Cux homeodomain transcription factor in regulating differentiation, cell growth and development

Gene 270 (2001) 1±15

www.elsevier.com/locate/gene

Review

Role of the multifunctional CDP/Cut/Cux homeodomain transcription factor in regulating differentiation, cell growth and development Alain Nepveu a,b,* a

Molecular Oncology Group, McGill University Health Center, 687 Pine Ave West, Montreal, Quebec, Canada, H3A 1A1 b Departments of Biochemistry, Medicine and Oncoloogy, McGill University, Montreal, Quebec, Canada Received 29 December 2000; received in revised form 26 March 2001; accepted 12 April 2001 Received by A.J. van Wijnen

Abstract CDP/Cux/Cut proteins are an evolutionarily conserved family of proteins containing several DNA binding domains: one Cut homeodomain and one, two or three Cut repeats. In Drosophila melanogaster, genetic studies indicated that Cut functions as a determinant of celltype speci®cation in several tissues, notably in the peripheral nervous system, the wing margin and the Malpighian tubule. Moreover, Cut was found to be a target and an effector of the Notch signaling pathway. In vertebrates, the same functions appear to be ful®lled by two cut-related genes with distinct patterns of expression. Cloning of the cDNA for the CCAAT-displacement protein (CDP) revealed that it was the human homologue of Drosophila Cut. CDP was later found be the DNA binding protein of the previously characterized histone nuclear factor D (HiNF-D). CDP and its mouse counterpart, Cux, were also reported to interact with regulatory elements from a large number of genes, including matrix attachment regions (MARs). CDP/Cut proteins were found generally to function as transcriptional repressors, although a participation in transcriptional activation is suggested by some data. Repression by CDP/Cut involves competition for binding site occupancy and active repression via the recruitment of a histone deacetylase activity. Various combinations of Cut repeats and the Cut homeodomains can generate distinct DNA binding activities. These activities are elevated in proliferating cells and decrease during terminal differentiation. One activity, involving the Cut homeodomain, is upregulated in S phase. CDP/Cut function is regulated by several post-translational modi®cation events including phosphorylation, dephosphorylation, and acetylation. The CUTL1 gene in human was mapped to 7q22, a chromosomal region that is frequently rearranged in various cancers. q 2001 Published by Elsevier Science B.V. All rights reserved. Keywords: Cut; CCAAT-displacement protein (CDP); Cux; Cux2; Clox; HiNF-D

1. CDP/Cut is conserved among metazoans The CDP/Cut family constitutes a unique group of homeoproteins, conserved among higher eukaryotes, and containing a Cut homeodomain as well as one or more Abbreviations: AER, apical ectodermal ridge; Antp, antennapedia; CBF, CCAAT binding factor; CR, Cut repeat; ct, cut wing; Cux, cut homeobox; CDP, CCAAT-displacement protein; CKII, casein kinase II; Clox, Cut-like homeobox; CNS, central nervous system; CUTL1, Cut-like 1; D±V, dorsalventral; E(spl)-C, enhancer of split complex; es, external sensory; HD, homeodomain; HiNF-D, histone nuclear factor D; Hnf-6, hepatocyte nuclear factor 6; HPV, human papillomavirus; kf, kinked femur; LT, large T; MARs, matrix attachment regions; MMTV, mouse mammary tumor virus; MMTV-LTR, mouse mammary tumor virus long terminal repeat; N, Notch; NCAM, neural cell adhesion molecule; pb, proboscipedia; PKC, protein kinase C; PNS, peripheral nervous system; poxn, poxneural; PyV, Polyomavirus; sno, strawberry notch; spa, sparkling; Su(H), suppresser of hairless; su(Hw), suppressor of hairy wing; Wg, Wingless * Tel.: 11-514-842-1231, ext. 5842; fax: 11-514-843-1478. E-mail address: [email protected]

`Cut repeat' DNA binding domain(s). The cDNAs for homologues of the Drosophila melanogaster Cut homeodomain protein have been isolated from several mammalian species including human (Neufeld et al., 1992), dog (Andres et al., 1992), mouse (Valarche et al., 1993) and rat (Yoon and Chikaraishi, 1994) and were, respectively, termed CDP (CCAAT displacement protein), Clox (Cut-like homeobox), Cux-1 (Cut homeobox), and CDP-2. More recently a second Cux gene, called Cux-2, was identi®ed in mouse and chicken (Quaggin et al., 1996; Tavares et al., 2000). CDP, Cux-1 and Cux-2 contain three Cut repeats and a Cut homeodomain and are referred to as the classical CDP/Cux/Cut proteins (the exact number of Cut repeats in the chicken Cux-1 and 2 genes is not yet known as these genes have not yet been completely characterized). In addition, other proteins contain one or two Cut repeats and a homeodomain: hepatocyte nuclear factor 6 (Hnf-6) (Lemaigre et al., 1996), OC-2 (Jacquemin et al., 1999), and SATB1 (Dickinson et al., 1997). Since a homologous Cut cDNA

0378-1119/01/$ - see front matter q 2001 Published by Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00485-1

2

A. Nepveu / Gene 270 (2001) 1±15

sequence has been reported in Caenorhabditis elegans (NCBI, accession number U28993), but no homologue has been found in yeast, Cut proteins appear to be conserved among metazoans. This review will focus on the classical CDP/Cut proteins. For simplicity, in this review the terms CDP/Cut and Cut will be used, respectively, to designate mammalian Cut proteins and Cut proteins in general. 2. Cut expression in Drosophila melanogaster In Drosophila embryos, Cut expression was observed in several tissues, and mutations affecting Cut expression or function caused developmental defects in these tissues (Table 1) (Bodmer et al., 1987; Blochlinger et al., 1988; Blochlinger et al., 1990). In the adult, cut expression was observed in the same, as well as in other, tissues (Table 1) (Blochlinger et al., 1993). Because of embryonic lethality caused by lethal cut mutations, it was not possible to determine whether cut is also required for the development or maintenance of these tissues in adults. However, the two classes of viable cut mutants, kinked femur (kf ) and cut wing (ct), exhibit malformations in the tissues where cut fails to be expressed. It is thus likely that cut is required for the development, and possibly also the maintenance, of the other tissues in which it is expressed. Tissue-speci®c expression is directed by a series of enhancers located at various distances upstream of the gene (Fig. 1) (Jack et al., 1991; Jack and DeLotto, 1995). In all these tissues, only a subset of cells corresponding to a speci®c cell-type expressed cut (Blochlinger et al., 1990; Blochlinger et al., 1993). Moreover, in all cases cut expression was detected in the precursor cells as well as in the differentiated progeny (Blochlinger et al., 1993). This pattern of expression is compatible with a role of cut in determining cell-type speci®city.

3. Genetic studies in Drosophila melanogaster A large number of viable as well as lethal cut mutations, ct, have been described in Drosophila and literature on this subject can be found as early as 1925 (reviewed in (Jack, 1985)); (Hertweck, 1931; Blanc, 1942; Braun, 1942; Bodmer et al., 1987; Jack et al., 1991; Liu et al., 1991; Jack and DeLotto, 1992; Liu and Jack, 1992). Mutations within the cut locus were divided into ®ve groups based on the tissues affected, viability, location within the locus and complementation with other cut mutations (Fig. 1). Viable cut mutations, kinked femur (kf) and cut wing (ct), cause limb malformations in the leg and wing, respectively, and are characterized by the loss of Cut expression speci®cally, and exclusively, in these tissues (Johnson and Judd, 1979; Jack, 1985; Jack et al., 1991). Lethal cut mutations were divided into three groups based on their positions and their capacity to complement other mutations (Liu et al., 1991). While lethal II mutations most likely represent point mutations or short deletions within coding sequences, kf and ct, lethal I and III mutations are caused by the insertion of a retrotransposon, most often gypsy, that prevents expression in a variable number of tissues. Consistent with this, lethal I and III can complement kinked femur, whereas lethal II cannot. In all cases, embryonic lethality occurs fairly late in development such that it is possible to investigate the tissues affected. Mutations result in defects in several tissues including the wings, legs, external sense organs, Malpighian tubules, tracheal system and some structures in the central nervous systems (Bodmer et al., 1987; Jack et al., 1991; Liu et al., 1991; Jack and DeLotto, 1992; Liu and Jack, 1992). The wing scalloping mutant phenotype could be rescued by ectopic expression of Cut, or its mammalian homologues, demonstrating the requirement for Cut expression in wing development (Ludlow et al., 1996).

Table 1 Cut expression in Drosophila melanogaster Embryos

Adults

Peripheral nervous system (PNS) es organs precursor cells and their progeny

Peripheral nervous system (PNS) es organ precursor cells and their progeny

Central nervous system

Central nervous system Cells of all cortical areas, cortices of the medulla and lobula complex, putative glial cells within the neuropil, thoracic ganglion. Malpighian tubules Tracheal histoblasts Cells surrounding the spiracles Cells of the wing margin Ovarian follicle cells Adepithelial cells Wing and leg discs Muscles thorax, head, abdomen Cone cells of the eye

Malpighian tubules Tracheal histoblasts Cells surrounding the spiracles Adepithelial cells Wing and leg discs, abdomen

A. Nepveu / Gene 270 (2001) 1±15

3

Fig. 1. The cut Locus in Drosophila melanogaster. A map of the cut locus is shown. White boxes represent enhancers, and the black box, the gene itself. Above the map are shown the positions of mutations, their names and the tissues affected in each case. Adapted from (Jack and DeLotto, 1995).

The mechanism by which transposable elements affect cut expression is itself very interesting. The gypsy retrotransposon was found to contain an insulator element that blocks the communication between upstream enhancers and a downstream promoter (Dorsett, 1993) (for reviews on insulators, see Kellum and Elgin, 1998; Bell and Felsenfeld, 1999). The phenotypes associated with gypsy insertion at various positions led to the suggestion that multiple tissue-speci®c enhancers were present over a large region upstream of the gene. The mapping of retrotransposon insertions eventually led to the identi®cation a series of tissue-speci®c enhancers (see Fig. 1) (Jack and DeLotto, 1995). Thus, gypsy insertions nearer the coding sequences caused defects in more tissues because they interfered with a larger number of upstream enhancers. The phenotypes associated with retrotransposon insertions were found early on to be suppressed by mutations in the suppressor of Hairy wing, {su(Hw)} gene (Modolell et al., 1983). This suppressive effect was later explained by the ®ndings that the transcription factor Su(Hw) is required for the gypsy insulator to mediate its effects (Dorsett, 1993; Kim et al., 1996).

