Interactive Fly, Drosophila

cut


EVOLUTIONARY HOMOLOGS

Characterization of mammalian Cut homologs

Clox (Cut-like homeo box) is a mammalian homeobox gene related to Drosophila cut. Both Clox and CDP have the same preferred DNA-binding specificity (Andres, 1992). Clox (Cut-like homeo box) and CDP (CCAAT displacement protein), two mammalian counterparts of the Drosophila Cut homeo protein, correspond to alternatively spliced products of the same gene. The human Cut homeodomain represses transcription from the c-myc promoter, suggesting a role for Cut in regulation of the cell cycle (Dufort, 1994). Not all CDP binding sites contain CCAAT boxes. Cut repeats function as novel DNA binding motifs. Each repeat exhibits sequence specific DNA binding, and the specificity of each repeat differs. Both the homeodomain and the Cut repeat bind to DNA (Aufiero, 1994).

Ectopic expression in the fly of the human protein CDP and the murine protein Cux as well as Cut, similarly affects embryonic sensory organ development and can rescue a wing scalloping mutant phenotype associated with loss of cut expression along the prospective wing margins. This suggests that the function of Cut is evolutionarily conserved (Ludlow, 1996).

Hepatocyte nuclear factor-6 (HNF-6) is a liver-enriched transcription factor that contains a single cut domain and a novel type of homeodomain. The developmental expression pattern of HNF-6 has been studied in the mouse. In situ hybridization experiments showed that HNF-6 mRNA is detected in the liver at embryonic day (E) 9, at the onset of liver differentiation. HNF-6 mRNA disappears transiently from the liver between E12.5 and E15. In transfection experiments, HNF-6 stimulates the expression of HNF-4 and of HNF-3 beta, two transcription factors known to be involved in liver development and differentiation. HNF-6 is detected in the pancreas from E10.5 onward, where it is restricted to the exocrine cells. HNF-6 is also detected in the developing nervous system. Both the brain and the spinal cord start to express HNF-6 at E9-9.5 in postmitotic neuroblasts. Later, HNF-6 is restricted to brain nuclei, to the retina, to the ventral horn of the spinal cord, and to dorsal root ganglia. The observations that HNF-6 contributes to the control of the expression of transcription factors and is expressed at early stages of liver, pancreas, and neuronal differentiation suggest that HNF-6 regulates several developmental programs (Landry, 1997).

DNA-binding of Cut homologs

A characteristic feature of the Cut homeo proteins is the presence of three evolutionarily conserved 73-amino-acid repeats of unknown function, the so-called Cut repeats. The Cut repeat II binds to Clox consensus targets independently of the DNA-binding activity of the homeodomain. In vitro selection of binding sites shows that the optimal targets for the Cut repeat II contain one or more CCAAT boxes and, like the homeodomain, an ATTA core. The Clox homeo domain and Cut repeat II interact in vitro in the absence of DNA. This interaction, which greatly enhances the DNA-binding activity of the binary complex, is specific to the Cut homeo proteins (Andres, 1994).

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 is increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo. Sequence analysis of Cut repeats reveals the presence of sequences that match the consensus phosphorylation site for casein kinase II (see Drosophila CKII). Therefore, an investigation was carried out to determine whether CKII can modulate the activity of mammalian Cut proteins. In vitro, a purified preparation of CKII efficiently phosphorylated Cut repeats, causing an inhibition of DNA binding. In vivo, overexpression of the CKII alpha and beta causes a decrease in DNA binding by Cut. The CKII phosphorylation sites within the murine Cut (mCut) protein have been identified by in vitro mutagenesis as residues Ser400, Ser789, and Ser972 within Cut repeats 1, 2, and 3, respectively. Cut homeodomain proteins are known to function as transcriptional repressors. Overexpression of CKII reduces transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites is not affected. Altogether these results indicate that the transcriptional activity of Cut proteins is modulated by CKII (Coqueret, 1998a).

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, a domain has been identified 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, it is concluded 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 are 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, 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 (Truscott, 2005).

