade tumors of breast and pancreatic cancers implicated CUX1 in tumor initiation and progression. Indeed, transgenic mouse models demonstrated a causal role of CUX1 in cancers originating from various cell types. These studies revealed that higher CUX1 expression or activity not only stimulates cell proliferation and motility, but also promotes genetic instability. CUX1 has also been implicated in the etiology of polycystic kidney diseases, both from a transgenic approach and the analysis of CUX1 activity in multiple mouse models of this disease. Studies in neurobiology have uncovered a potential implication of CUX1 in cognitive disorders, neurodegeneration and obesity. CUX1 was shown to be expressed specifically in pyramidal neurons of the neocortex upper layers where it regulates dendrite branching, spine development, and synapse formation. In addition, modulation of CUX1 expression in neurons of the hypothalamus has been associated with changes in leptin receptor trafficking in the vicinity of the primary cilium resulting in altered leptin signaling and ultimately, eating behavior. Overall, studies in various fields have allowed the development of several cell-based assays to monitor CUX1 function and have extended the range of organs in which CUX1 plays an important role in development and tissue homeostasis.

Chromatin immunoprecipitation (ChIP) has become an essential assay in the field of transcriptional regulation. It is currently the most popular method to monitor the in vivo interaction between a protein and specific genomic sites. The method can also serve to identify novel transcriptional targets when the immunoprecipitated chromatin, sometimes called chipped DNA, is used either as a probe in hybridization experiments with microarrays of genomic DNA (ChIP-chip) or as template in DNA sequencing (ChIP-Seq). ChIP assays rely on the availability of good antibodies that can specifically and efficiently immunoprecipitate the protein under study even after cross-linking. However, good antibodies are not always available. To circumvent this problem, we have developed and validated the method of chromatin affinity purification (ChAP). The subsequent microarray analysis is then referred to as ChAP-chip. In brief, the protein under study is expressed together with two tags in order to allow the purification of chromatin by tandem affinity purification. To ensure that only true targets are identified, it is important to express the recombinant tagged-protein at physiological level. This requirement is not trivial as most expression vectors are designed to express proteins at high levels. We found most convenient to use an inducible retroviral vector in the absence of inducer and transactivator protein. We describe the procedure to generate cells stably expressing recombinant tagged-proteins at physiological level and then to purify the associated chromatin by affinity purification. Targets identified in this manner were validated in independent ChAP assays as well as in ChIP assays using antibodies against the endogenous protein.

α-fetoprotein (AFP) is not only a widely used biomarker in hepatocellular carcinoma (HCC) surveillance, but is also clinically recognized as linked with aggressive tumour behaviour. Here we show that deregulation of microRNA122, a liver-specific microRNA, is a cause of both AFP elevation and a more biologically aggressive phenotype in HCC. We identify CUX1, a direct target of microRNA122, as a common central mediator of these two effects. Using liver tissues from transgenic mice in which microRNA122 is functionally silenced, an orthotopic xenograft tumour model, and human clinical samples, we further demonstrate that a microRNA122/CUX1/microRNA214/ZBTB20 pathway regulates AFP expression. We also show that the microRNA122/CUX1/RhoA pathway regulates the aggressive characteristics of tumours. We conclude that microRNA122 and associated signalling proteins may represent viable therapeutic targets, and that serum AFP levels in HCC patients may be a surrogate marker for deregulated intracellular microRNA122 signalling pathways in HCC tissues.

Background Lats1 (large tumor suppressor 1) codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog wts (warts) in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of Lats1 inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation. Results Genome-wide location arrays in cell lines of various cell types revealed that Lats1 was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of Lats1. Expression of Lats1 was reduced in cux1-/- MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of Lats1 was sufficient to confer transcriptional activation by CUX1. Lats1 was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1. Conclusion While inactivation of Lats1/wts in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that Lats1 is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of LATS1 in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be associated with either loss-of-function or overexpression of Lats1.

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.

p27(Kip1) is a cyclin-dependent kinase inhibitor that regulates the G(1)/S transition. Increased degradation of p27(Kip1) is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCF(Skp2) ubiquitinate p27(Kip1) in G(1) and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27(Kip1) levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G(0)/G1 to S phase, and accumulated p27(Kip1). This increase in p27(Kip1) was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27(-/-) cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G(1) to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion.