4. Cut plays a role in cell type speci®cation in Drosophila Genetic studies in Drosophila melanogaster indicated that cut plays an important role in determining cell-type speci®city in several tissues. Defects caused by Cut mutations appear to result from the fact that some cells have enrolled in the wrong developmental program (Bodmer et al., 1987; Blochlinger et al., 1991; Liu et al., 1991; Liu and Jack, 1992). The role of cut as a determinant of cell type speci®city was most thoroughly demonstrated in the peripheral nervous system (Bodmer et al., 1987; Blochlinger et al., 1991; Liu et al., 1991; Ludlow et al., 1996). Embryonic lethal cut mutations caused the transformation of external sensory (es) organs into internal (chordotonal) sensory organs (Bodmer et al., 1987). In contrast, when Cut expression was arti®cially elevated in embryos, all precursor cells gave rise to external sensory organs (Blochlinger et al., 1991; Ludlow et al., 1996). Another organ that fails to develop in lethal I and II cut mutants is the Malpighian tubules, which normally form as four outgrowths of the hindgut primordium at the junction between the anterior hindgut and the posterior midgut. Interestingly, in cut

4

A. Nepveu / Gene 270 (2001) 1±15

mutants a thickening of the gut wall was observed where the Malpighian tubule should normally form, suggesting that cells mistakenly differentiated into gut wall cells (Liu et al., 1991; Liu and Jack, 1992). In other tissues, cut was found to produce non-autonomous effects and to affect cells adjacent to those expressing cut. In cut wing mutants, loss of cut expression in the presumptive wing margin resulted in the loss of some adjacent epithelial cells (probably by apoptosis), thereby producing the truncated wing phenotype (Blanc, 1942; Fristrom, 1969; Jack et al., 1991; Blochlinger et al., 1993). This effect may be caused by the failure to maintain wingless expression in the wing margin (Micchelli et al., 1997). In ovarioles, the site of oogenesis in Drosophila, genetic manipulations to reduce cut expression caused a decrease in the number of follicle cells and defective packaging of germline-derived cysts into egg chambers (Jackson and Blochlinger, 1997). As cut is expressed in somatic follicle cells but not in germline-derived cells, it was proposed that defects affecting the latter cells were the consequence of altered interactions between follicle cells and germline cells. In these two tissues, non-autonomous effects suggest that cut regulate the expression of molecules involved in intercellular communications: either secreted cytokines, cell surface proteins or alternatively, proteins of the cytoskeleton. Altogether these ®ndings strongly implicate cut as an important determinant of cell-type speci®city in several tissues. How cut in¯uences cell fate is not clear, but sustained cut expression in several tissues of the adult ¯y, which is composed essentially of post-mitotic cells, raised the possibility that cut is required not only for the acquisition but also the maintenance of the differentiated phenotype (Blochlinger et al., 1993; Ludlow et al., 1996). In agreement with this notion, functional complementation of certain mutant cut phenotypes necessitated the continuous ectopic expression of Cut proteins (Ludlow et al., 1996). The latter study also provided evidence for an evolutionarily conserved function of Cut proteins, since the murine Cux and human CDP proteins were able to complement, at least to some extent, certain cut mutants in Drosophila. 5. The role of cut in the nervous system While the function of cut in the central nervous system remains to be de®ned, the analysis of loss-of-function and gain-of-function cut mutants has revealed a role for cut in the speci®cation of neuronal subtype in the peripheral nervous system. The formation of the peripheral nervous system in Drosophila involves several steps (reviewed in (Jan and Jan, 1994; Chan and Jan, 1999)) (Fig. 2). Sensory organs derive from one common neuronal precursor cell, which divides and differentiates to give rise to one neuron and three supporting cells. Two types of sensory organs can be anatomically distinguished: external sensory (es) organs and internal (chordotonal) sensory organs. In wild type ¯ies,

cut is expressed in each cell of the es organ as well as in their sensory precursor cell (Blochlinger et al., 1990), whereas in chordotonal organs cut expression is repressed by the product of the proneural gene, atonal (Jarman and Ahmed, 1998). In the lethal cut mutants, either the protein was not detected or a truncated form was seen in the cytoplasm only (Blochlinger et al., 1990). As a consequence, the neuron and the support cells adopted morphological and antigenic characteristics of chordotonal organs (Bodmer et al., 1987; Blochlinger et al., 1990; Merritt et al., 1993). In contrast, forced expression of cut in Drosophila embryos caused the transformation of chordotonal organs into external sensory organs (Blochlinger et al., 1991). Thus, cut plays a role in the last step of the neurogenic process by instructing the neuronal precursor cell to divide and differentiate into an external sensory organ. Another neuronal-type selector gene, poxneural (poxn), speci®es the poly-innervation of external sensory organs; in addition, the product of poxn was found to bind to the A3 enhancer (see Fig. 1) and induce cut expression (Vervoort et al., 1995). 6. The role of cut in limb development: genetic interactions between cut, Notch and Wingless Mutations that affect the expression or function of Cut, Wingless or Notch in the wing affect wing development and produce phenotypes that can be observed easily. The similitude between some of these phenotypes suggested that the products of these genes might play a role in a common process required for proper wing formation. Indeed, multiple genetic interactions have been reported between cut, and both the Notch (N) and Wingless (Wg) signaling pathways during the development of the wing imaginal discs. In Drosophila, the limbs originate from undifferentiated epithelial cells called imaginal discs. In the wing imaginal disc, signaling between ventral and dorsal cells result in the formation of a band about 4±5 cells wide at the dorsalventral boundary. These D±V boundary cells are also referred to as the D±V organizer, since they produce signals that promote the proximal-distal outgrowth of the limb. The boundary cells will eventually form the edge of the adult wing, the wing margin. The Notch and Wingless signaling pathways are believed to be responsible for the signaling between the dorsal and ventral compartments which determine the fate of those cells at the D/V boundary (Couso et al., 1994; de Celis et al., 1996; Neumann and Cohen, 1996; de Celis and Bray, 1997; Micchelli et al., 1997). These signals not only de®ne the boundary region, but also play an important role later in generating further subregions that will provide each of the speci®c cell types within the wing margin. The available data suggest a complex regulatory cascade Fig. 3. At the end of the third larval instar, Notch activation is no longer dependent on interactions between dorsal and ventral cells, but depends on signaling between boundary cells and cells

A. Nepveu / Gene 270 (2001) 1±15

5

Fig. 2. The development of sensory organs in Drosophila. The formation of the peripheral nervous system in Drosophila involves several steps: (a) the formation of proneural clusters from the neurogenic ectoderm, (b) the speci®cation of a single neural precursor cell through lateral inhibition, (c) asymetric cell divisions of the neural precursor cell accompanied by differentiation and maturation of the daughter cells. Speci®cation of the neuronal subtype occurs during the last step. In the presence of Cut, the neuronal precursor cell divides and differentiates into an external sensory (es) organ. In contrast, when Cut expression is repressed by atonal, an internal (chordotonal) sensory organ is formed. The product of the poxneural (poxn)gene speci®es the polyinnervation (as opposed to monoinnervation) of external sensory organs and also activates Cut expression. It should be noted that some proneural genes, in particular achaete, scute and atonal, can also participate in the speci®cation of neuronal subtypes. Modi®ed from (Jan and Jan, 1994).

¯anking the border on either side. Flanking cells express the Notch ligands, Serrate and Delta that activate Notch in boundary cells. Activation of Notch somehow results in the induction of Cut expression in boundary cells. The evidence for this can be summarized as follows. Expression of Cut was reduced in N hypomorphs (Jack and DeLotto, 1992), and was lost in Suppresser of Hairless (Su(H)) clones (Neumann and Cohen, 1996). (Su(H)) encodes for the DNA binding partner of Notch (reviewed in (Artavanis-Tsakonas et al., 1999)). Loss of reduction of the Notch ligands, Delta and Serrate, also caused a reduction in Cut expression (Jack and DeLotto, 1992; Thomas et al., 1995; Micchelli et al., 1997). Conversely, ectopic expression of these molecules, or expression of an activated form of Notch, induced higher Cut expression (de Celis et al., 1996; Doherty et al., 1996; Neumann and Cohen, 1996; Micchelli et al., 1997). The fact that Notch and Su(H) were found to be autonomously required for Cut expression suggested that Cut might be a target of Notch. However, Cut may not be directly induced by Notch, since loss of function of strawberry notch (sno), itself believed to be a downstream target of Notch, abolished

Fig. 3. Simpli®ed view of the interactions between Cut and the Notch signaling pathway at the dorsal-ventral border of the Drosophila wing. At the end of the third larval instar, Notch is expressed both in dorsalventral (D±V) border cells and ¯anking cells, however, the Notch ligands, Delta (Dl) and Serrate (Ser), are expressed only in ¯anking cells. Thus, the Notch pathway is activated in D±V border cells, but not in ¯anking cells. Following Notch activation, the expression of Cut and Wg is induced. Cut represses expression of Serrate and Delta and sustains Wg expression. Wg acts to maintain expression of Serrate and Delta in ¯anking cells, most likely through interaction with its receptor Frizzled-2. Modi®ed from (de Celis and Bray, 1997).