Mutation of Cut homologs

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. This study analyzed genetically targeted mice (Cutl1tm2Ejn, referred to as DeltaC) 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 exhibits significantly reduced nuclear localization. Consistent with these data, DNA binding activity of HiNF-D is lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (DeltaC-/-) 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 DeltaC-/- 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 Cutl1tm2Ejn/tm2Ejn mice suggest that CDP/Cux is required for the developmental control of dermal and reproductive functions (Luong, 2002).

Cut regulation by cell cycle mediators

Cut is a homeodomain transcription factor which has the unusual property of containing several DNA-binding domains: three regions (termed Cut repeats) and the Cut homeodomain. Genetic studies in Drosophila indicate that cut plays important roles in the determination and maintenance of cell-type specificity. Mammalian Cut proteins may yet play another biological role, specifically in proliferating cells. The binding of Cut to a consensus binding site is found to vary during the cell cycle. Binding is virtually undetectable in G0 and early G1, but becomes very strong as cells reach S phase. This results both from an increase in Cut expression and dephosphorylation of the Cut homeodomain by the Cdc25A phosphatase. Cdc25A has been found to play a role in the G1-S transition. It becomes active in late G1, activated by the cdk2-cyclin E complex and blocks the inhibitory effect of p21 (See Drosophila Dacapo) on cyclin-cdk complexes. Microinjection of anti-Cdc25A antibodies in G1 cells is known to prevent progression into S phase, and Cdc25A expression is upregulated by c-Myc. Thus, Cdc25A plays an important role in the control of cell-cycle progression; however, apart from cdk2 and cdk4, its physiological targets have remained unknown. The increase in Cut activity coincides with a decrease in p21 mRNAs. In co-transfection experiments, Cut proteins repress p21 gene expression through binding to a sequence that overlaps the TATA box. Moreover, p21 expression is repressed equally well by either Cdc25A or Cut. Together, these results suggest a model by which Cdc25A activates the Cut repressor, which then downregulates transcription of p21 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 (Coqueret, 1998b).

Previous experiments with peptide fusion proteins suggested that cyclin A/Cdk1 and Cdk2 might exhibit similar yet distinct phosphorylation specificities. Using a physiological substrate, CDP/Cux, this notion has been confirmed. Proteolytic processing of CDP/Cux by cathepsin L generates the CDP/Cux p110 isoform at the beginning of S phase. CDP/Cux p110 makes stable interactions with DNA during S phase but is inhibited in G2 following the phosphorylation of serine 1237 by cyclin A/Cdk1. It is proposed that differential phosphorylation by cyclin A/Cdk1 and cyclin A/Cdk2 enables CDP/Cux p110 to exert its function as a transcriptional regulator specifically during S phase. Like cyclin A/Cdk1, cyclin A/Cdk2 interacts efficiently with recombinant CDP/Cux proteins that contain the Cut homeodomain and an adjacent cyclin-binding motif (Cy). In contrast to cyclin A/Cdk1, however, cyclin A/Cdk2 does not efficiently phosphorylate CDP/Cux p110 on serine 1237 and does not inhibit its DNA binding activity in vitro. Accordingly, co-expression with cyclin A/Cdk2 in cells does not inhibit the DNA binding and transcriptional activities of CDP/Cux p110. To confirm that the sequence surrounding serine 1237 is responsible for the differential regulation by Cdk1 and Cdk2, four amino acids flanking the phosphorylation site were replaced to mimic a known Cdk2 phosphorylation site present in the Cdc6 protein. Both cyclin A/Cdk2 and Cdk1 efficiently phosphorylates the CDP/Cux(Cdc6) mutant and inhibits its DNA binding activity. Altogether these results help explain why the DNA binding activity of CDP/Cux p110 is maximal during S phase and decreases in G2 phase (Santaguida, 2005).