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.

It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non–cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus. (Mol Cancer Res 2007;5(9):899–907)

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 p75 CCAAT-displacement protein/Cut homeobox (CDP/Cux) isoform was previously reported to be overexpressed in human breast cancers. To investigate its oncogenic potential, we engineered two transgenic mouse lines expressing p75 CDP/Cux under the control of the mouse mammary tumor virus-long terminal repeat. The FVB strain of mouse is generally used in the generation of mouse models for breast cancer. The transgene was introduced into the hprt locus of 129/Ola embryonic stem cells and, following germ line passage, was backcrossed onto the FVB and C57BL/6 mouse strains. Here, we describe the phenotype of p75 CDP/Cux transgenic virgin female mice of the first backcross generations. We report that after a long latency period, ∼33% of mice from two independent transgenic lines and from backcrosses into either the FVB or the C57BL/6 strains succumbed to a similar disease characterized by splenomegaly, hepatomegaly, and frequent infiltration of leukocytes into nonhematopoietic organs like the kidneys and lungs. Although an excess of B or T cells was observed in three diseased mice, in 17 other cases, histologic and flow cytometry analyses revealed the expansion of a population of neutrophils in the blood, spleen, and bone marrow. The increase in neutrophils correlated with signs of anemia and thrombocytopenia, whereas there was no indication of a reactive process. Therefore, p75 CDP/Cux transgenic mice displayed heightened susceptibility to a disease defined as a myeloproliferative disease–like myeloid leukemia. These results indicate that the overexpression of p75 CDP/Cux could alter homeostasis in the hematopoietic compartment. (Cancer Res 2006; 66(19): 9492-501)

The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.

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.

An important mechanism of regulation that controls progression through the cell cycle involves the timely degradation of specific regulatory proteins. In parallel to the main degradative pathways, it appears that the function of certain proteins may also be modulated by a process called limited proteolysis. We have recently shown that the CDP/Cux transcription factor is proteolytically processed at the G(1)/S transition by the cathepsin L protease. Two aspects of these findings are discussed in the context of the cell cycle. Firstly, together with the cohesin subunit Scc1 and the HCF-1 factor, CDP/Cux represents a third example whereby the process of "limited proteolysis" plays a role in the control of cell cycle progression. Secondly, our findings provides compelling evidence that the cathepsin L protease, which was believed to be obligatorily targeted through the endoplasmic reticulum to the lysosomes or the extra-cellular milieu, could also be present in the nucleus and modulate the function of transcription factors.

The subclass of cysteine proteases termed lysosomal cathepsins has long been thought to be primarily involved in end-stage protein breakdown within lysosomal compartments. Furthermore, few specific protein substrates for these proteases have been identified. We show here that cathepsin L functions in the regulation of cell cycle progression through proteolytic processing of the CDP/Cux transcription factor. CDP/Cux processing in situ was increased following ectopic expression of cathepsin L but was reduced in Cat L−/− cells. Furthermore, catalytically active cathepsin L was localized to the nucleus during the G1-S transition as detected by immunofluorescence imaging and labeling using activity-based probes. Trafficking of cathepsin L to the nucleus is accomplished through a mechanism involving translation initiation at downstream AUG sites and the synthesis of proteases that are devoid of a signal peptide. Overall, these results uncover an as yet unsuspected role for cysteine proteases in the control of cell cycle progression.