6

A. Nepveu / Gene 270 (2001) 1±15

expression of a reporter driven by the cut wing enhancer (Majumdar et al., 1997). Moreover, another target of Notch, the Enhancer of split Complex {E(spl)-C} genes, has been implicated in the activation of Cut expression. The {E(spl)C} genes encode a family of seven basic-helix-loop-helix transcriptional repressors (reviewed in (Lecourtois and Schweisguth, 1997). Overexpression of {E(spl)} proteins, in particular E(spl)mg, was shown to induce Cut expression (Ligoxygakis et al., 1999). Altogether, these results suggest that both Sno and {E(spl)] proteins may serve as regulatory effectors between Notch activation and Cut expression. Following its activation downstream of Notch, Cut is thought to inhibit Serrate and Delta expression (de Celis and Bray, 1997), and to maintain Wingless expression in these cells (Micchelli et al., 1997). In turn, the Wingless morphogen would trigger cells of both ¯anks, presumably through its receptor Frizzle, to express the Notch ligands, Delta and Serrate (Micchelli et al., 1997). These cells would signal back to activate the Notch pathway (Neumann and Cohen, 1996). Thus, a positive regulatory feedback loop would be established to maintain and reinforce differences between the two cell compartments. In summary, it is thought that Cut is one of the downstream effectors of Notch, and would act during late larval and pupal stages to maintain Wingless expression and suppress that of Serrate and Delta in the presumptive wing margin. 7. Other genetic interactions A number of genetic interactions with cut have been reported in addition to those involved in the development of the wing. Mutations in Notch were shown to affect egg chamber formation in the ovary, however in this case a cut null mutation suppressed the notch phenotype, suggesting that the two genes have antagonistic effects in this process (Jackson and Blochlinger, 1997). In contrast, a heterozygous mutation in the Pka-C1 gene enhanced the phenotype of a cut hypomorph (Jackson and Blochlinger, 1997). Since Pka-C1 is required in germ cells and cut in follicle cells, these results suggested that the two genes function in a signaling pathways involving these two cell types. Elsewhere, a hypomorphic cut mutation was found to aggravate the phenotype caused by the heterozygous deletions of two homeotic genes of the Antennapedia complex, proboscipedia (pb) and antennapedia (Antp) (Johnston et al., 1998). In ct;pb/1 mutant ¯ies, labial structures were transformed to both antennal and leg structures, while in ct;Antp/1 mutant ¯ies, prothoracic outgrowths appeared at the position of the anterior spiracle. In both cases, the abnormalities resemble the phenotype caused by pb and Antp loss-of-function, raising the possibility that cut positively regulates the function of these homeotic genes and, perhaps, plays a role in the control of segment identity. In agreement with this, ectopic expression of cut in a stripe of cells within the eye-antennal disc caused uniform activation of Antp expression through-

out the disc. This latter observation further supports the notion that cut may ful®ll some non-autonomous function as in the wing imaginal disc. 8. Regulators of cut Expression of cut in various tissues is affected by loss-offunction or ectopic expression of a number of other genes. The role of notch in the activation of cut in the wing imaginal discs has been discussed earlier. Mutations in strawberry notch (sno) (Majumdar et al., 1997), scalloped (Jack and DeLotto, 1992), mastermind (Morcillo et al., 1996; Rollins et al., 1999), Nipped-B (Rollins et al., 1999), Chip (Morcillo et al., 1997) and mod(mdg4) (Cai and Levine, 1997) were found to affect the function of the wing enhancer and thus to affect cut expression in the wing. Among these, Chip and mod(mdg4) appear to mediate their effect through longrange action on chromatin. Early expression of cut in cone cells of the eye was shown to depend on the function of the sparkling (spa) gene, a homologue of the vertebrate Pax2 gene that is required for the development of cone and pigment cells (Fu and Noll, 1997). In the peripheral nervous system, the product of the gene poxn was shown to bind to the A3 enhancer (see Fig. 1) and induce expression of cut in embryonic sensory organs (Vervoort et al., 1995). In contrast, ectopic expression of the proneural gene atonal was able to prevent activation of cut in sense organ precursors and consequently to drive differentiation towards chordotonal sensory organs (Jarman and Ahmed, 1998). Finally, ectopic expression of Cut itself, or of its human and mouse homologues CDP and Cux, was found to activate expression of the endogenous cut gene, suggesting the existence of a positive autoregulatory loop (Blochlinger et al., 1991; Ludlow et al., 1996). In the chick limb bud, cux2 expression was found to be regulated by retinoic acid, Sonic hedgehog and the posterior AER (Tavares et al., 2000). 9. Biochemical activities of Cut in Drosophila Very little information is available regarding the molecular functions of Cut proteins in Drosophila. However, Cut proteins of 215 and 230 kDa were shown to mediate ventral repression of the zen gene by binding to a sequence, AT2, containing a CDP/Cut binding site (Valentine et al., 1998). The mechanism of repression in this case is not entirely clear but may involve cooperation with the dead ringer gene product, Dri, to recruit the co-repressor Groucho. 10. The CAAT-displacement activity The CCAAT-displacement activity was ®rst described in the context of the sperm histone H2B-1 gene of the sea urchin Psammechinus miliaris (Barberis et al., 1987). This gene is expressed in testis but not in the rest of the embryo.

A. Nepveu / Gene 270 (2001) 1±15

Its lack of expression in tissues other than testis was found to correlate with the presence of a factor that was capable of displacing the ubiquitous CCAAT binding factor (CBF) from the promoter. Analysis of the g-globin gene promoter, that differs from the other globin gene promoters in that it includes a duplicated CCAAT site, revealed the existence of a similar factor in mammalian cells (Superti-Furga et al., 1988; Mantovani et al., 1989; Ottolenghi et al., 1989; Superti-Furga et al., 1989). This factor was termed CCAAT-displacement protein, CDP, since retarded complexes of identical, slow, mobility were generated using probes derived from either the H2B-1 or g-globin gene promoter and extracts from sea urchin embryos and mammalian cell lines (Superti-Furga et al., 1988). A thorough analysis of the myelomonocytic-speci®c cytochrome b heavy chain (gp91-phox) gene promoter clearly established the role of CDP as a transcriptional repressor (Skalnik et al., 1991; Neufeld et al., 1992; Lievens et al., 1995; Luo and Skalnik, 1996). CDP activity was present in non-expressing cells and induction of endogenous gp91-phox expression upon terminal differentiation of myelomonocytic cells was found to correlate with the disappearance of CDP activity (Skalnik et al., 1991). Moreover, deletion of a CDP binding region within gp91-phox reporter constructs led to increased expression in undifferentiated cells. Since derepression was independent of CBF, it was proposed that CDP repressed transcription by blocking the interaction of multiple activators (Skalnik et al., 1991; Luo and Skalnik, 1996). CDP was puri®ed from HeLa cells by fractionation and DNA af®nity chromatography, antibodies were raised, and overlapping cDNAs were cloned by immunoscreening of an expression library (Neufeld et al., 1992). Sequence comparison revealed a close relationship with Drosophila Cut and binding analysis revealed that both proteins exhibited similar DNA binding speci®cities. In later experiments, transient co-transfections established that CDP/Cut can repress reporter plasmids containing gp91phox gene promoter sequences; moreover, constitutive CDP/Cut expression in a stable transformant of HL-60 myeloid cells caused a reduction in gp91-phox expression during cellular differentiation (Lievens et al., 1995). Many other promoters have been reported to be repressed by CDP/Cut (Table 2, and see below). Evidence for the involvement of CDP/Cut included correlative associations between CDP/Cut activity and reduced expression of the targets, mapping of regulatory DNA sequences, EMSA and transient transfections. 11. CDP/Cut DNA binding activity in mammals generally correlates with cellular proliferation The bulk of the results in mammals suggested that CDP/ Cut expression or activity might be restricted to proliferating cells. Expression of the mouse CDP/Cut protein, Cux1, in the kidney was found to be inversely related to the

7

degree of cellular differentiation (Heuvel et al., 1996). CDP/Cut binding activity, initially characterized as the CCAAT displacement activity (Skalnik et al., 1991; Lievens et al., 1995) or histone nuclear factor D (HiNFD) (van Wijnen et al., 1989), was found to disappear upon differentiation of myeloid cells. Furthermore, the level of CDP/Cut binding activity (i.e. HiNF-D) was found to be downregulated during fetal liver development (van Wijnen et al., 1991), and osteoblast differentiation (Holthuis et al., 1990; van Gurp et al., 1999). Consistent with the ubiquitous downregulation of CDP/Cut binding activity in many cell types, many of the identi®ed targets of CDP/Cut are genes that are repressed in proliferating precursor cells and are turned on as cells become terminally differentiated and CDP/Cut activity ceases (see Table 2). This pattern may also extend to viruses as CDP/Cut was shown to repress human papillomavirus (HPV) gene expression and replication in undifferentiated epithelial cells but not in keratinocytes (Pattison et al., 1997; Ai et al., 1999; O'Connor et al., 2000). In addition, a role for CDP/Cut in cell cycle progression was suggested from the ®ndings that CDP/Cut DNA binding activity oscillates during the cell cycle, reaching a maximum at the end of G1 and during the S phase (Coqueret et al., 1998a). CDP/Cut (HiNF-D) binding interactions with the histone H4, H3 and H1 promoters are also regulated with respect to S phase (Holthuis et al., 1990; Wright et al., 1992; van den Ent et al., 1994; van Wijnen et al., 1997). CDP/Cut (HiNF-D) binding activity is barely detectable in cells deprived of serum, and dramatically upregulated when cells enter the cell cycle following growth factor stimulation (Wright et al., 1992; van Wijnen et al., 1997). The regulation of CDP-cut binding activity during the cell cycle indicates that this factor may repress speci®c target genes in a cell cycle dependent manner. The concept of CDP/Cut as a cell cycle dependent repressor is consistent with studies on the p21 promoter. For example, the increase in CDP/Cut activity coincided with a decrease in p21 WAF1/CIP1/SDI1 mRNAs, and in co-transfection experiments, a reporter controlled by the p21 promoter was repressed by CDP/Cut and was shut off in S phase, whereas antisense Cut restored expression. Moreover, p21 WAF1/CIP1/SDI1 expression was repressed equally well by either Cdc25A or CDP/Cut. Altogether, these results led to a model whereby Cdc25A activates the CDP/Cut repressor which then down-regulates transcription of p21 WAF1/CIP1/ SDI1 in S phase. The same study also established that, at least in ®broblastic cells, there was no correlation between CDP/Cut DNA binding activity and repression of c-Myc, a target that was previously identi®ed through biochemical studies (Dufort and Nepveu, 1994; Coqueret et al., 1998a). We cannot, however, exclude the possibility that in certain physiological situations yet to be identi®ed CDP/Cut may contribute to the down-regulation of c-Myc. We note that the persistence of cut expression in differentiated cells in Drosophila is in apparent contrast with the