Cut interactions as a transcriptional repressor

The CCAAT displacement protein/cut homolog (CDP/cut) is a divergent homeodomain protein that is highly conserved through evolution and has properties of a potent transcriptional repressor. CDP/cut contains three conserved cut-repeat domains and a conserved homeobox, each involved in directing binding specificity to unique nucleotide sequence elements. Furthermore, CDP/cut may play a role as a structural component of chromatin through its direct interaction with nucleosomal DNA and association with nuclear matrix attachment regions. CDP/cut is cell-cycle regulated through interactions with Rb, p107, specific kinases and phosphatases directing the transcriptional activity of CDP/cut on such genes encoding p21WAF1,CIP1, c-myc, thymidine kinase, and histones. CDP/cut is associated with histone deacetylase activity and is associated with a corepressor complex through interactions with histone deacetylases. The interaction of CDP/cut with CBP and p300/CREB-binding protein-associated factor (PCAF) is reported along with the modification of CDP/cut by the histone acetyltransferase PCAF. Acetylation of CDP/cut by PCAF is directed at conserved lysine residues near the homeodomain region and regulates CDP/cut function. These observations are consistent with the ability of CDP/cut to regulate genes as a transcriptional repressor, suggesting acetylation as a mechanism that regulates CDP/cut function (Li, 2000).

CCAAT displacement protein/cut homolog (CDP/cut) is a highly conserved homeodomain protein that contains three cut repeat sequences. 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. 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. This study identifies 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 (Nishio, 2004).

Mammalian Cut homologs regulation histone expression

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B and H1 (See Drosophila Histone H4) is activated at the G1/S phase transition of the cell cycle. The promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors, cyclin A, CDC2, and a member of the retinoblastoma protein family. The intrinsic DNA-binding subunit for HiNF-D is the ubiquitous protein CDP/cut. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and RB protein, whereas the CDP/cut complex with a nonhistone promoter reacts only with CDP and p107 (another member of the RB protein family) antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct RB-related proteins. CDP/cut can be shown to modulate H4 promoter activity via the HiNF-D binding site. Overexpression of CDP/cut represses H4 promoter activity via the HiNF-D(CDP/cut) site, suggesting that HiNF-D may repress or activat H4 transcription dependent on the availability of associated proteins. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclinA/CDC2/RB-related proteins (van Wijnen, 1996).

The histone H4 gene promoter provides a paradigm for defining transcriptional control operative at the G1/S phase transition point in the cell cycle. Transcription of the cell cycle-dependent histone H4 gene is upregulated at the onset of S phase; the cell cycle control element that mediates this activation has been functionally mapped to a proximal promoter domain designated Site II. Activity of Site II is regulated by an E2F-independent mechanism involving binding of the oncoprotein IRF2 and the multisubunit protein HiNF-D, which contains the following four subunits: the homeodomain protein CDP/cut, CDC2, cyclin A, and the tumor suppressor pRb. To address mechanisms that define interactions of Site II regulatory factors with this cell cycle control element, these determinants of transcriptional regulation at the G1/S phase transition have been investigated in FDC-P1 hematopoietic progenitor cells. The representation and activities of histone gene regulatory factors were examined as a function of FDC-P1 growth stimulation. Striking differences in expression of the pRb-related growth regulatory proteins (pRb/p105, pRb2/p130, and p107) were found following the onset of proliferation. pRb2/p130 is present at elevated levels in quiescent cells and declines following growth stimulation. By contrast, pRb and p107 are minimally represented in quiescent FDC-P1 cells but are upregulated at the G1/S phase transition point. A dramatic upregulation of the cellular levels of pRb2/p130-associated protein kinase activity is observed when S phase is initiated. Selective interactions of pRb and p107 with CDP/cut are observed during the FDC-P1 cell cycle and suggest functional linkage to competency for DNA binding and/or transcriptional activity. These results are particularly significant in the context of hematopoietic differentiation where stringent control of the cell cycle program is requisite for expanding the stem cell population during development and tissue renewal (van Wijnen, 1997).

The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, it has been shown that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor. Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, it has been found that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters (Gupta, 2003).

