Cut Repeats Research Articles

Clinical and Biological Implications of CUX1 Mutations in Myeloid Neoplasms

Recurrent somatic mutations of CUX1 are described in myeloid neoplasms. CUX1 is located at chromosome 7q22.1; -7/del(7q) involving CUX1 locus are common abnormalities in myelodysplastic syndromes (MDS). Mutations and loss of heterozygosity involving CUX1 have been also described in breast, lung and uterine cancers. Preliminary functional studies, lack of a mutational hotspot and coincidental deletions suggest loss of function/hypomorphic consequences of these molecular defects. CUX1 (p200), contains 4 evolutionarily conserved DNA-binding domains, including 3 CUT repeats and a CUT homeodomain. Functionally, CUX1 regulates many genes involved in DNA replication and chromosome segregation. Cell-based assays have established a role for CUX1 in the control of cell-cycle progression, cell motility, and invasion .The objective of this study is to assess the molecular context and clinical significance of CUX1 mutations and deletions in myeloid neoplasms.We analyzed a subset of 1478 patients [24% lower-risk MDS, 17% higher-risk MDS, 22% primary (p)AML, 14% secondary AML, 14% MDS/myeloproliferative neoplasms (MPN) and 9% MPN] for the presence of CUX1 mutations and deletions. No CUX1 mutations were found in core binding factor AML. We correlated the presence of these lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and associated somatic mutations. Copy number variation and their boundaries were analyzed by Single Nucleotide Polymorphism (SNP) arrays and mutations by multiamplicon deep sequencing utilizing a panel targeting 60 most commonly mutated genes in myeloid neoplasms.In total cohort 4 % of patients had CUX1 mutations and 6% had locus deletions (affecting ch 7q commonly deleted region: 7q22.1) including 90% of del (7q) cases. Expression of CUX1 is significantly lower in AML with -7/del(7q) compared to AML with normal cytogenetics (p<.00001) and also in MDS with -7/del(7q) compared tohealthy controls (p=.004). Additionally, decreased expression of CUX1 was found in 15% of MDS and 8% of AML patients without -7/del(7q) or related mutations. Cases with lower expression had worse OS compared to patients with higher expression (p=.002). In terms of configuration, most mutations were heterozygous, 5% of mutations were hemizygous and 4% were homozygous (due to UPD). Among 75 somatic CUX1mutations; 72% were missense, 20% where frame shift and 8% where non sense.CUX1 mutations were associated with either lower-risk MDS (p=.0001) and pAML (p=.04) while deletions involving the CUX1 locus were significantly related to higher-risk MDS (p=.05). Heterozygous CUX1 mutations were more commonly associated with normal cytogenetics (p=.01). Patients with -7/del(7q) frequently represented del(5q) (p=.04) and thrombocytopenia (p=.001). The OS of patients with CUX1 mutations was shorter (p=.04) as was that of patients with CUX1/deletions (p=.02) when compared to wild type.We subsequently studied the molecular background of CUX1 alterations. CUX1 mutations (vs. wild type) were associated with TET2 (31% vs. 14%, p=.006), ASXL1 (29% vs. 9%, p=.0005), BCOR (28% vs. 8%, p=.0004), and cohesion mutations (26%, vs. 5%, p=.0005), while NPM1 mutations showed the reverse relationship (1% vs. 7%, p=.03). RAS and CUX1 mutations were mutually exclusive (0% vs. 6%, p=.03).When we analyzed clonal hierarchy in the context of CUX1 mutations; dominant CUX1 mutations (24%; mean VAF=49%); were accomplished by ASXL1 (21%) and SRSF2 (14%) mutations which were the most common secondary events in this context. Phenotypically, dominant CUX1 mutations were associated with MDS/MPN (42%) and MDS (33%). 14% of CUX1 mutant cases did not harbor any other alterations and were not associated with a discernable phenotype. Secondary CUX1 lesions (62%; mean VAF=22%) were found in the context of dominant TET2 mutations (16%). The pathomorphologic context of secondary CUX1 mutation did not differ from that of primary lesions. AML seemed to be underrepresented (p=.006) and MPN overrepresented (p=.019) among dominant CUX1 mutant cases.In conclusion, CUX1 lesions including locus deletions with haploinsuffciency, mutations and a fraction of cases with decreased CUX1 expression can be encountered in MDS and related neoplasms, chiefly AML. CUX1 dysfunction is associated with poor survival likely due to its distinct molecular background. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

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.