8

A. Nepveu / Gene 270 (2001) 1±15

Table 2 Gene targets of CDP/Cut Genes repressed in proliferating precursor cells

References

g-globin (human) b-globin (Xenopus laevis) g-globulin gp91-phox b-MHC gene NCAM (neural cell adhesion molecule) gene Tyrosine hydroxylase CD8a gene, matrix attachment region (MAR) Lactoferrin gene Human Papillomavirus Type 6 Long Control Region Neutrophil collagenase p21 Waf1/Cip1/Sdi1 T cell receptor b gene (MAR) Cystic ®brosis transmembrane conductance regulator gene Osteocalcin gene Immunoglobulin heavy chain enhancer Tryptophan hydroxylase Genes expressed in proliferating cells Histone H2B-1 Histone H2A and H2B Histone H4 c-myc Thymidine kinase c-mos

Mantovani et al., 1989; Ottolenghi et al., 1989 Patient et al., 1989 Superti-Furga et al., 1989 Skalnik et al., 1991; Neufeld et al., 1992; Lievens et al., 1995; Luo and Skalnik, 1996 Andres et al., 1992 Valarche et al., 1993 Yoon and Chikaraishi, 1994 Banan et al., 1997 Khanna-Gupta et al., 1997 Pattison et al., 1997; Ai et al., 1999; O'Connor et al., 2000 Lawson et al., 1998 Coqueret et al., 1998a Chattopadhyay et al., 1998 Li et al., 1999 van Gurp et al., 1999 Wang et al., 1999 Teerawatanasuk et al., 1999 References Barberis et al., 1987 el-Hodiri and Perry, 1995 van Wijnen et al., 1996; van Wijnen et al., 1997; Aziz et al., 1998b; Last et al., 1998 Dufort and Nepveu, 1994 Kim et al., 1997 Higgy et al., 1997

situation in mammals, where CDP/Cut DNA binding activity disappears as cells become fully differentiated in mammals. It should be stressed that Cut DNA binding activity has not been investigated in Drosophila, whereas in mammals in most cases it is not known whether the decrease in CDP/Cut DNA binding activity as cells differentiate is due to a drop in expression or to some post-translational modi®cations. Whether CDP/Cut in mammals also serves as a determinant of cell type speci®city, as suggested by the complementation studies using CDP and Cux in Drosophila, remains to be assessed in mammals (Ludlow et al., 1996). Another related question is whether the role of CDP/ Cut in cell fate determination could be a consequence of its role in cell proliferation. 12. Mechanisms of repression by CDP/Cut CDP/Cut proteins were found generally to function as transcriptional repressors (Superti-Furga et al., 1989; Skalnik et al., 1991; Andres et al., 1992; Valarche et al., 1993; Dufort and Nepveu, 1994; Lievens et al., 1995; Mailly et al., 1996; Li et al., 1999; van Gurp et al., 1999). Repression by CDP/Cut involves at least two mechanisms. In most cases, CDP/Cut appears to compete with some activators for the occupancy of a binding site. It is this property that bestowed upon this protein the name of `CCAAT displacement protein' (Barberis et al., 1987; Skalnik et al., 1991; Neufeld et al., 1992). In addition, two active repression domains have been identi®ed within the carboxy-terminal domain

of CDP/Cut suggesting that once stably bound to a promoter CDP/Cut can actively repress gene expression (Mailly et al., 1996). This function has been referred to as `active repression' to distinguish it from the `passive repression' resulting from competitive DNA binding (Hanna-Rose and Hansen, 1996). It was recently shown that immunoprecipitates of CDP/Cut contain deacetylase activity and that the deacetylase HDAC1 can be pulled down by a GST-CDP/Cut fusion protein containing the Cut homeodomain as well as the carboxy-terminal region (Li et al., 1999). These results suggest that the mechanism of active repression by CDP/ Cut involves the direct recruitment of the HDAC1 deacetylase. 13. CDP/Cut may also function as an activator The role of CDP/Cut in proliferating cells may not be limited to that of a transcriptional repressor, as certain results suggest that CDP/Cut may also be able to participate in gene activation. A number of groups have identi®ed binding sites for CDP/Cut in the regulatory sequences of genes encoding for histones and the thymidine kinase (Barberis et al., 1987; el-Hodiri and Perry, 1995; van Wijnen et al., 1996; Kim et al., 1997). The peak of expression of these genes coincides with or closely precedes DNA replication, a time at which CDP/Cut DNA binding activity is the highest (Coqueret et al., 1998a). Moreover, CDP/Cut was found to be a component of the promoter complex HiNF-D, which is believed to contribute to the transcriptional induction of

A. Nepveu / Gene 270 (2001) 1±15

several histone genes at the G1/S phase transition of the cell cycle (van Wijnen et al., 1996; van Wijnen et al., 1997; Aziz et al., 1998b). Assuming that CDP/Cut can also function as an activator, the logical conclusion would be that CDP/Cut does not repress these genes but instead contributes to their activation. It is equally conceivable (based on the currently available data) that the protein is an attenuator of the cell cycle dependent activation that is mediated by other histone gene transcription factors (i.e. IRF proteins, HiNF-P) (Aziz et al., 1998a). The molecular basis for the opposite action of CDP/Cut on different promoters is currently unknown, however, it was suggested that the regulatory effect of CDP/Cut on transcription might vary depending on the proteins with which it interacts (Yoon and Chikaraishi, 1994; van Wijnen et al., 1996). In particular, supershift assays using antibodies against pRb-related proteins suggested that Cut might alternatively interact with pRb or p107 on different DNA sequences (van Wijnen et al., 1996). A somewhat analogous situation was observed in the case of the tyrosine hydroxylase gene: CDP/Cut alone could not activate the corresponding reporter plasmid but did so when transfected in conjunction with the rat ITF2 transcription factor (Yoon and Chikaraishi, 1994). 14. Matrix attachment regions (MAR) A number of reports have described the interaction of CDP/Cut with matrix attachment regions (MARs) that are believed to play an important role in gene regulation. These include the MARs upstream of the CD8a (Banan et al., 1997) and T cell receptor b genes (Chattopadhyay et al., 1998), the MARs ¯anking the immunoglobulin heavy chain intronic enhancer (Wang et al., 1999), the negative regulatory elements within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) (Liu et al., 1997), and the MARs ¯anking the long control region (LCR) of human papillomavirus type 16 (Stunkel et al., 2000). The af®nity of CDP/Cut for MARs may result from the fact that these regions are rich in ATC nucleotides. However, we cannot exclude the possibility that CDP/Cut can recognize a DNA structure that is speci®c to base-unpairing regions. Such preference for certain DNA structures would likely not be revealed through PCR-mediated site selection. Interestingly, studies of MARs-binding proteins in thymocytes have also revealed the existence of a CDP/Cut-related protein, SATB1, which contains two Cut repeats and an atypical homeodomain (Dickinson et al., 1997). SATB1 and CDP/Cut were shown to interact with overlapping sites and compete for MAR binding (Banan et al., 1997; Chattopadhyay et al., 1998). Moreover, SATB1 and CDP/Cut were found to interact with each other in a DNA-independent manner, and this interaction was shown to inhibit their DNA binding activities (Liu et al., 1999). Overall, the data suggested

9

a model whereby CDP/Cut would downmodulate receptor genes in non-terminally differentiated B and T cells, by preventing the interaction between MARs and nuclear matrix proteins like SATB1 and Bright (Banan et al., 1997; Chattopadhyay et al., 1998; Wang et al., 1999). In contrast, CDP/Cut was postulated to positively regulate MMTV expression by counteracting the effect of SATB1 (Liu et al., 1999). Although the exact role of MARs in gene regulation is still not clear, the fact that CDP/Cut bound to multiple MARs and could effectively complete with various MAR-binding proteins suggests that CDP/Cut may regulate transcription within certain loci in part by modulating their association of with structural components of the nucleus. 15. DNA binding domains of CDP/Cut The high degree of conservation of Cut repeats suggested that they might have an important biochemical function (Blochlinger et al., 1988; Neufeld et al., 1992). Indeed, Cut repeats were found to function as speci®c DNA binding domains (Andres et al., 1994; Au®ero et al., 1994; Harada et al., 1994; Harada et al., 1995). Cut proteins therefore are unique in that they contain four DNA binding domains: the Cut homeodomain and the three Cut repeats. How exactly the CDP/Cut protein interacts with DNA has not yet been entirely resolved, but the available data suggest that several modes of interactions may exist. We will review the results obtained with puri®ed fusion proteins, and then with nuclear extracts. The original studies with bacterially expressed glutathione S-transferase (GST) fusion proteins suggested that each Cut repeat and the Cut homeodomain were able individually to bind to DNA (Andres et al., 1994; Au®ero et al., 1994; Harada et al., 1994; Catt et al., 1999). PCR-mediated site selection with GST/Cut repeat fusion proteins led to the isolation of several types of sequences, which could be aligned to either ATNNAT (mainly ATCGAT and ATCAAT) or CCAAT. However, a sizable fraction of the selected sequences (,20%) diverged greatly from any consensus and yet represented excellent binding sites (Andres et al., 1994; Au®ero et al., 1994; Harada et al., 1995). These results suggested that CDP/Cut could tolerate a certain degree of ¯exibility in their DNA targets. This ¯exibility was also re¯ected by DNA recognition motifs identi®ed for CDP/Cut (HiNF-D) binding to histone H4, H3 and H1 gene promoters by methylation interference analysis of protein/ DNA complexes using endogenous CDP/Cut protein (van den Ent et al., 1994). This property may not be unique to CDP/Cut proteins, as similar relaxed sequence speci®city was found for the GATA factors when submitted to PCRmediated site selection (Ko and Engel, 1993; Merika and Orkin, 1993). Other studies demonstrated that GST/Cut repeat fusion proteins exist as dimers (Harada et al., 1995), and that indi-