Other transcriptional target of mammalian Cut homologs

Murine hepatocyte nuclear factor-3 beta (HNF-3 beta) protein (see Drosophila Forkhead) is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3 beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelial cells, and pancreatic acinar cells. A liver-enriched transcription factor, HNF-6, is required for HNF-3 beta promoter activity and also recognizes the regulatory region of numerous hepatocyte-specific genes. In this study the yeast one-hybrid system was used to isolate the HNF-6 cDNA, which encodes a cut-homeodomain-containing transcription factor that binds with the same specificity as the liver HNF-6 protein. Cotransfection assays demonstrate that HNF-6 activates expression of a reporter gene driven by the HNF-6 binding site from either the HNF-3 beta or transthyretin (TTR) promoter regions. Interspecific backcross analysis was used to determine that the murine Hnf6 gene is located in the middle of mouse chromosome 9. In situ hybridization studies of staged specific embryos demonstrate that HNF-6 and its potential target gene, HNF-3 beta, are coexpressed in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3 beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelial, and in the pancreatic ductal epithelial and exocrine acinar cells. The expression patterns of these two transcription factors do not overlap in other endoderm-derived tissues or the neurotube. HNF-6 is also abundantly expressed in the dorsal root ganglia, the marginal layer, and the midbrain. At day 18 of gestation and in the adult pancreas, HNF-6 and HNF-3 beta transcripts colocalize in the exocrine acinar cells, but their expression patterns diverge in other pancreatic epithelium. HNF-6 expression, but not the expression of HNF-3 beta, continues in the pancreatic ductal epithelium, whereas only HNF-3 beta becomes restricted to the endocrine cells of the islets of Langerhans (Rausa, 1997).

Developmental control of bone tissue-specific genes requires positive and negative regulatory factors to accommodate physiological requirements for the expression or suppression of the encoded proteins. Osteocalcin (OC) gene transcription is restricted to the late stages of osteoblast differentiation. OC gene expression is suppressed in nonosseous cells and osteoprogenitor cells and during the early proliferative stages of bone cell differentiation. The rat OC promoter contains a homeodomain recognition motif within a highly conserved multipartite promoter element (OC box I) that contributes to tissue-specific transcription. The CCAAT displacement protein (CDP), a transcription factor related to the Cut homeodomain protein in Drosophila, may regulate bone-specific gene transcription in immature proliferating osteoblasts. Using gel shift competition assays and DNase I footprinting, CDP/cut is shown to recognize two promoter elements (TATA and OC box I) of the bone-related rat OC gene. Overexpression of CDP/cut in ROS 17/2.8 osteosarcoma cells results in repression of OC promoter activity; this repression is abrogated by mutating OC box I. Gel shift immunoassays show that CDP/cut forms a proliferation-specific protein/DNA complex in conjunction with cyclin A and p107, a member of the retinoblastoma protein family of tumor suppressors. These findings suggest that CDP/cut may represent an important component of a cell signaling mechanism that provides cross-talk between developmental and cell cycle-related transcriptional regulators to suppress bone tissue-specific genes during proliferative stages of osteoblast differentiation (van Gurp, 1999).

Nuclear matrix attachment regions (MARs) flanking the immunoglobulin heavy chain intronic enhancer (Emu) are the targets of the negative regulator, NF-muNR, found in non-B and early pre-B cells. Expression library screening with NF-muNR binding sites yields a cDNA clone encoding an alternatively spliced form of the Cux/CDP homeodomain protein. Cux/CDP fulfills criteria required for NF-muNR identity. It is expressed in non-B and early pre-B cells but not mature B cells. It binds to NF-muNR binding sites within Emu with appropriate differential affinities. Antiserum specific for Cux/CDP recognizes a polypeptide of the predicted size in affinity-purified NF-muNR preparations and binds NF-muNR complexed with DNA. Cotransfection with Cux/CDP represses the activity of Emu via the MAR sequences in both B and non-B cells. Cux/CDP antagonizes the effects of the Bright (B cell regulator of IgH transcription) transcription activator at both the DNA binding and functional levels. It is proposed that Cux/CDP regulates cell-type-restricted, differentiation stage-specific Emu enhancer activity by interfering with the function of nuclear matrix-bound transcription activators (Wang, 1999).