The function of CUX1 in oxidative DNA damage repair is needed to prevent premature senescence of mouse embryo fibroblasts.

Despite having long telomeres, mouse embryo fibroblasts (MEFs) senesce more rapidly than human diploid fibroblasts because of the accumulation of oxidative DNA damage. The CUX1 homeodomain protein was recently found to prevent senescence in RAS-driven cancer cells that produce elevated levels of reactive-oxygen species. Here we show that Cux1-/- MEFs are unable to proliferate in atmospheric (20%) oxygen although they can proliferate normally in physiological (3%) oxygen levels. CUX1 contains three domains called Cut repeats. Structure/function analysis established that a single Cut repeat domain can stimulate the DNA binding, Schiff-base formation, glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase 1, OGG1. Strikingly and in contrast to previous reports, OGG1 exhibits efficient AP-lyase activity in the presence of a Cut repeat. Repair of oxidative DNA damage and proliferation in 20% oxygen were both rescued in Cux1-/- MEFs by ectopic expression of CUX1 or of a recombinant Cut repeat protein that stimulates OGG1 but is devoid of transcription activation potential. These findings reinforce the causal link between oxidative DNA damage and cellular senescence and suggest that the role of CUX1 as an accessory factor in DNA repair will be critical in physiological situations that generate higher levels of reactive oxygen species.

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Novel CUX1 missense mutation in association with 7q− at leukemic transformation of MPN

Novel <i>CUX1</i> missense mutation in association with 7q− at leukemic transformation of MPN

Courts will screen offenders to help cut repeat crimes

Courts will screen offenders to help cut repeat crimes

CUX1 Transcription Factor Is a Downstream Effector of the Proteinase-activated Receptor 2 (PAR2)

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.

A novel proteolytically processed CDP/Cux isoform of 90 kDa is generated by cathepsin L

The Cut-like genes code for multiple isoforms of the CDP/Cux transcription factor. The full-length protein contains four DNA-binding domains: Cut repeats 1, 2 and 3 (CR1, CR2 and CR3) and the Cut homeodomain (HD). The p75 isoform is expressed from an mRNA that is initiated within intron 20 and contains only CR3 and HD. The p110 isoform is generated by proteolytic processing by cathepsin L and contains CR2, CR3 and HD. In the present study, we show that an additional isoform of 90 kDa is expressed in many cell lines of epithelial origin. Mapping experiments with deletion mutants indicated that the N-terminus of p90 is located upstream of CR2, between amino acids 918 and 938. Indeed, p90 and p110 displayed similar DNA-binding and transcriptional activities. The p90 isoform, like p110, was found to be generated by proteolytic processing. The steady-state level of both p90 and p110 correlated with the level of cathepsin L activity. Importantly, co-expression with a cathepsin L mutant that is initiated at downstream AUG sites also stimulated the generation of p90 and p110. These results strongly suggest that p90, like p110, is generated by cathepsin L isoforms that are devoid of a signal peptide.

CUTL1 Is Phosphorylated by Protein Kinase A, Modulating Its Effects on Cell Proliferation and Motility

CUTL1, also known as CDP (CCAAT Displacement Protein), Cut, or Cux-1, is a homeodomain transcription factor known to play an essential role in development and cell cycle progression. Previously, we identified CUTL1 as modulator of cell motility and invasiveness. Here we report that protein kinase A (PKA), known to inhibit tumor progression in various tumor types, directly phosphorylates CUTL1 at serine 1215 in NIH3T3 fibroblasts. The PKA-induced phosphorylation results in decreased DNA binding affinity of CUTL1 and diminished CUTL1-mediated cell cycle progression and cell motility. Furthermore, the expression of several CUTL1 target genes involved in proliferation and migration, such as DNA polymerase A and DKK2, was modulated by PKA-induced phosphorylation. These data identify CUTL1 as a novel target of PKA through which this protein kinase can modulate tumor cell motility and tumor progression.