10

A. Nepveu / Gene 270 (2001) 1±15

vidual Cut repeats are unable to bind to DNA with high af®nity when expressed as monomers with either the maltose binding protein (MBP) or a histidine-tag (Moon et al., 2000). These results suggest that the use of GST fusion proteins in DNA binding experiments is likely to lead to artifacts because of the propensity of the GST moiety to dimerize. Thus, the results from site selection experiments with GST/Cut repeats should be taken with caution. Using his-tagged fusion proteins, various combinations of domains were found to bind to DNA with distinct af®nities and kinetics: CR1CR2, CR3HD, CR1HD, CR2HD and CR2CR3HD (Moon et al., 2000). In contrast, the hisCR2CR3 fusion protein did not ef®ciently bind to DNA. Interestingly, hisCR1CR2 displayed rapid on and off-rates, and bound preferably to two CA/GAT sites, organized as direct or inverted repeats and separated by a variable number of nucleotides (Fig. 4). The maximum distance between two CA/GAT sites that can be accommodated by CR1CR2 has not been determined, but could be fairly large if DNA looping is allowed. In contrast, any combination of a Cut repeat with the Cut homeodomain exhibited slower binding kinetics and a preference for sequences conforming to the ATNNAT consensus (Fig. 4). While hisCR1HD preferred ATCAAT, CR3HD selected ATCGAT and ATCAAT. Because of the closely related DNA binding speci®cities of these combinations, it is not possible to verify whether CDP/Cut may exist in different conformational states. Surprisingly, a his-tagged full length CDP/Cut protein puri®ed from insect cells exhibited DNA binding kinetics similar to that of CR1CR2, suggesting that the Cut homeodomain may not be active in the context of the full-length protein. However, these binding assays were performed with a small number of relatively short oligonucleotides and it remains to be determined whether the full-

Fig. 4. Schematic diagrams of two modes of DNA binding by CDP/Cut. DNA binding by CDP/Cut must involve at least two DNA binding domains: either two Cut repeats or one Cut repeat and the Cut homeodomain. While several combinations of domains have been shown to function in vitro using puri®ed recombinant proteins, so far two modes of DNA binding have been documented in mammalian cells. The CCAAT-displacement activity involves primarily CR1 1 2, whereas DNA binding to ATCGAT involves CR3HD. CR1 1 2 and CR3HD exhibits fast and slow `on' and `off' rates, respectively. Thus, while CR1 1 2 binds to DNA rapidly but only transiently, CR3HD makes a stable interaction with DNA.

length CDP/Cut protein can make a stable interaction with other and, in particular, larger pieces of DNA. In one study, site selection was performed by immunoprecipitation using cell extracts from COS cells expressing a recombinant full length protein CDP/Cut protein. Interestingly, two types of sequences were pulled down: one set contained sequences conforming to an ATCGATTA consensus, the other set contained a CCAAT site in addition to this consensus (Andres et al., 1994). The author concluded that one Cut repeat made contact with the ATCG sequence and the homeodomain interacted with the ATTA motif, while another Cut repeat could bind a CCAAT site positioned upstream or downstream and in either orientation. This notion is in agreement with the ®ndings that hisCR2CR2 selected dimers of the CA/GAT motif in either orientation. However, an ATTA motif was not found in other studies and direct comparison of the ATCGATTA consensus with one lacking ATTA (ATCGATCGCC) demonstrated that the latter was better recognized by endogenous as well as recombinant CDP/Cut proteins (Harada et al., 1995). The available data suggest that CDP/Cut has the capability to bind to a wide range of DNA sequences. This is evident from the number of studies that have reported interaction between CDP/Cut and apparently unrelated sequences. Two types of retarded complexes have been observed in EMSA. One slowly migrating species is observed in the reports on the CCAAT-displacement activity. Another retarded complex migrates with faster mobility and exhibit higher af®nity for the ATCGAT sequence. Recent data indicate that this faster migrating species is a shorter CDP/Cut protein that is generated by proteolytic processing of CDP/Cut, speci®cally in S phase (Moon et al., unpublished data). In contrast to CR1CR2 and the full length CDP/Cut, this shorter species makes a stable interaction with DNA. Thus proteolytic processing would yet represent another post-translational modi®cation that modulates CDP/Cut activity. Which DNA binding domains of CDP/Cut mediate the CCAAT-displacement activity may depend on the particular promoter sequence. In vitro studies with CDP/Cut fusion proteins indicated that CR1CR2, but not CR3HD, was able to bind with high af®nity to a CCAAT site provided that a second C(A/G)AT motif was present at close proximity (Moon et al., 2000). In agreement with these ®ndings, only CR1CR2 was able to displace the NF-Y factor. Moreover, the CDP activity from nuclear extracts was found to exhibit a fast off-rate, a property shared both by CR1CR2 and the puri®ed full-length CDP/Cut protein, but not by CR3HD (Skalnik et al., 1991; Moon et al., 2000). Yet, an amino-terminally truncated CDP/Cut protein containing CR2CR3HD was shown to repress as ef®ciently as fulllength CDP/Cut a reporter construct containing the g-globin gene promoter (Lievens et al., 1995). It should be stressed that the DNA binding properties of CR2CR3HD have not yet been thoroughly investigated.

A. Nepveu / Gene 270 (2001) 1±15

16. CDP/Cut DNA binding is inhibited by posttranslational modi®cations of either cut repeats or the cut homeodomain Comparison with the sequence of Drosophila Cut revealed that Cut repeats contain evolutionarily conserved consensus phosphorylation sites for protein kinase C (PKC) and casein kinase II (CKII). PKC (Coqueret et al., 1996) and CKII (Coqueret et al., 1998b) were shown to phosphorylate Cut repeats, in vitro and in vivo. Phosphorylation of Cut repeats caused an inhibition of DNA binding and, consequently, the transcriptional repression activity of CDP/Cut. In ®broblastic cells, CDP/Cut DNA binding activity was found to be regulated in a cell-cycle dependent manner (Coqueret et al., 1998a). Only weak DNA binding was detected in G0 and early G1, unless cell extracts were previously treated with alkaline phosphatase. In contrast, strong DNA binding was observed in S phase and was shown to result both from an increase in CDP/Cut expression and dephosphorylation by the Cdc25A phosphatase. Interestingly, mapping of the phosphorylation sites indicated that inhibition of DNA binding in this situation was caused by serine phosphorylation within the Cut homeodomain. The kinase(s) responsible for this regulation have yet to be identi®ed. More recently, PCAFmediated acetylation of the Cut homeodomain was shown to inhibit DNA binding (Li et al., 2000).

17. The existence of Cux-1 and Cux-2 genes in vertebrates suggests some degree of redundancy and specialization A second Cux gene, called Cux-2, was identi®ed in mouse and chicken (Quaggin et al., 1996; Tavares et al., 2000). An almost identical human cDNA sequence has been isolated from a human brain library, suggesting that a second, neuronal speci®c, CDP/Cut gene exists in all vertebrates (NCBI, accession number AB006631). In contrast to mouse Cux-1 which is expressed in most tissues (including the nervous system), Cux-2 was found to be expressed primarily in nervous tissues (Quaggin et al., 1996). In the adult brain, Cux-2 expression was highest in neurons of the thalamus and limbic system; in the embryo, Cux-2 was expressed in the central and peripheral nervous systems, in particular in the teleencephalon and peripheral ganglia of the trigeminal and glossopharyngeal nerves. These results suggest that some of the function of Drosophila cut in the nervous system may be carried by the Cux-2 gene in vertebrates. The signaling processes involved in limb development in Drosophila and vertebrates have been found to be remarkably similar, as related proteins appear to mediate analogous events in the two systems (Irvine and Vogt, 1997). The role of Drosophila cut in the dorsal-ventral (D±V) boundary cells during the development of the wing imaginal discs has been described in Section 6. In vertebrates, similar events occurring at the apex of the limb buds result in the formation of

11

the apical ectodermal ridge (AER), which produce signals promoting outgrowth of the limb bud (see below) (Irvine and Vogt, 1997). Interestingly, cux1 and cux2 in chicken are expressed in different compartments of the developing limb bud (Tavares et al., 2000). While cux1 expression was detected in the ectoderm outside the AER, as well as around ridge-like structures, cux2 expression was observed in the pre-limb lateral plate mesoderm, posterior limb bud and ¯ank mesenchyme, a pattern reminiscent of the distribution of polarizing activity. Their expression pattern and the phenotypes resulting from cux1 overexpression as well as grafting experiments led the authors to suggest that chick Cux1 and Cux2 may act by modulating proliferation versus differentiation in the limb ectoderm and polarizing activity regions, respectively. Altogether these ®ndings suggest that the role of Drosophila cut in the development of the limb and the peripheral nervous system has been conserved in evolution. However, in vertebrates this role appears to be ful®lled by two genes with distinct patterns of expression. To what extent the two genes carry redundant or complementary functions will remain to be investigated. 18. Genetic studies in mouse An early attempt to generate a knock-out mouse resulted in the generation of a mouse expressing a Cux protein with an internal deletion of 246 amino acids encompassing Cut repeat 1. DeltaCR1 homozygous mutant mice displayed a mild phenotype characterized by curly vibrissae, wavy hair, and a high degree of pup loss resulting probably from feeding problems (Tufarelli et al., 1998). More recently, the group of Dr Ellis J. Neufeld has generated another knockout, designated DeltaHD, in which the Cut homeodomain was replaced with a neomycin cassette. The mutant mice are currently being characterized in a collaborative effort with the laboratory of Dr Andre Van Wijnen. The DeltaHD homozygous mutant mice are reported to display severely reduced reproductive potential, high perinatal lethality and a pronounced fur defect (A. VanWijnen, personal communication). The fur defect appears to be due to abnormalities in hair follicle development. At the molecular level, the Delta HD mouse were found to be phenotypically null for the HiNF-D complex, that has been implicated in the activation of several histone genes (see Section 13). 19. CUTL1 is located in a chromosomal region that is frequently rearranged in cancers The CUTL1 gene was mapped to chromosome 7, band 7q22 (Scherer et al., 1993; Lemieux et al., 1994). Cytogenetic analyses revealed that rearrangements or deletions of 7q22 occur frequently in uterine leiomyomas (reviewed in (Ozisik et al., 1993), acute myeloid leukemia (Fenaux et al., 1989; Swansbury et al., 1994) and myelodysplastic