CDP/Cux (CCAAT-displacement protein/cut homeobox) contains four DNA binding domains, namely, three Cut repeats (CR1, CR2, and CR3) and a Cut homeodomain. CCAAT-displacement activity involves rapid but transient interaction with DNA. More stable DNA binding activity is up-regulated at the G(1)/S transition and was previously shown to involve an N-terminally truncated isoform, CDP/Cux p110, that is generated by proteolytic processing. CDP/Cux has been previously characterized as a transcriptional repressor. However, here we show that expression of reporter plasmids containing promoter sequences from the human DNA polymerase alpha (pol alpha), CAD, and cyclin A genes is stimulated in cotransfections with N-terminally truncated CDP/Cux proteins but not with full-length CDP/Cux. Moreover, expression of the endogenous DNA pol alpha gene was stimulated following the infection of cells with a retrovirus expressing a truncated CDP/Cux protein. Chromatin immunoprecipitation (ChIP) assays revealed that CDP/Cux was associated with the DNA pol alpha gene promoter specifically in the S phase. Using linker scanning analyses, in vitro DNA binding, and ChIP assays, we established a correlation between binding of CDP/Cux to the DNA pol alpha promoter and the stimulation of gene expression. Although we cannot exclude the possibility that stimulation of gene expression by CDP/Cux involved the repression of a repressor, our data support the notion that CDP/Cux participates in transcriptional activation. Notwithstanding its mechanism of action, these results establish CDP/Cux as an important transcriptional regulator in the S phase.

Two isoforms of the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor have been characterized thus far. The full length protein, p200, which contains four DNA binding domains, transiently binds to DNA and carries the CCAAT-displacement activity. The p110 isoform is generated by proteolytic processing at the G1-S transition and is capable of stable interaction with DNA. Here we demonstrate the existence of a shorter CDP/Cux isoform, p75, which contains only two DNA binding domains, Cut repeat 3 and the Cut homeodomain, and binds more stably to DNA. CDP/Cux p75 was able to repress a reporter carrying the promoter for the cyclin-dependent kinase inhibitor p21 gene and to activate a DNA polymerase alpha gene reporter. Expression of CDP/Cux p75 involved a novel mechanism: transcription initiation within intron 20. The intron 20-initiated mRNA (I20-mRNA) was expressed at higher level in the thymus and in CD4+/CD8+ and CD4+ T cells. I20-mRNA was expressed only weakly or not at all in normal human mammary epithelial cells and normal breast tissues but was detected in many breast tumor cells lines and breast tumors. In invasive tumors a significant association was established between higher I20-mRNA expression and a diffuse infiltrative growth pattern (n = 41, P = 0.0137). In agreement with these findings, T47D breast cancer cells stably expressing p75 could not form tubule structures in collagen but rather developed as solid undifferentiated aggregates of cells. Taken together, these results suggest that aberrant expression of the CDP/Cux p75 isoform in mammary epithelial cells may be associated with the process of tumorigenesis in breast cancer.

Genetic analyses and mRNA expression studies have implicated CUTL1 as a candidate tumor-suppressor gene in uterine leiomyomas and breast cancers. However, modulation of CDP/Cux, the protein encoded by CUTL1, does not agree with this notion. The activity of CDP/Cux, which is the DNA binding subunit of HiNF-D, was upregulated as normal cells progressed into S phase and constitutively elevated in several tumor cell lines. Activation of CDP/Cux at the G(1)/S transition involved the proteolytic processing of the protein to generate a shorter isoform. Uterine leiomyomas represent a unique reagent for molecular analysis because they are resected as homogeneous tumor tissue together with the adjacent normal myometrium and they are often very large. In the present study, proteins were isolated from 16 pairs of matched tumors and adjacent myometrium and analyzed by Western blot and electrophoretic mobility shift assays. Strikingly, in 11/16 tumors, the steady-state level of small CDP/Cux isoforms was increased compared to normal control tissue. Where tested, a corresponding increase in CDP/Cux stable DNA binding activity was observed. DNA sequencing analysis of CUTL1 cDNAs from 6 leiomyomas, including 4 with LOH of CUTL1, did not reveal any gross rearrangement or point mutations. Altogether these findings suggest that CUTL1 is probably not the tumor suppressor on 7q22. Moreover, the frequent increase in smaller CDP/Cux isoforms indicates that molecular events associated with the truncation of CDP/Cux proteins may be selected in uterine leiomyomas.