Human cystic fibrosis transmembrane conductance regulator gene (CFTR) transcription is tightly regulated by nucleotide sequences upstream of the initiator sequences. This analysis human CFTR transcription focuses on identifying transcription factors that bind to an inverted CCAAT consensus or 'Y-box element'. The human homeodomain CCAAT displacement protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis of stably transfected cell lines with wild-type and mutant human CFTR-directed reporter genes demonstrates that human histone acetyltransferase GCN5 and transcription factor ATF-1 can potentiate CFTR transcription through the Y-box element. Human CDP/cut acts as a repressor of CFTR transcription through the Y-box element by competing for the sites of transactivators hGCN5 and ATF-1. The ability of CDP/cut to repress activities of hGCN5 and ATF-1 activity is contingent on the amount of CDP/cut expression. Histone acetylation may have a role in the regulation of gene transcription by altering the accessibility of the CFTR Y-box for sequence-specific transcription factors. Trichostatin A, an inhibitor of histone deacetylase activity, activates transcription of CFTR through the Y-box element, and the inhibition of histone deacetylase activity leads to an alteration of local chromatin structure requiring an intact Y-box sequence in CFTR. Immunocomplexes of CDP/cut possess an associated histone deacetylase activity and the carboxyl region of CDP/cut, responsible for the transcriptional repressor function, interacts with the histone deacetylase, HDAC1. It is proposed that CFTR transcription may be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted CCAAT (Y-box) element of the CFTR promoter (Li, 1999).

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 involves an N-terminally truncated isoform, CDP/Cux p110, that is generated by proteolytic processing. CDP/Cux has been characterized as a transcriptional repressor. However, this study shows 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 is stimulated following the infection of cells with a retrovirus expressing a truncated CDP/Cux protein. Chromatin immunoprecipitation (ChIP) assays revealed that CDP/Cux is associated with the DNA pol alpha gene promoter specifically in the S phase. Using linker scanning analyses, in vitro DNA binding, and ChIP assays, a correlation has been established between binding of CDP/Cux to the DNA pol alpha promoter and the stimulation of gene expression. Although the possibility that stimulation of gene expression by CDP/Cux involves the repression of a repressor cannot be excluded, the 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 (Truscott, 2003).

The CCAAT-displacement protein (CDP) has been implicated in developmental and cell-type-specific regulation of many cellular and viral genes. 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, whether binding mutations could alter viral expression was tested. Infection of mice with MMTV proviruses containing CDP binding site mutations elevates 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 eliminates the entire C terminus and the homeodomain (DeltaC mice) show increased levels of MMTV, beta-casein, WAP, and alpha-lactalbumin RNA in virgin mammary glands compared to those from wild-type animals. These data show independent contributions of different CDP domains to negative regulation of differentiation-specific genes in the mammary gland (Zhu, 2004).

Cut and development

In vertebrates, the apical ectodermal ridge (AER) is a specialized epithelium localized at the dorsoventral boundary of the limb bud that regulates limb outgrowth. In Drosophila, the wing margin is also a specialized region located at the dorsoventral frontier of the wing imaginal disc. The wingless and Notch pathways have been implicated in positioning both the wing margin and the AER. One of the nuclear effectors of the Notch signal in the wing margin is the transcription factor cut. Two chick homologs of the Cut/Cux/CDP family that are expressed in the developing limb bud have been identified. Chick cux1 is expressed in the ectoderm outside the AER, as well as around ridge-like structures induced by beta-catenin, a downstream target of the Wnt pathway. cux1 overexpression in the chick limb results in scalloping of the AER and limb truncations, suggesting that Cux1 may have a role in limiting the position of the AER by preventing the ectodermal cells around it from differentiating into AER cells. The second molecule of the Cut family identified in this study, cux2, is expressed in the pre-limb lateral plate mesoderm, posterior limb bud and flank mesenchyme, a pattern reminiscent of the distribution of polarizing activity. The polarizing activity is determined by the ability of a certain region to induce digit duplications when grafted into the anterior margin of a host limb bud. Several manipulations of the chick limb bud show that cux2 expression is regulated by retinoic acid, Sonic hedgehog and the posterior AER. These results suggest that Cux2 may have a role in generating or mediating polarizing activity. Taking into account the probable involvement of Cut/Cux/CDP molecules in cell cycle regulation and differentiation, these results raise the hypothesis that chick Cux1 and Cux2 may act by modulating proliferation versus differentiation in the limb ectoderm and polarizing activity regions, respectively (Tavares, 2000).