12

A. Nepveu / Gene 270 (2001) 1±15

syndrome (Yunis et al., 1988; Heim, 1992). The fact that such a high proportion of some cancers present a cytogenetically detectable deletion of 7q22 suggested that a tumor suppressor gene was located within this chromosomal region. Using three polymorphic markers within CUTL1 (Zeng et al., 2000) as well as a panel of markers within 7q, CUTL1 was shown to be present in the chromosomal region that is deleted in 15% human uterine leiomyomas (Zeng et al., 1997) and 18% of breast cancers (Zeng et al., 1999). In the latter cancers, LOH of 7q22 was associated with increased tumor size. Interestingly, in 5 out of 12 breast tumors with LOH of 7q22, deletions included the two markers within introns 3 and 6, but not the marker within intron 20, leaving intact the 3 0 end of the gene. In an animal model, female transgenic mice expressing the Polyomavirus (PyV) Large T (LT) antigen under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) frequently developed, in addition to mammary tumors, uterine leiomyomas (Webster et al., 1998). The results of coimmunoprecipitation analyses revealed that speci®c complexes of CDP/Cut and PyV LT antigen could be detected in both leiomyomas and mammary tumors (Webster et al., 1998). Although the functional signi®cance of this interaction remains to be determined, the existence of complexes between CDP/Cut and a viral oncoprotein suggest that alterations in the function of CDP/Cut may be an important event in the etiology of breast cancer. Therefore, two sets of data, LOH mapping analysis and proteinprotein interaction studies, point towards CUTL1 as a candidate cancer gene. Clearly, additional work will be needed to assess the implication of CUTL1 in cancer. At the minimum mapping of the centromeric boundary of the commonly deleted chromosomal region in breast cancer to the region in between introns 6 and 20 will provide a useful start point for positional cloning approaches to identify the critical cancer gene at 7q22. In this regards, it should be noted that CUTL1 appears to cover a very large distance, over 350 Kbp, and that several putative genes have already been identi®ed within this chromosomal region (Glockner et al., 1998).

References Ai, W., Toussaint, E., Roman, A., 1999. CCAAT displacement protein binds to and negatively regulates human papillomavirus type 6 E6, E7, and E1 promoters. J. Virol. 73, 4220±4229. Andres, V., Chiara, M.D., Mahdavi, V., 1994. A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins. Genes Dev. 8, 245±257. Andres, V., Nadal-Ginard, B., Mahdavi, V., 1992. Clox, a mammalian homeobox gene related to Drosophila cut, encodes DNA-binding regulatory proteins differentially expressed during development. Development 116, 321±334. Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: cell fate control and signal integration in development. Science 284, 770± 776. Au®ero, B., Neufeld, E.J., Orkin, S.H., 1994. Sequence-speci®c DNA bind-

ing of individual Cut repeats of the human CCAAT displacement/Cut homeodomain protein. Proc. Natl. Acad. Sci. USA 91, 7757±7761. Aziz, F., van Wijnen, A.J., Stein, J.L., Stein, G.S., 1998a. HiNF-D (CDPcut/CDC2/cyclin A/pRB-complex) in¯uences the timing of IRF- 2dependent cell cycle activation of human histone H4 gene transcription at the G1/S phase transition. J. Cell. Physiol. 177, 453±464. Aziz, F., van Wijnen, A.J., Vaughan, P.S., Wu, S.J., Shakoori, A.R., Lian, J.B., Soprano, K.J., Stein, J.L., Stein, G.S., 1998b. The integrated activities of Irf-2 (Hinf-M), Cdp/Cut (Hinf-D) and H4tf-2 (Hinf-P) regulate transcription of a cell cycle controlled human histone H4 gene ± mechanistic differences between distinct H4 genes. Mol. Biol. Rep. 25, 1±12. Banan, M., Rojas, I.C., Lee, W.H., King, H.L., Harriss, J.V., Kobayashi, R., Webb, C.F., Gottlieb, P.D., 1997. Interaction of the nuclear matrixassociated region (MAR)-binding proteins, SATB1 and CDP/Cux, with a MAR element (L2a) in an upstream regulatory region of the mouse CD8a gene. J. Biol. Chem. 272, 18440±18452. Barberis, A., Superti-Furga, G., Busslinger, M., 1987. Mutually exclusive interaction of the CCAAT-binding factor and of a displacement protein with overlapping sequences of a histone gene promoter. Cell 50, 347± 359. Bell, A.C., Felsenfeld, G., 1999. Stopped at the border: boundaries and insulators. Curr. Opin. Genet. Dev. 9, 191±198. Blanc, R., 1942. The production of wing scalloping in Drosophila melanogaster. Univ. Calif. Publ. Zool., 49. Blochlinger, K., Bodmer, R., Jack, J., Jan, L.Y., Jan, Y.N., 1988. Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature 333, 629±635. Blochlinger, K., Bodmer, R., Jan, L.Y., Jan, Y.N., 1990. Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes Dev. 4, 1322± 1331. Blochlinger, K., Jan, L.Y., Jan, Y.N., 1991. Transformation of sensory organ identity by ectopic expression of Cut in Drosophila. Genes Dev. 5, 1124±1135. Blochlinger, K., Jan, L.Y., Jan, Y.N., 1993. Postembryonic patterns of expression of cut, a locus regulating sensory organ identity in Drosophila. Development 117, 441±450. Bodmer, R., Barbel, S., Shepherd, S., Jack, J.W., Jan, L.Y., Jan, Y.N., 1987. Transformation of sensory organs by mutations of the cut locus of D. melanogaster. Cell 51, 293±307. Braun, W., 1942. The effect of puncture on the developing wing of several mutants of Drosophila melanogaster. J. Exp. Zool. 84, 325±350. Cai, H.N., Levine, M., 1997. The gypsy insulator can function as a promoter-speci®c silencer in the Drosophila embryo. EMBO J. 16, 1732± 1741. Catt, D., Luo, W., Skalnik, D.G., 1999. DNA-binding properties of CCAAT displacement protein cut repeats [In Process Citation]. Cell. Mol. Biol. (Noisy-Le-Grand) 45, 1149±1160. Chan, Y.M., Jan, Y.N., 1999. Conservation of neurogenic genes and mechanisms. Curr. Opin. Neurobiol. 9, 582±588. Chattopadhyay, S., Whitehurst, C.E., Chen, J., 1998. A nuclear matrix attachment region upstream of the T cell receptor beta gene enhancer binds Cux/CDP and SATB1 and modulates enhancer-dependent reporter gene expression but not endogenous gene expression. J. Biol. Chem. 273, 29838±29846. Coqueret, O., Berube, G., Nepveu, A., 1996. DNA binding By Cut homeodomain proteins Is down-modulated by protein kinase C. J. Biol. Chem. 271, 24862±24868. Coqueret, O., Berube, G., Nepveu, A., 1998a. The mammalian Cut homeodomain protein functions as a cell-cycle-dependent transcriptional repressor which downmodulates P21(Waf1/Cip1/Sdi1) In S phase. EMBO J. 17, 4680±4694. Coqueret, O., Martin, N., Berube, G., Rabbat, M., Litch®eld, D.W., Nepveu, A., 1998b. DNA binding by Cut homeodomain proteins is down-modulated by casein kinase II. J. Biol. Chem. 273, 2561±2566. Couso, J.P., Bishop, S.A., Martinez Arias, A., 1994. The wingless signal-

A. Nepveu / Gene 270 (2001) 1±15 ling pathway and the patterning of the wing margin in Drosophila. Development 120, 621±636. de Celis, J.F., Bray, S., 1997. Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124, 3241±3251. de Celis, J.F., Garcia-Bellido, A., Bray, S.J., 1996. Activation and function of Notch at the dorsal-ventral boundary of the wing imaginal disc. Development 122, 359±369. Dickinson, L.A., Dickinson, C.D., Kohwi-Shigematsu, T., 1997. An atypical homeodomain in SATB1 promotes speci®c recognition of the key structural element in a matrix attachment region. J. Biol. Chem. 272, 11463±11470. Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L.Y., Jan, Y.N., 1996. Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation. Genes Dev. 10, 421±434. Dorsett, D., 1993. Distance-independent inactivation of an enhancer by the suppressor of Hairy-wing DNA-binding protein of Drosophila. Genetics 134, 1135±1144. Dufort, D., Nepveu, A., 1994. The human cut homeodomain protein represses transcription from the c-myc promoter. Mol. Cell. Biol. 14, 4251±4257. el-Hodiri, H.M., Perry, M., 1995. Interaction of the CCAAT displacement protein with shared regulatory elements required for transcription of paired histone genes. Mol. Cell. Biol. 15, 3587±3596. Fenaux, P., Preudhomme, C., Lai, J.L., Morel, P., Beuscart, R., Bauters, F., 1989. Cytogenetics and their prognostic value in de novo acute myeloid leukaemia: a report on 283 cases. Br. J. Haemat. 73, 61±67. Fristrom, D., 1969. Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster. Mol. Gen. Genetics 103, 363± 379. Fu, W., Noll, M., 1997. The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye [see comments]. Genes Dev 11, 2066±2078. Glockner, G., Scherer, S., Schattevoy, R., Boright, A., Weber, J., Tsui, L.C., Rosenthal, A., 1998. Large-scale sequencing of two regions in human chromosome 7q22: analysis of 650 kb of genomic sequence around the EPO and CUTL1 loci reveals 17 genes. Genome Res. 8, 1060±1073. Hanna-Rose, W., Hansen, U., 1996. Active repression mechanisms of eukaryotic transcription repressors. Trends Genet. 12, 229±234. Harada, R., Berube, G., Tamplin, O.J., Denis-Larose, C., Nepveu, A., 1995. DNA-binding speci®city of the cut repeats from the human cut-like protein. Mol. Cell. Biol. 15, 129±140. Harada, R., Dufort, D., Denis-Larose, C., Nepveu, A., 1994. Conserved cut repeats in the human cut homeodomain protein function as DNA binding domains. J. Biol. Chem. 269, 2062±2067. Heim, S., 1992. Cytogenetic ®ndings in primary and secondary MDS. Leukemia Res. 16, 43±46. Hertweck, H., 1931. Anatomie und variabilitaet des nerven-systems und der sinneorgane von Drosophila melanogaster. J. Exp. Zool. 139, 559±663. Heuvel, G.B.V., Bodmer, R., McConnell, K.R., Nagami, G.T., Igarashi, P., 1996. Expression of a Cut-related homeobox gene in developing and polycystic mouse kidney. Kidney Int. 50, 453±461. Higgy, N.A., Tarnasky, H.A., Valarche, I., Nepveu, A., Vanderhoorn, F.A., 1997. Cux/CDP homeodomain protein binds to an enhancer in the rat cMos locus and represses its activity. Biochim. Biophys. Acta ± Gene Struct. Express. 1351, 313±324. Holthuis, J., Owen, T.A., van Wijnen, A.J., Wright, K.L., Ramsey-Ewing, A., Kennedy, M.B., Carter, R., Cosenza, S.C., Soprano, K.J., Lian, J.B., et al., 1990. Tumor cells exhibit deregulation of the cell cycle histone gene promoter factor HiNF-D. Science 247, 1454±1457. Irvine, K.D., Vogt, T.F., 1997. Dorsal-ventral signaling in limb development. Curr. Opin. Cell. Biol. 9, 867±876. Jack, J., DeLotto, Y., 1992. Effect of wing scalloping mutations on cut expression and sense organ differentiation in the Drosophila wing margin. Genetics 131, 353±363. Jack, J., DeLotto, Y., 1995. Structure and regulation of a complex locus: the cut gene of Drosophila. Genetics 139, 1689±1700.