Knock-Down of CCAAT Displacement Protein (CDP) Restores Specific Granule Protein (SGP) Gene Expression in NB4 Acute Promyelocytic Leukemia Cells.

CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes involved in neutrophil maturation, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε ). It also represses expression of neutrophil secondary granule proteins (SGPs), including LF, which are downstream targets of C/EBPε . We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. Additionally, we have demonstrated that overexpression of CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Conversely, knocking down CDP expression by RNA interference in 32D cells upregulates levels of LF mRNA in uninduced cells. NB4 cells are an acute promyelocytic cell line that contains the t(15;17) PML-RARα translocation and can be induced toward neutrophil maturation with all-trans-retinoic Acid (ATRA). We have previously demonstrated that NB4 cells uniformly fail to express LF and all the SGP genes upon ATRA induction. In further studies, we showed that this failure of expression occurs despite appropriate binding of C/EBP factors known to upregulate SGP expression. We further demonstrated persistent binding of CDP to the LF promoter in NB4 cells upon ATRA induction, suggesting that failure of LF expression was due to persistent binding of CDP rather than a failure of C/EBP transactivation. In order to test this hypothesis, we knocked down CDP expression in NB4 cells using short hairpin RNA (shRNA) constructs and examined the effect of reduced CDP levels on SGP expression. ShRNA CDP constructs were introduced into NB4 cells by nucleofection using an Amaxa® nucleofector. Half the transfected cells were induced with ATRA, while the other half remained uninduced. After 24-hours of induction, total RNA was extracted from the cells and RTqPCR performed to assess the levels of SGP mRNA. We demonstrate that decreased CDP expression in ATRA- induced NB4 cells is sufficient to restore LF, collagenase, and gelatinase expression in the leukemic cell line. SGP expression remains absent in uninduced CDP-shRNA NB4 cells, as well as in both uninduced and induced cells transfected with an empty vector. Our observations reaffirm the critical role played by CDP in regulating the expression of LF and other SGP proteins during neutrophil development. How the PML-RARα gene product functions to alter CDP activity in NB4 cells at the molecular level, thereby restricting SGP expression, is currently being addressed.

Biochemical characterization of the mammalian Cux2 protein

The CCAAT displacement protein (CDP) and cux (Cut homeobox) genes were originally identified as the human and mouse orthologs of Drosophila melanogaster cut. More recently, vertebrates were found to possess a second cut orthologs that was generated by gene duplication: Cux2. We report the initial biochemical characterization of the Cux2 protein in tissue culture and in vitro. We generated four polyclonal antibodies that were able to recognize the human and mouse Cux2 protein but displayed little or no cross-reactivity towards CDP1 and Cux1. The expression of the Cux2 protein was convincingly detected in only one among 19 neuronal cell lines: the SH-SY5Y human neuroblastoma cell line. CDP/Cux proteins contain four DNA binding domains, three Cut repeat (CR1, CR2 and CR3) and one Cut homeodomain (HD). Purified fusion proteins containing either CR1CR2, CR2CR3HD or CR3HD exhibited similar DNA binding specificities as the corresponding domains of Cux1, but their DNA binding kinetics were much more rapid. Similarly, the full-length Cux2 protein made rapid but transient interactions with DNA. We did not observe an N-terminally processed Cux2 isoform equivalent to the Cux1 p110 isoform. Whereas Cux1 can function as a repressor or activator in a promoter-specific manner, Cux2 functioned exclusively as a transcriptional repressor in NIH3T3 cells. Overall, our results suggest that the Cux1 and Cux2 proteins carry distinct biochemical functions. Cux2 is able, like Cux1, to perform the CCAAT-displacement activity. However, Cux2 is unlikely to execute transcriptional regulatory functions that require stable interaction with DNA.

The Role of CCAAT Displacement Protein in Neutrophil-Specific Gene Expression.

CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε)and its downstream targets the neutrophil secondary granule proteins (SGPs), including lactoferrin (LF). We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Moreover, LF is not expressed in NB4 cells, a human acute promyelocytic cell line which contains the t(15;17) PML-RARα translocation. CDP has been found to bind persistently to the LF promoter in NB4 cells after morphological differentiation following ATRA induction. Several attempts at generating a CDP knockout mouse have been performed, but all have produced incomplete knockouts. We have generated short hairpin RNA (shRNA) constructs to knock down mouse CDP in myeloid cells by RNA interference. Single cell clones stably expressing the mouse shRNA have been generated in 32Dwt18 cells, and have been shown to decrease CDP expression. Knockdown of CDP does not change the phenotype of the cells, which remain IL3 dependent and undergo phenotypic maturation upon G-CSF induction. However, increased LF gene expression can be seen in uninduced cells expressing CDP shRNA, and the level of LF expression in single cell clones expressing the shRNA for CDP appears to correlate with the level of CDP repression. Control clones do not express LF until induced with G-CSF for several days. The knockdown of CDP does not appear to affect the expression of C/EBPε, suggesting that LF expression reflects direct modulation of CDP binding to its promoter and is not an indirect effect of increased C/EBPε expression. This suggests that CDP can function as the sole negative regulatory element for LF gene expression, and that relief of CDP repression can increase LF expression independent of increased binding of positive regulatory factors.

The N-terminal Region of the CCAAT Displacement Protein (CDP)/Cux Transcription Factor Functions as an Autoinhibitory Domain that Modulates DNA Binding

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.

CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription.

CCAAT displacement protein/cut homolog (CDP/cut) is a highly conserved homeodomain protein that contains three cut repeat sequences. CDP/cut is a transcriptional factor for many diverse cellular and viral genes that are involved in most cellular processes, including differentiation, development, and proliferation. Here, we report that CDP/cut interacts with a histone lysine methyltransferase (HKMT), G9a, in vivo and in vitro. The deletion of the cut repeats within CDP/cut abrogates the interaction with G9a. The transcriptional repressor function of CDP/cut is mediated through HKMT activity of G9a associated with CDP/cut. We show that the recruitment of G9a to the human p21(waf1/cdi1) promoter is contingent on the interaction with CDP/cut, and CDP/cut is directly associated with an increase in the methylation in vivo of Lys-9 in histone H3 within the CDP/cut-regulatory region of the p21(waf1/cdi1) promoter. The endogenous level of p21(waf1/cdi1) expression is repressed through CDP/cut and mediated by HKMT activity of G9a. Furthermore, we report the identification of G9a as a component of CDP/cut complex. G9a colocalizes with CDP/cut in the nucleus. These results indicate that G9a functions as a transcriptional corepressor in association with a CDP/cut complex. These studies now reveal the interaction of G9a with a sequence-specific transcription factor that regulates gene repression through CDP/cut.

The homeodomain protein CDP regulates mammary-specific gene transcription and tumorigenesis.

The CCAAT-displacement protein (CDP) has been implicated in developmental and cell-type-specific regulation of many cellular and viral genes. We previously have shown that CDP represses mouse mammary tumor virus (MMTV) transcription in tissue culture cells. Since CDP-binding activity for the MMTV long terminal repeat declines during mammary development, we tested whether binding mutations could alter viral expression. Infection of mice with MMTV proviruses containing CDP binding site mutations elevated viral RNA levels in virgin mammary glands and shortened mammary tumor latency. To determine if CDP has direct effects on MMTV transcription rather than viral spread, virgin mammary glands of homozygous CDP-mutant mice lacking one of three Cut repeat DNA-binding domains (DeltaCR1) were examined by reverse transcription-PCR. RNA levels of endogenous MMTV as well as alpha-lactalbumin and whey acidic protein (WAP) were elevated. Heterozygous mice with a different CDP mutation that eliminated the entire C terminus and the homeodomain (DeltaC mice) showed increased levels of MMTV, beta-casein, WAP, and alpha-lactalbumin RNA in virgin mammary glands compared to those from wild-type animals. No differences in amounts of WDNM1, epsilon-casein, or glyceraldehyde-3-phosphate dehydrogenase RNA were observed between the undifferentiated mammary tissues from wild-type and mutant mice, indicating the specificity of this effect. These data show independent contributions of different CDP domains to negative regulation of differentiation-specific genes in the mammary gland.