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBPepsilon) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBPepsilon activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. Overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBPepsilon-/- mice and in SGD patients. Based on these observations, potential interactions between C/EBPepsilon and CDP/cut during neutrophil maturation were investigated. Inducible expression of C/EBPepsilon in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells has revealed the absence of C/EBPepsilon mRNA. It is therefore hypothesized that C/EBPepsilon positively regulates SGP gene expression, and that C/EBPepsilon is itself negatively regulated by CDP/cut during neutrophil maturation. The C/EBPepsilon promoter has been shown to be regulated by CDP/cut during myeloid differentiation (Khanna-Gupta, 2001).

It has been suggested that CDP/cut activity is restricted to proliferating cells, and that CDP/cut target genes are repressed in proliferating cells and are up-regulated as cells undergo cell cycle arrest and terminal differentiation. Target genes of CDP/cut include c-myc, c-mos, thymidine kinase (TK), cyclin-dependent kinase (cdk) inhibitor p21WAF1/CIP1, CFTR, TGFß-type II receptor, gp91phox, MHC class 1 locus, and the neutrophil SGP genes. Recently, CDP/cut has been shown to function as a repressor of transcription involving chromatin modification through recruitment of histone deacetylases (HDAC), consistent with the notion that transcriptional silencing is associated with hypoacetylated histones. Both acetylation of CDP/cut via p300/CBP and phosphorylation of CDP/cut are posttranscriptional modifications that have been postulated to regulate CDP/cut function. It is speculated that CDP/cut uses a different combination of its four binding elements to bind to the CDP/cut motifs in the promoters of C/EBPepsilon and the SGP genes, respectively, during neutrophil maturation. Differential modification, involving either phosphorylation and/or acetylation, of CDP/cut-DNA complexes in the promoters of C/EBPepsilon and SGP genes could result in the differential repression exerted by CDP/cut during neutrophil development (Khanna-Gupta, 2001 and references therein).

The mammalian Cutl1 gene codes for the CCAAT displacement protein (CDP), which has been implicated as a transcriptional repressor in diverse processes such as terminal differentiation, cell cycle progression, and the control of nuclear matrix attachment regions. To investigate the in vivo function of Cutl1, the C-terminal Cut repeat 3 and homeodomain exons were replaced with an in-frame lacZ gene by targeted mutagenesis in the mouse. The CDP-lacZ fusion protein is retained in the cytoplasm and fails to repress gene transcription, indicating that the Cutl1lacZ allele corresponds to a null mutation. Cutl1 mutant mice on inbred genetic backgrounds are born at Mendelian frequency, but die shortly after birth because of retarded differentiation of the lung epithelia, which indicates an essential role of CDP in lung maturation. A less pronounced delay in lung development allows Cutl1 mutant mice on an outbred background to survive beyond birth. These mice are growth-retarded and develop an abnormal pelage because of disrupted hair follicle morphogenesis. The inner root sheath (IRS) is reduced, and the transcription of Sonic hedgehog and IRS-specific genes is deregulated in Cutl1 mutant hair follicles, consistent with the specific expression of Cutl1 in the progenitors and cell lineages of the IRS. These data implicate CDP in cell-lineage specification during hair follicle morphogenesis, which resembles the role of the related Cut protein in specifying cell fates during Drosophila development (Ellis, 2001).

Cux-1 is a murine homeobox gene that is highly expressed in the developing kidney with expression restricted to the nephrogenic zone. Cux-1 is highly expressed in cyst epithelium of polycystic kidneys from C57BL/6J-cpk/cpk mice, but not in kidneys isolated from age-matched phenotypically normal littermates. To further elucidate the role of Cux-1 in renal development, transgenic mice were generated expressing Cux-1 under the control of the CMV immediate early gene promoter. Mice constitutively expressing Cux-1 develop multiorgan hyperplasia and organomegaly, but not an overall increase in body size. Transgenic kidneys were enlarged 50% by 6 weeks of age, with the increased growth primarily restricted to the cortex. Proliferating cells were found in proximal and distal tubule epithelium throughout the cortex, and the squamous epithelium that normally lines Bowman's capsule was replaced with proximal tubule epithelium. However, the total number of nephrons was not increased. In the developing kidneys of transgenic mice, Cux-1 is ectopically expressed in more highly differentiated tubules and glomeruli, and this is associated with reduced expression of the cyclin kinase inhibitor, p27. Transient transfection experiments have revealed that Cux-1 is an inhibitor of p27 promoter activity. These results suggest that Cux-1 regulates cell proliferation during early nephrogenesis by inhibiting expression of p27 (Ledford, 2002).