13

Jack, J., Dorsett, D., Delotto, Y., Liu, S., 1991. Expression of the cut locus in the Drosophila wing margin is required for cell type speci®cation and is regulated by a distant enhancer. Development 113, 735±747. Jack, J.W., 1985. Molecular organization of the cut locus of Drosophila melanogaster. Cell 42, 869±876. Jackson, S.M., Blochlinger, K., 1997. cut interacts with Notch and protein kinase A to regulate egg chamber formation and to maintain germline cyst integrity during Drosophila oogenesis. Development 124, 3663± 3672. Jacquemin, P., Lannoy, V.J., Rousseau, G.G., Lemaigre, F.P., 1999. OC-2, a novel mammalian member of the ONECUT class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6. J. Biol. Chem. 274, 2665±2671. Jan, Y.N., Jan, L.Y., 1994. Genetic control of cell fate speci®cation in Drosophila peripheral nervous system. Annu. Rev. Genet. 28, 373±393. Jarman, A.P., Ahmed, I., 1998. The speci®city of proneural genes in determining Drosophila sense organ identity. Mech. Dev. 76, 117±125. Johnson, T.K., Judd, B.H., 1979. Analysis of the cut locus of Drosophila melanogaster. Genetics 92, 485±502. Johnston, L.A., Ostrow, B.D., Jasoni, C., Blochlinger, K., 1998. The homeobox gene Cut interacts genetically with the homeotic genes Proboscipedia and Antennapedia. Genetics 149, 131±142. Kellum, R., Elgin, S.C., 1998. Chromatin boundaries: punctuating the genome. Curr. Biol. 8, R521±R524. Khanna-Gupta, A., Zibello, T., Kolla, S., Neufeld, E.J., Berliner, N., 1997. CCAAT displacement protein (CDP/cut) recognizes a silencer element within the lactoferrin gene promoter. Blood 90, 2784±2795. Kim, E.C., Lau, J.S., Rawlings, S., Lee, A.S., 1997. Positive and Negative Regulation Of the Human Thymidine Kinase Promoter Mediated By CCAAT Binding Transcription Factors Nf-Y/Cbf, Dbpa, and Cdp/Cut. Cell Growth Diff. 8, 1329±1338. Kim, J., Shen, B., Rosen, C., Dorsett, D., 1996. The DNA-binding and enhancer-blocking domains of the Drosophila suppressor of Hairywing protein. Mol. Cell. Biol. 16, 3381±3392. Ko, L.J., Engel, J.D., 1993. DNA-binding speci®cities of the GATA transcription factor family. Mol. Cell. Biol. 13, 4011±4022. Last, T.J., Birnbaum, M., van Wijnen, A.J., Stein, G.S., Stein, J.L., 1998. Repressor elements in the coding region of the human histone H4 gene interact with the transcription factor CDP/cut. Gene 221, 267±277. Lawson, N.D., Khannagupta, A., Berliner, N., 1998. Isolation and characterization of the cDNA for mouse neutrophil collagenase - demonstration of shared negative regulatory pathways for neutrophil secondary granule protein gene expression. Blood 91, 2517±2524. Lecourtois, M., Schweisguth, F., 1997. Role of suppressor of hairless in the delta-activated Notch signaling pathway. Perspect. Dev. Neurobiol. 4, 305±311. Lemaigre, F.P., Durviaux, S.M., Truong, O., Lannoy, V.J., Hsuan, J.J., Rousseau, G.G., 1996. Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single Cut domain. Proc. Natl. Acad. Sci. USA 93, 9460±9464. Lemieux, N., Zhang, X.X., Dufort, D., Nepveu, A., 1994. Assignment of the human homologue of the Drosophila Cut homeobox gene (CUTL1) to band 7q22 by ¯uorescence in situ hybridization. Genomics 24, 191± 193. Li, S., Au®ero, B., Schiltz, R.L., Walsh, M.J., 2000. Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)- associated factor and CBP. Proc. Natl. Acad. Sci. USA 97, 7166±7171. Li, S., Moy, L., Pittman, N., Shue, G., Au®ero, B., Neufeld, E.J., LeLeiko, N.S., Walsh, M.J., 1999. Transcriptional repression of the cystic ®brosis transmembrane conductance regulator gene, mediated by CCAAT displacement protein/cut homolog, is associated with histone deacetylation. J. Biol. Chem. 274, 7803±7815. Lievens, P.M.J., Donady, J.J., Tufarelli, C., Neufeld, E.J., 1995. Repressor activity of CCAAT displacement protein in HL-60 myeloid leukemia cells. J. Biol. Chem. 270, 12745±12750. Ligoxygakis, P., Bray, S.J., Apidianakis, Y., Delidakis, C., 1999. Ectopic

14

A. Nepveu / Gene 270 (2001) 1±15

expression of individual E(spl) genes has differential effects on different cell fate decisions and underscores the biphasic requirement for notch activity in wing margin establishment in Drosophila. Development 126, 2205±2214. Liu, J., Barnett, A., Neufeld, E.J., Dudley, J.P., 1999. Homeoproteins CDP and SATB1 interact: potential for tissue-speci®c regulation. Mol. Cell. Biol. 19, 4918±4926. Liu, J., Bramblett, D., Zhu, Q., Lozano, M., Kobayashi, R., Ross, S.R., Dudley, J.P., 1997. The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-speci®c gene expression. Mol. Cell. Biol. 17, 5275±5287. Liu, S., Jack, J., 1992. Regulatory interactions and role in cell type speci®cation of the Malpighian tubules by the cut, kruppel, and caudal genes of Drosophila. Dev. Biol. 150, 133±143. Liu, S., McLeod, E., Jack, J., 1991. Four distinct regulatory regions of the cut locus and their effect on cell type speci®cation in Drosophila. Genetics 127, 151±159. Ludlow, C., Choy, R., Blochlinger, K., 1996. Functional analysis of Drosophila and mammalian Cut proteins in ¯ies. Dev. Biol. 178, 149±159. Luo, W., Skalnik, D.G., 1996. CCAAT displacement protein competes with multiple transcriptional activators for binding to four sites in the proximal gp91(phox) promoter. J. Biol. Chem. 271, 18203±18210. Mailly, F., Berube, G., Harada, R., Mao, P.L., Phillips, S., Nepveu, A., 1996. The human cut homeodomain protein can repress gene expression by two distinct mechanisms: active repression and competition for binding site occupancy. Mol. Cell. Biol. 16, 5346±5357. Majumdar, A., Nagaraj, R., Banerjee, U., 1997. strawberry notch encodes a conserved nuclear protein that functions downstream of Notch and regulates gene expression along the developing wing margin of Drosophila. Genes Dev. 11, 1341±1353. Mantovani, R., Superti-Furga, G., Gilman, J., Ottolenghi, S., 1989. The deletion of the distal CCAAT box region of the A gamma-globin gene in black HPFH abolishes the binding of the erythroid speci®c protein NFE3 and of the CCAAT displacement protein. Nucleic Acids Res. 17, 6681±6691. Merika, M., Orkin, S.H., 1993. DNA-binding speci®city of GATA family transcription factors. Mol. Cell. Biol. 13, 3999±4010. Merritt, D.J., Hawken, A., Whitington, P.M., 1993. The role of the cut gene in the speci®cation of central projections by sensory axons in Drosophila. Neuron 10, 741±752. Micchelli, C.A., Rulifson, E.J., Blair, S.S., 1997. The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development 124, 1485±1495. Modolell, J., Bender, W., Meselson, M., 1983. Drosophila melanogaster mutations suppressible by the suppressor of Hairy-wing are insertions of a 7.3-kilobase mobile element. Proc. Natl. Acad. Sci. USA 80, 1678± 1682. Moon, N.S., Berube, G., Nepveu, A., 2000. CCAAT displacement activity involves Cut repeats 1 and 2, not the Cut homeodomain. J. Biol. Chem. 275, 31325±31334. Morcillo, P., Rosen, C., Baylies, M.K., Dorsett, D., 1997. Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila. Genes Dev. 11, 2729± 2740. Morcillo, P., Rosen, C., Dorsett, D., 1996. Genes regulating the remote wing margin enhancer in the Drosophila cut locus. Genetics 144, 1143±1154. Neufeld, E.J., Skalnik, D.G., Lievens, P.M., Orkin, S.H., 1992. Human CCAAT displacement protein is homologous to the Drosophila homeoprotein, cut. Nat. Genet. 1, 50±55. Neumann, C.J., Cohen, S.M., 1996. A hierarchy of cross-regulation involving Notch, wingless, vestigial and cut organizes the dorsal/ventral axis of the Drosophila wing. Development 122, 3477±3485. O'Connor, M.J., Stunkel, W., Koh, C.H., Zimmermann, H., Bernard, H.U., 2000. The differentiation-speci®c factor CDP/Cut represses transcrip-