CDP/Cux stimulates transcription from the DNA polymerase alpha gene promoter.

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.

The Paired-like Homeodomain Protein, Arix, Mediates Protein Kinase A-stimulated Dopamine β-Hydroxylase Gene Transcription through Its Phosphorylation Status

The homeodomain transcription factor Arix/Phox2a plays a critical role in the specification of noradrenergic neurons by inducing the expression of dopamine beta-hydroxylase (DBH), the terminal enzyme for noradrenaline biosynthesis. In reporter assays, Arix together with activation of cAMP-dependent protein kinase (PKA) potentiates DBH gene transcription. We have evaluated whether post-translational modification of Arix regulates PKA-mediated DBH gene transcription. We found that Arix is constitutively phosphorylated in vivo at the basal level and that the phosphorylation level is substantially decreased upon stimulation of the PKA pathway. The change in the Arix phosphorylation state coincides with DNA binding activity of Arix. Treatment of cells with forskolin results in a robust enhancement of the DNA binding of Arix, which is reversed by treatment with serine/threonine and tyrosine phosphatase inhibitors. Consistent with the DNA binding activity of Arix, treatment of cultured cells with phosphatase inhibitors diminishes transcriptional activation with Arix plus forskolin. Amino acid analysis demonstrates the presence of phosphoserine within Arix. The results collectively suggest that dephosphorylation of Arix is a necessary event to fully activate PKA-mediated DBH transcription. Thus, the present study demonstrates that Arix can integrate extrinsic signals through post-translational modification, regulating DBH gene transcription in response to activation of the PKA pathway.

Genetic ablation of the CDP/Cux protein C terminus results in hair cycle defects and reduced male fertility.

Murine CDP/Cux, a homologue of the Drosophila Cut homeoprotein, modulates the promoter activity of cell cycle-related and cell-type-specific genes. CDP/Cux interacts with histone gene promoters as the DNA binding subunit of a large nuclear complex (HiNF-D). CDP/Cux is a ubiquitous protein containing four conserved DNA binding domains: three Cut repeats and a homeodomain. In this study, we analyzed genetically targeted mice (Cutl1(tm2Ejn), referred to as Delta C) that express a mutant CDP/Cux protein with a deletion of the C terminus, including the homeodomain. In comparison to the wild-type protein, indirect immunofluorescence showed that the mutant protein exhibited significantly reduced nuclear localization. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Delta C(-/-)) mice, indicating the functional loss of CDP/Cux protein in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wild-type and Delta C(-/-) MEFs in culture. However, specific histone genes (H4.1 and H1) containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice have stunted growth (20 to 50% weight reduction), a high postnatal death rate of 60 to 70%, sparse abnormal coat hair, and severely reduced fertility. The deregulated hair cycle and severely diminished fertility in Cutl1(tm2Ejn/tm2Ejn) mice suggest that CDP/Cux is required for the developmental control of dermal and reproductive functions.

CDP and AP-2 mediated repression mechanism of the replication-dependent hamster histone H3.2 promoter.

The replication-dependent hamster histone H3.2 promoter contains two tandem CCAAT repeats located upstream of the TATA element. It has been shown that the NF-Y/CBF complex binds to a single CCAAT motif with high affinity, whereas the CCAAT displacement protein (CDP) binds to at least two CCAAT motifs in close proximity. Here, we report that the two CCAAT motifs within the H3.2 promoter confer transcriptional repression of the promoter during the cell cycle. While we cannot detect direct association of CDP with Rb in vitro, we discover that CDP can bind AP-2, a ubiquitous factor that interacts with Rb. The interaction domains between CDP and AP-2 are mapped to the highly conserved cut repeats of CDP as well as the basic and dimerization region of AP-2. Further, in transfection assays, CDP and AP-2 act synergistically to suppress the H3.2 promoter. Together, these data support a repression mechanism mediated by CDP and AP-2 that regulates H3.2 gene expression during the mammalian cell cycle.

S phase-specific proteolytic cleavage is required to activate stable DNA binding by the CDP/Cut homeodomain protein.

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.