Joint formation, the onset of which is characterized by the segmentation of continuous skeletal rudiments into two or more separate elements, is a fundamental aspect of limb pattern formation, playing a critical role in determining the size, shape, and number of individual skeletal elements. Joint formation is initiated by conversion of differentiated chondrocytes at sites of presumptive joints into densely packed nonchondrogenic cells of the joint interzone. This conversion is accompanied by loss of Alcian blue-staining cartilage matrix and downregulation of cartilage-specific gene expression. Cux1 is highly expressed at all of the discrete sites of incipient joint formation in the developing limb concomitant with conversion of differentiated chondrocytes into interzone tissue. Moreover, differentiated limb chondrocytes in micromass cultures infected with a Cux1 retroviral expression vector are converted into nonchondrogenic cells that exhibit loss of Alcian blue cartilage matrix and downregulation of cartilage-specific gene expression as occurs at the onset of normal joint formation. These results suggest that Cux1 is involved in regulating the onset of joint formation by facilitating conversion of chondrocytes into nonchondrogenic cells of the interzone (Lizarraga, 2002).

Neurogenesis requires the coordination of neural progenitor proliferation and differentiation with cell-cycle regulation. However, the mechanisms coordinating these distinct cellular activities are poorly understood. This study demonstrates that a Cut-like homeodomain transcription factor family member, Cux2 (Cutl2), regulates cell-cycle progression and development of neural progenitors. Cux2 loss-of-function mouse mutants exhibit smaller spinal cords with deficits in neural progenitor development as well as in neuroblast and interneuron differentiation. These defects correlate with reduced cell-cycle progression of neural progenitors coupled with diminished NeuroD and p27Kip1 activity. Conversely, in Cux2 gain-of-function transgenic mice, the spinal cord is enlarged in association with enhanced neuroblast formation and neuronal differentiation, particularly with respect to interneurons. Furthermore, Cux2 overexpression induces high levels of NeuroD and p27Kip1. Mechanistically, it was discovered through chromatin immunoprecipitation assays that Cux2 binds both the NeuroD and p27Kip1 promoters in vivo, indicating that these interactions are direct. These results therefore show that Cux2 functions at multiple levels during spinal cord neurogenesis. Cux2 initially influences cell-cycle progression in neural progenitors but subsequently makes additional inputs through NeuroD and p27Kip1 to regulate neuroblast formation, cell-cycle exit and cell-fate determination. Thus this work defines novel roles for Cux2 as a transcription factor that integrates cell-cycle progression with neural progenitor development during spinal cord neurogenesis (Iulianella, 2008).

Cux2 functions downstream of Notch signaling to regulate dorsal interneuron formation in the spinal cord

Obtaining the diversity of interneuron subtypes in their appropriate numbers requires the orchestrated integration of progenitor proliferation with the regulation of differentiation. This study demonstrates through loss-of-function studies in mice that the Cut homeodomain transcription factor Cux2 (Cutl2) plays an important role in regulating the formation of dorsal spinal cord interneurons. Furthermore, Notch regulates Cux2 expression. Although Notch signaling can be inhibitory to the expression of proneural genes, it is also required for interneuron formation during spinal cord development. These findings suggest that Cux2 might mediate some of the effects of Notch signaling on interneuron formation. Together with the requirement for Cux2 in cell cycle progression, this work highlights the mechanistic complexity in balancing neural progenitor maintenance and differentiation during spinal cord neurogenesis (Iulianella, 2009).