tion and replication of human papillomaviruses through a conserved silencing element [In Process Citation]. J. Virol 74, 401±410. Ottolenghi, S., Mantovani, R., Nicolis, S., Ronchi, A., Giglioni, B., 1989. DNA sequences regulating human globin gene transcription in nondeletional hereditary persistence of fetal hemoglobin. Hemoglobin 13, 523±541. Ozisik, Y.Y., Meloni, A.M., Surti, U., Sandberg, A.A., 1993. Deletion 7q22 in uterine leiomyoma. A cytogenetic review. Cancer Genet. Cytogenet. 71, 1±6. Patient, R., Leonard, M., Brewer, A., Enver, T., Wilson, A., Walmsley, M., 1989. Activation mechanisms of the Xenopus beta globin gene. Prog. Clin. Biol. Res., 105±116. Pattison, S., Skalnik, D.G., Roman, A., 1997. CCAAT displacement protein, a regulator of differentiation-speci®c gene expression, binds a negative regulatory element within the 5 0 end of the human papillomavirus type 6 long control region. J. Virol. 71, 2013±2022. Quaggin, S.E., Vandenheuvel, G.B., Golden, K., Bodmer, R., Igarashi, P., 1996. Primary Structure. Neural-Speci®c Expression, and Chromosomal Localization Of Cux-2, a Second Murine Homeobox Gene Related to Drosophila Cut. J. Biol. Chem. 271, 22624±22634. Rollins, R.A., Morcillo, P., Dorsett, D., 1999. Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes. Genetics 152, 577±593. Scherer, S.W., Neufeld, E.J., Lievens, P.M., Orkin, S.H., Kim, J., Tsui, L.C., 1993. Regional localization of the CCAAT displacement protein gene (CUTL1) to 7q22 by analysis of somatic cell hybrids. Genomics 15, 695±696. Skalnik, D.G., Strauss, E.C., Orkin, S.H., 1991. CCAAT displacement protein as a repressor of the myelomonocytic-speci®c gp91-phox gene promoter. J. Biol. Chem. 266, 16736±16744. Stunkel, W., Huang, Z., Tan, S.H., O'Connor, M.J., Bernard, H.U., 2000. Nuclear matrix attachment regions of human papillomavirus type 16 repress or activate the E6 promoter, depending on the physical state of the viral DNA. J. Virol. 74, 2489±2501. Superti-Furga, G., Barberis, A., Schaffner, G., Busslinger, M., 1988. The 117 mutation in greek HPFH affects the binding of the three nuclear factors to the CCAAT region of the g-globulin gene. EMBO J. 7, 3099± 3107. Superti-Furga, G., Barberis, A., Schreiber, E., Busslinger, M., 1989. The protein CDP, but not CP1, Footprints on the CCAAT region of the gglobulin gene in unfractionated B-cell extracts. Biochim. Biophys. Acta 1007, 237±242. Swansbury, G.J., Lawler, S.D., Alimena, G., Arthur, D., Berger, R., Van Den Berghe, H., Bloom®eld, C.D., de la Chappelle, A., Dewald, G., Garson, O.M., Hagemeijer, A., Mitelman, F., Rowley, J.D., Sakurai, M., 1994. Long-term survival in acute myelogenous leukemia: a second follow-up of the Fourth International Workshop on Chromosomes in Leukemia. Cancer Genet. Cytogenet. 73, 1±7. Tavares, A.T., Tsukui, T., Izpisua Belmonte, J.C., 2000. Evidence that members of the Cut/Cux/CDP family may be involved in AER positioning and polarizing activity during chick limb development. Development 127, 5133±5144. Teerawatanasuk, N., Skalnik, D.G., Carr, L.G., 1999. CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene. J. Neurochem. 72, 29±39. Thomas, U., Jonsson, F., Speicher, S.A., Knust, E., 1995. Phenotypic and molecular characterization of SerD, a dominant allele of the Drosophila gene Serrate. Genetics 139, 203±213. Tufarelli, C., Fujiwara, Y., Zappulla, D.C., Neufeld, E.J., 1998. Hair defects and pup loss in mice with targeted deletion of the ®rst cut repeat domain of the Cux/CDP homeoprotein gene. Dev. Biol. 200, 69±81. Valarche, I., Tissier-Seta, J.P., Hirsch, M.R., Martinez, S., Goridis, C., Brunet, J.F., 1993. The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype. Development 119, 881± 896.

A. Nepveu / Gene 270 (2001) 1±15 Valentine, S.A., Chen, G., Shandala, T., Fernandez, J., Mische, S., Saint, R., Courey, A.J., 1998. Dorsal-mediated repression requires the formation of a multiprotein repression complex at the ventral silencer. Mol. Cell. Biol. 18, 6584±6594. van den Ent, F.M., van Wijnen, A.J., Lian, J.B., Stein, J.L., Stein, G.S., 1994. Cell cycle controlled histone H1, H3, and H4 genes share unusual arrangements of recognition motifs for HiNF-D supporting a coordinate promoter binding mechanism. J. Cell. Physiol. 159, 515±530. van Gurp, M.F., Pratap, J., Luong, M., Javed, A., Hoffmann, H., Giordano, A., Stein, J.L., Neufeld, E.J., Lian, J.B., Stein, G.S., van Wijnen, A.J., 1999. The CCAAT displacement protein/cut homeodomain protein represses osteocalcin gene transcription and forms complexes with the retinoblastoma protein-related protein p107 and cyclin A. Cancer Res. 59, 5980±5988. van Wijnen, A.J., Choi, T.K., Owen, T.A., Wright, K.L., Lian, J.B., Jaenisch, R., Stein, J.L., Stein, G.S., 1991. Involvement of the cell cycle-regulated nuclear factor HiNF-D in cell growth control of a human H4 histone gene during hepatic development in transgenic mice. Proc. Natl. Acad. Sci. USA 88, 2573±2577. van Wijnen, A.J., Cooper, C., Odgren, P., Aziz, F., De Luca, A., Shakoori, R.A., Giordano, A., Quesenberry, P.J., Lian, J.B., Stein, G.S., Stein, J.L., 1997. Cell cycle-dependent modi®cations in activities of pRbrelated tumor suppressors and proliferation-speci®c CDP/cut homeodomain factors in murine hematopoietic progenitor cells. J. Cell. Biochem. 66, 512±523. van Wijnen, A.J., van Gurp, M.F., de Ridder, M.C., Tufarelli, C., Last, T.J., Birnbaum, M., Vaughan, P.S., Giordano, A., Krek, W., Neufeld, E.J., Stein, J.L., Stein, G.S., 1996. CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F. Proc. Natl. Acad. Sci. USA 93, 11516±11521. van Wijnen, A.J., Wright, K.L., Lian, J.B., Stein, J.L., Stein, G.S., 1989. Human H4 histone gene transcription requires the proliferation-speci®c nuclear factor HiNF-D. Auxiliary roles for HiNF-C (Sp1-like) and HiNF-A (high mobility group-like). J. Biol. Chem. 264, 15034±15042. Vervoort, M., Zink, D., Pujol, N., Victoir, K., Dumont, N., Ghysen, A., Dambly-Chaudiere, C., 1995. Genetic determinants of sense organ

15

identity in Drosophila: regulatory interactions between cut and poxn. Development 121, 3111±3120. Wang, Z., Goldstein, A., Zong, R.T., Lin, D., Neufeld, E.J., Scheuermann, R.H., Tucker, P.W., 1999. Cux/CDP homeoprotein is a component of NF-muNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the bright transcription activator. Mol. Cell. Biol. 19, 284±295. Webster, M., Martin-Soudant, N., Nepveu, A., Cardiff, R.D., Muller, W., 1998. The induction of uterine leiomyomas and mammary tumors in transgenic mice expressing the Polyomavirus (PyV) large T (LT) antigen is associated with ability of PyV LT antigen to form speci®c complexes with Retinoblastoma and CUTL1 family members. Oncogene 16, 1963±1972. Wright, K.L., Dell'Orco, R.T., van Wijnen, A.J., Stein, J.L., Stein, G.S., 1992. Multiple mechanisms regulate the proliferation-speci®c histone gene transcription factor HiNF-D in normal human diploid ®broblasts. Biochemistry 31, 2812±2818. Yoon, S.O., Chikaraishi, D.M., 1994. Isolation of two E-box binding factors that interact with the rat tyrosine hydroxylase enhancer. J. Biol. Chem. 269, 18453±18462. Yunis, J.J., Lobell, M., Arnesen, M.A., Oken, M.M., Mayer, M.G., Rydell, R.E., Brunning, R.D., 1988. Re®ned chromosome study helps de®ne prognostic subgroups in most patients with primary myelodysplastic syndrome and acute myelogenous leukemia. Br. J. Haemat. 68, 189± 194. Zeng, R.W., Soucie, E., Sung Moon, N., Martin-Soudant, N., BeÂrubeÂ, G., Leduy, L., Nepveu, A., 2000. Exon/intron structure and alternative transcripts of the CUTL1 gene. Gene 241, 75±85. Zeng, W.R., Scherer, S.W., Koutsilieris, M., Huizenga, J.J., Filteau, F., Tsui, L.C., Nepveu, A., 1997. Loss of heterozygosity and reduced expression of the Cutl1 gene In uterine leiomyomas. Oncogene 14, 2355±2365. Zeng, W.R., Watson, P., Lin, J., Jothy, S., Lidereau, R., Park, M., Nepveu, A., 1999. Re®ned mapping of the region of loss of heterozygosity on the long arm of chromosome 7 in human breast cancer de®nes the location of a second tumor suppressor gene at 7q22 in the region of the CUTL1 gene. Oncogene 18, 2015±2021.