The CCAAT displacement protein (CDP), the homologue of the Drosophila melanogaster Cut protein, contains four DNA binding domains that function in pairs. Cooperation between Cut repeat 3 and the Cut homeodomain allows stable DNA binding to the ATCGAT motif, an activity previously shown to be upregulated in S phase. Here we showed that the full-length CDP/Cut protein is incapable of stable DNA binding and that the ATCGAT binding activity present in cells involves a 110-kDa carboxy-terminal peptide of CDP/Cut. A vector expressing CDP/Cut with Myc and hemagglutinin epitope tags at either end generated N- and C-terminal products of 90 and 110 kDa, suggesting that proteolytic cleavage was involved. In vivo pulse/chase labeling experiments confirmed that the 110-kDa protein was derived from the full-length CDP/Cut protein. Proteolytic processing was weak or not detectable in G(0) and G(1) but increased in populations of cells enriched in S phase, and the appearance of the 110-kDa protein coincided with the increase in ATCGAT DNA binding. Interestingly, the amino-truncated and the full-length CDP/Cut isoforms exhibited different transcriptional properties in a reporter assay. We conclude that proteolytic processing of CDP/Cut at the G(1)/S transition generates a CDP/Cut isoform with distinct DNA binding and transcriptional activities. These findings, together with the cleavage of the Scc1 protein at mitosis, suggest that site-specific proteolysis may play an important role in the regulation of cell cycle progression.

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.

Resistance to the growth inhibitory effects of transforming growth factor beta (TGFbeta) has been associated with decreased levels of the TGFbeta type II receptor (TbetaR-II) and has been correlated with tumorigenicity. Previously, we reported an A --G mutation at position -364 in the TbetaR-II promoter in A431 tumor cells which results in reduced TbetaR-II promoter activity. In this study, we show that the CDP/Cut (CCAAT displacement protein) transcription factor, a transcriptional repressor, binds both the wild type and the mutant TbetaR-II promoter. We also demonstrate that the A --G mutation increases CDP/Cut binding affinity, and that overexpression of CDP/Cut reduces transcription from TbetaR-II promoter reporter constructs. Increased binding of the CDP/Cut repressor protein, as a result of a mutation at position -364, represents a novel mechanism of regulation in a neoplastic cell of the promoter of a tumor suppressor gene, TbetaR-II.

The T-cell protein tyrosine phosphatase (TC PTP) is expressed ubiquitously at all stages of mammalian development. However, mRNA levels fluctuate in a cell-cycle-dependent manner, reaching peak levels in late G1, and rapidly decreasing in S phase. Furthermore, TC PTP being present in higher amounts in lymphoid tissues, we have recently shown that it is essential for proper maintenance of both the bone marrow micro-environment and B- and T-cell functions. In order to better understand the elements controlling the expression pattern of this gene, we have isolated and characterized approx. 4kb of the murine TC PTP promoter. DNA sequencing of the proximal 5' region revealed the absence of both TATAA and CAAT boxes. Primer extension analysis and S1 nuclease mapping techniques identified multiple transcription initiation sites. Functional promoter activity was determined using transfection experiments of promoter deletion constructs fused to a CAT reporter construct. Our results indicate that the minimal promoter sequence required for functional expression is contained within the first 147bp of the TC PTP promoter. In addition, consistent with the cell-cycle-dependent expression of TC PTP, we localized a domain between 492 and 1976bp from the transcription initiation site through which repression occurs. In conclusion, although initiator-driven transcription allows for ubiquitous expression of TC PTP, we define general transcription motifs present within the promoter that may mediate specific modulations of the TC PTP gene.

Cut is a homeodomain transcription factor which has the unusual property of containing several DNA-binding domains: three regions called Cut repeats and the Cut homeodomain. Genetic studies in Drosophila melanogaster indicate that cut plays important roles in the determination and maintenance of cell-type specificity. In the present study, we show that mammalian Cut proteins may yet play another biological role, specifically in proliferating cells. We found that the binding of Cut to a consensus binding site varies during the cell cycle. Binding was virtually undetectable in G0 and early G1, but became very strong as cells reached S phase. This was shown to result both from an increase in Cut expression and dephosphorylation of the Cut homeodomain by the Cdc25A phosphatase. We also show that the increase in Cut activity coincides with a decrease in p21WAF1/CIP1/SDI1 mRNAs. In co-transfection experiments, Cut proteins repressed p21WAF1/CIP1/SDI1 gene expression through binding to a sequence that overlaps the TATA box. Moreover, p21WAF1/CIP1/SDI1 expression was repressed equally well by either Cdc25A or Cut. Altogether, these results suggest a model by which Cdc25A activates the Cut repressor which then downregulates transcription of p21WAF1/CIP1/SDI1 in S phase. Thus, in addition to their role during cellular differentiation, Cut proteins also serve as cell-cycle-dependent transcriptional factors in proliferating cells.