Although NICD signaling positively regulates Cux2 in spinal cord progenitors, the constitutive activation of NICD overrides Cux2 promotion of neuronal differentiation through the aberrant activation of Hes1. However, Notch signaling is normally only transiently activated in dividing progenitor populations, resulting in stochastic cell fate determination. This process is subsequently stabilized by lateral inhibition among neighboring cells and results in the acquisition of asymmetric cell fate, such as the formation of a Hes1-positive progenitor cell alongside a proneural daughter. Although the Notch pathway is involved in the initial regulation of proneural gene expression, other mechanisms are required to increase or maintain the levels of proneural gene expression in selected progenitors so as to stabilize the neuronal differentiation program. Interestingly, Cux2 is expressed in a salt-and-pepper manner in the developing nervous system, as is the case for several Notch1 target genes, including Dll1 and Hes1. The data imply that Notch activity, which is normally transient, results in the induction of a Cux2-positive interneuron progenitor in the vz, which then goes on to promote neuronal maturation. Continued Cux2 activity then acts to force cell cycle withdrawal of these nascent neurons through p27Kip1 and p57Kip2 activation, resulting in interneuron maturation (Iulianella, 2009).

Cux2 has been shown to regulate both cell cycle progression and the balance between interneuron and motoneuron differentiation in the ventral spinal cord. Notch signaling also regulates the formation of interneurons in the developing spinal cord, and might do so at least in part via the regulation of Cux2. These findings suggest that Cux2 acts downstream of the Notch pathway to stabilize the neurogenic program and promote cell cycle exit in dorsal interneuron progenitors (Iulianella, 2009).

Cux2 acts as a critical regulator for neurogenesis in the olfactory epithelium of vertebrates

Signaling pathways and transcription factors are crucial regulators of vertebrate neurogenesis, exerting their function in a spatial and temporal manner. Despite recent advances in understanding of the molecular regulation of embryonic neurogenesis, little is known regarding how different signaling pathways interact to tightly regulate this process during the development of neuroepithelia. This study has investigated the events lying upstream and downstream of a key neurogenic factor, the Cut-like homeodomain transcription factor-2, during embryonic neurogenesis in chick and mouse. By using the olfactory epithelium as a model for neurogenesis this study analyzed mouse embryos deficient in Cux2, as well as chick embryos exposed to Cux2 silencing (si) RNA or a Cux2 over-expression construct. Evidence is provided that enhanced BMP activity increases Cux2 expression and suppresses olfactory neurogenesis in the chick olfactory epithelium. In addition, the results show that up-regulation of Cux2, either BMP-induced or ectopically over-expressed, reduces Delta1 expression and suppresses proliferation. Interestingly, the loss of Cux2 activity, using mutant mice or siRNA in chick, also diminishes neurogenesis, Notch activity and cell proliferation in the olfactory epithelium. The results suggest that controlled low levels of Cux2 activity are necessary for proper Notch signaling, maintenance of the proliferative pool and ongoing neurogenesis in the olfactory epithelium. Thus, this study demonstrates a novel conserved mechanism in vertebrates in which levels of Cux2 activity play an important role for ongoing neurogenesis in the olfactory epithelium (Wittmann, 2014).

Cut proteins and cancer

CUTL1, also known as CDP, Cut, or Cux-1, is a homeodomain transcriptional regulator known to be involved in development and cell cycle progression. CUTL1 activity is associated with increased migration and invasiveness in numerous tumor cell lines, both in vitro and in vivo. Furthermore, CUTL1 has been identified as a transcriptional target of transforming growth factor ß and a mediator of its promigratory effects. CUTL1 activates a transcriptional program regulating genes involved in cell motility, invasion, and extracellular matrix composition. CUTL1 expression is significantly increased in high-grade carcinomas and is inversely correlated with survival in breast cancer. This suggests that CUTL1 plays a central role in coordinating a gene expression program associated with cell motility and tumor progression (Michl, 2005).

A major challenge in cancer genetics is to determine which low-frequency somatic mutations are drivers of tumorigenesis. This study interrogated the genomes of 7,651 diverse human cancers, and inactivating mutations were found in the homeodomain transcription factor gene CUX1 (cut-like homeobox 1) in ~1-5% of various tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, CUX1 was validated as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. It was demonstrated that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth and susceptibility to PI3K-AKT inhibition. Thus, these complementary approaches identify CUX1CUX1 as a pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors (Wong 2014).


cut: Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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