The inactivation of certain tumor suppressor genes is thought to play an important role in the genesis of a number of tumor types. For example, inactivation of the Retinoblastoma (Rb) tumor suppressor is frequently observed in a proportion of sporadic human breast cancers. While these studies suggest that inactivation of key tumor suppressor genes may play an important role in the induction of mammary cancers, direct evidence supporting this contention is lacking. Because polyomavirus (PyV) Large T (LT) antigen is known to associate with and inactivate certain members of the Rb family (p105Rb, p107, p130), we have derived transgenic mice which express PyV LT antigen in the mammary epithelium. As expected mammary epithelial-specific expression of PyV LT antigen resulted in the induction of mammary tumors which correlated with their capacity to associate with Rb family members. In addition to mammary carcinomas, female transgenic mice expressing the PyV LT transgene frequently develop uterine leiomyomas. Because loss of heterozygosity involving the human CUTL1 (Cut like 1) gene located at chromosomal position 7q22 has been recently implicated in sporadic human uterine leiomyomas, we tested the hypothesis that PyV LT antigen may also form specific complexes with CUTL1. The results of these analyses revealed that specific complexes of CUTL1 and PyV LT antigen could be detected in both leiomyomas and mammary tumors. Taken together, these observations suggest that PyV LT antigen may be involved in inducing these tumors by sequestering both CUTL1 and Rb growth regulatory proteins.

Cytogenetic analyses has revealed deletions and/or rearrangments at several chromosomal positions in approximately half of uterine leiomyomas. The most frequent genetic alteration, deletion of 7q22, was found in approximately 35% of studied cases with cytogenetic abnormalities (128/366=35%). The same chromosomal band was also found to be deleted in a fraction of acute myeloid leukemias and myelodysplastic syndromes. The frequent deletion of 7q22 in some tumors suggest that a tumor suppressor gene may be located in this region. The human Cut-like homeobox gene, CUTL1, is one of the genes localized to 7q22 and it was shown previously to encode a transcriptional repressor that down-modulates the expression of c-Myc. Activation of the c-Myc oncogenic potential has been shown in many cancers to result from alterations in one or the other of its several mechanisms of regulation. These observations led us to hypothesize that CUTL1 could act as a tumor suppressor gene. In the present study, we have identified polymorphic markers within and directly adjacent to CUTL1 at 7q22 and demonstrated that these markers are present in a commonly deleted region in seven out of 50 uterine leiomyomas samples examined. Furthermore, Northern blot analysis revealed that CUTL1 mRNA levels were reduced in eight tumors out of 13. These results suggest that CUTL1 may act as a tumor suppressor gene whose inactivation could be of pathological importance in the etiology of uterine leiomyomas.

Chromosome 7 lesions are common and are associated almost uniformly with a deleterious outcome in MDS and related myeloid neoplasms. We analyzed a large cohort of these patients (pts) (n=1595) and identified those with loss of heterozygosity (LOH) of chromosome 7, including del(7q), monosomy7 (-7) and UPD(7q) in 171 cases. Using single nucleotide polymorphism (SNP)-array karyotyping, 3 commonly deleted regions (CDR) have been isolated, including 7q22, 7q34, and 7q35-36. CDR 7q22 was involved in 119 cases and spanned among others PLOD3, RBL5 and CUX1 genes, which could play a role in del(7q) pathogenesis. To further investigate the molecular pathogenesis of -7/del(7q), we performed whole exome new generation sequencing (NGS) in 428 pts with MDS and related conditions; this cohort included 72 -7/del(7q) or UPD7. When we studied the mutational status of genes in 7q22, we noted 5 cases of CUX1mutations, including 2 with heterozygous and 3 with homozygous mutations (associated with UPD7q). These mutations were validated by Sanger sequencing and targeted deep sequencing of DNA from both tumor and normal cells. All CUX1 mutations revealed critical structural and functional determinants such as nonsense mutations resulting in premature stop codons (n=3), nonsense mutation located in the DNA-binding Cut homeodomain (p.R1296K, n=1) and splice site mutation (n=1). CUX1 mutation occurred in MDS (n=2) and MDS/MPN (n=3), and concomitant TET2 mutations were seen in 4 cases. No hemizygous CUX1mutations were found in cases with del(7q). CUX1 is haploinsufficient in cases with -7/del(7q). In total 166 cases (10.9%) had -7/del(7q) involving CUX1, in addition to pts in whom CUX1 was affected by likely hypomorphic/inactivating mutations. Pts with decreased expression of CUX1 had poor survival compared to pts without CUX1 (p In addition to somatic mutations, LOH of CUX1 has been reported in several cancers. A role of CUX1 as a haploinsufficient tumor suppressor cannot be explained by the known functions of CUX1 in stimulating cell proliferation, motility and resistance to apoptosis. We recently identified a novel molecular function of CUX1 in DNA repair that may explain how haplo-deficient expression of CUX1 contributes to leukemic transformation. We used single cell gel electrophoresis (comet assay) to show that Cux1-/- mouse embryo fibroblasts (MEFs) are deficient, while Cux1+/- MEFs are haploinsufficient, in the repair of oxidative DNA damage. Using an inverse-PCR assay, following etoposide exposure, the frequency of chromosomal translocations involving the mixed lineage leukemia (Mll) gene is significantly and progressively increased in Cux1+/- and Cux1-/- MEFs compared to Cux1+/+ MEFs. We then performed comet assays using primary leukemic cells that harbor a frameshift mutation predicted to inactivate CUX1 by producing a C-terminally truncated protein devoid of a nuclear localization signal. Repair of oxidative DNA damage was delayed in leukemic cells compared to bone marrow cells from a healthy donor. Using an in vitrobase excision repair assay, we show that repair of 8-oxoguanine is reduced in a cell line displaying LOH of CUX1. Similarly, repair of 8-oxoguanine is reduced following siRNA-mediated CUX1 knockdown, but is rescued by the addition of a purified CUX1 protein. Together these results demonstrate that CUX1 plays a direct role in DNA repair and that inactivation of one CUX1 allele reduces the DNA repair capability of cells. In conclusion, novel somatic mutations of CUX1 as a candidate gene are associated with poor prognosis in MDS pts with -7/del(7q) and UPD7. Somatic events constitute loss of function of CUX1, resulting in insufficiency of DNA repair mechanisms, which is associated with leukemogenesis and could be considered as a new therapeutic target. CUX1 mutations may affect base excision repair, and dysfunction of CUX1 could theoretically predispose to chromosomal translocations and complex karyotype, seen in conjunction with del(7q) cases. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

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.

By analogy with other homeodomain proteins conserved in evolution, mammalian Cut proteins are believed, as in Drosophila melanogaster, to play an important role in determining cell type specificity in several tissues. At the molecular level, Cut proteins appear to serve as transcriptional repressors. In this study, we have examined the mechanism by which the human Cut (hCut) protein down-regulates gene expression. The homeodomain and the three regions called Cut repeats are evolutionarily conserved and were previously shown to function as DNA binding domains. The carboxy-terminal region, although it does not show amino acid sequence homology per se, in all cases is enriched in alanine and proline residues, a distinctive feature of some transcriptional repression domains. Our results reveal two distinct modes of repression: competition for binding site occupancy and active repression. On one hand, the composite DNA binding domain formed by Cut repeat 3 and the Cut homeodomain was shown to bind to CCAAT and Sp1 sites within the tk gene promoter and to reduce gene expression, presumably by preventing activation by the corresponding transcription factors. On the other hand, the carboxy-terminal region of mammalian Cut proteins was found to function as an active repression domain in a distance-independent manner. We have further narrowed this activity to two subdomains that can independently repress activated transcription. Finally, we present a model to illustrate the two mechanisms by which Cut proteins repress gene expression.

Three cDNA probes that were used for the mapping. The regions covered by these probes comprise nucleotides 2492 to 5374 for probe 1, 2492 to 3936 for probe 2, and 3936 to 5374 for probe 3. The chromosomal localization of the CUTL1 gene was determined by FISH (fluorescence in situ hybridization). Probes were labeled by nick-translation using biotin-11-dUTP. After hybridization, slides were rinsed once with 2 x SSC. Probes were visualized by an indirect immunofluorescence system without any amplification.

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.

RmI is a circular DNA molecule that consists of a complete polyomavirus genome with an insertion (Ins) of mouse cellular DNA. This polyomavirus genome carries a mutation which renders its replication, but not its transforming ability, temperature sensitive. Ins contains both unique and repetitive cellular DNA sequences. We transfected RmI into rat cells at the permissive and nonpermissive temperatures for replication and isolated clones that had integrated RmI in their genomes. In this paper, we describe detailed mapping of the integrated RmI sequences present in 37 different cell clones. Our results indicated that transfection at the permissive temperature resulted in a random integration pattern, whereas transfection at the nonpermissive temperature resulted in a nonrandom integration pattern. The nonrandom insertions had a preferential length and preferential endpoints. We argue from these results that the nonrandom integration pattern is related to the presence of Ins and that the switch between nonrandom integration and random integration reflects a modification of the integrating substrate. When both are active, the random mechanism dominates the nonrandom mechanism.

We have previously shown that integration of a polyoma vector containing rodent repetitive elements into rat cellular DNA is non-random (Wallenburg et al. J. Virol. 50: 678-683). Junctions between the polyoma vector and the host DNA occur in the repetitive sequences of the vector about ten times more frequently than would be expected if sequences from the vector were used randomly for integration. In this paper we looked at the host sequences involved in these junctions. Our analysis did not reveal any repetitive or specific sequences and we presume therefore that the repetitive sequences of the vector acted as hot spots for illegitimate recombination. We also analysed the integration mechanism and found that: First, even though the polyoma vector was transfected in the presence of carrier DNA, integration did not involve the formation of a transgenome. Second, in at least one of the clones analysed, integration resulted in deletion of host DNA sequences. Third, the host DNA displaced at the integration site was considerably longer than the integrated segment.

We have observed that failure to remove certain restriction enzymes after digestion reduced the transforming ability of DNA from 10- to 50-fold. The DNA found integrated in the transformed cells isolated under these conditions had lost little or no sequences. We interpret these results as indicating that certain restriction enzymes remain bound to the DNA ends after digestion, thus generating a substrate unfavorable both for integration and exonucleolytic degradation. As expected from this interpretation, removal of the restriction enzymes before transfection restored the full transforming ability of linear DNA, but also resulted in the integrated sequences being significantly shorter than the transfected DNA. These findings strongly argue for the hypothesis that integration of linear DNA by illegitimate recombination requires free ends and further suggest that exonucleolytic degradation of such ends may generate a preferred substrate for integration. Finally, a comparison of the sequences found integrated after transfection with circular or linear molecules, led us to conclude that circular molecules need not be linearized to become integrated.

Chromosomal rearrangements involving the c-myc oncogene are a prevalent feature of plasmacytomas that arise after inoculating BALB/c mice with pristane and Abelson murine leukaemia virus (A-MuLV). With this observation in mind, we decided to determine if any genetic alterations of the c-myc locus could be observed in cells of a different type, when transformed in vitro by A-MuLV. Here we have analysed three independent A-MuLV-transformed NIH 3T3 lines (ANN-I, 54c12 and N25), and found that the c-myc locus is amplified 8-19-fold in each transformant. Quantitative S1 nuclease mapping performed on ANN-I and 54c12 RNAs demonstrated that: (1) c-myc messenger RNAs accumulated to double the levels found in NIH 3T3 cells; and (2) a shift in the use of the two normal c-myc transcription initiation sites (P1 and P2) occurred in favour of the 3' site, P2. Analysis of c-myc chromatin by DNase I treatment of 54c12 nuclei revealed that most, if not all, of the c-myc gene copies were transcriptionally competent. We present alternative ideas to explain why amplification of the c-myc gene occurs repeatedly in A-MuLV-transformed fibroblasts. Finally, we discuss our results in relation to the hypothesis linking the phenomenon of tumour progression with the amplification of oncogenes.