CCAAT Displacement Protein Research Articles

Overexpression of CCAAT Displacement Protein Represses the Promiscuously Active Proximal gp91phox Promoter

AbstractCCAAT displacement protein (CDP) is a transcriptional repressor that restricts expression of the gp91phox gene to mature myeloid cells. CDP interacts with multiple sites within the −450 to +12 bp human gp91phox promoter, and down-regulation of CDP DNA-binding activity is required for induction of gp91phox transcription in mature phagocytes. Truncation of the gp91phox promoter to −102 to +12 bp removes 4 CDP-binding sites and reveals a promiscuous promoter activity that is active in some nonphagocytic cells. A cis-element at −90 bp is required for derepressed transcription and serves as a binding site for multiple transcriptional activators. We now report that this element also serves as a binding site for CDP. The affinity of CDP for this element is relatively weak compared with upstream CDP-binding sites within the promoter, consistent with the promiscuous transcriptional activity exhibited by the −102 to +12 bp gp91phox promoter fragment. Further analysis of the proximal promoter reveals an additional weak-affinity CDP-binding site centered at approximately −20 bp. Overexpression of cloned CDP represses the −102 to +12 bp gp91phox promoter, indicating that these proximal CDP-binding sites are functionally significant. The constellation of transcriptional activators and a repressor that interacts with the −90 bp cis-element is identical to that observed for a promoter element at −220 bp, reflecting the highly modular organization of the gp91phoxpromoter. These studies illustrate the complex interplay between transcriptional activators and a repressor that contribute to the myeloid-restricted expression of the gp91phox gene.

Homeoproteins CDP and SATB1 interact: potential for tissue-specific regulation.

Homeoproteins are known to participate in development and cell type specification. The homeoproteins CCAAT displacement protein (CDP) and special AT-rich sequence binding protein 1 (SATB1) have been shown to bind to nuclear matrix-associated regions and to act as repressors of many cellular genes. Moreover, binding of SATB1 to the mouse mammary tumor virus (MMTV) promoter region dramatically affects the tissue-specific transcription of this retrovirus. Because protein-protein interactions are a common means of regulating homeoprotein function, we tested whether SATB1 and CDP interact in vivo and in vitro. SATB1 interacted with CDP through its DNA-binding domain, as demonstrated by glutathione S-transferase (GST) pull-down assays. GST pull-down assays also showed that CDP associated with SATB1 through three of its four DNA-binding domains (CR1, CR2, and the homeodomain). SATB1-specific antisera, but not preimmune sera, precipitated CDP from nuclear extracts, and CDP-specific antisera precipitated SATB1 from the same extracts. Far-Western blotting detected interaction of SATB1 and CDP in several different tissue extracts. Association of purified SATB1 and CDP in vitro resulted in the inability of each protein to bind to DNA in gel retardation assays. CDP overexpression in cultured T cells led to a loss of detectable SATB1 binding to the MMTV promoter region, as measured by gel shift experiments. CDP overexpression also elevated MMTV long terminal repeat reporter gene activity in transient-transfection assays, a result consistent with neutralization of the SATB1 repressor function in T cells. SATB1 is very abundant in certain tissues, particularly thymus, whereas CDP is relatively ubiquitous, except in certain terminally differentiated cell types. Because of the tissue and cell type distribution of SATB1 and CDP, we propose that the SATB1-to-CDP ratio in different tissues is a novel mechanism for homeoproteins to control gene expression and differentiation in mammals.

CCAAT displacement protein binds to and negatively regulates human papillomavirus type 6 E6, E7, and E1 promoters.

Expression of human papillomavirus genes increases as the target cell, the keratinocyte, differentiates. CCAAT displacement protein (CDP) is a cellular protein which has been shown in other cell types to negatively regulate gene expression in undifferentiated cells but not in differentiated cells. We have previously shown that a 66-bp purine-thymidine-rich sequence (the 66-mer) binds CDP and negatively regulates the human papillomavirus type 6 (HPV-6) E6 promoter (S. Pattison, D. G. Skalnik, and A. Roman, J. Virol. 71:2013-2022, 1997). Cotransfection experiments with a plasmid expressing luciferase from the HPV-6 E6, E7, or E1 regulatory region and a plasmid carrying the CDP gene indicate that CDP represses transcription from all three HPV-6 promoters. Using electrophoretic mobility shift assays (EMSAs), we have shown that CDP binds HPV-6 both upstream and downstream of the E6, E7, and E1 transcription initiation start sites. Furthermore, when keratinocytes were induced to differentiate, all three promoter activities increased. Consistent with this, immunoblotting and EMSAs revealed that endogenous nucleus CDP and, correspondingly, DNA binding activity decreased when keratinocytes were induced to differentiate. The elevated promoter activities were abrogated by exogenously transfected CDP. Our data demonstrate that CDP fulfills the requirement of a differentiation-dependent negative regulator that could tie the HPV life cycle to keratinocyte differentiation.

CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric illnesses. The mechanism of transcriptional regulation of the human TPH gene is largely unknown. We have identified a negative regulatory element located between nucleotides -310 and -220 in the human TPH (hTPH) gene. Electromobility shift analyses performed with the -310/-220 hTPH probe and nuclear extract from P815-HTR (a TPH-expressing cell line) revealed two slow migrating protein-DNA complexes, designated I and II. CCAAT displacement protein (CDP/Cut) is involved in complex I formation as shown in electromobility shift analysis, using consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut binding site not only disrupted the CDP-DNA complex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance of these protein-DNA interactions was assessed by transiently transfecting wild-type and mutant pTPH/luciferase reporter constructs into P815-HTR cells. Mutations in the core CDP/Cut site resulted in an approximately fourfold increase in relative luciferase activities. Because CDP/Cut has been shown to repress transcription of many target genes, we speculate that disruption of the CDP/Cut binding was responsible, at least in part, for the activation of hTPH gene.

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.

We have sequenced and annotated two genomic regions located in the Giemsa negative band q22 of human chromosome 7. The first region defined by the erythropoietin (EPO) locus is 228 kb in length and contains 13 genes. Whereas 3 genes (GNB2, EPO, PCOLCE) were known previously on the mRNA level, we have been able to identify 10 novel genes using a newly developed automatic annotation tool RUMMAGE-DP, which comprises >26 different programs mainly for exon prediction, homology searches, and compositional and repeat analysis. For precise annotation we have also resequenced ESTs identified to the region and assembled them to build large cDNAs. In addition, we have investigated the differential splicing of genes. Using these tools we annotated 4 of the 10 genes as a zonadhesin, a transferrin homolog, a nucleoporin-like gene, and an actin gene. Two genes showed weak similarity to an insulin-like receptor and a neuronal protein with a leucine-rich amino-terminal domain. Four predicted genes (CDS1-CDS4) CDS that have been confirmed on the mRNA level showed no similarity to known proteins and a potential function could not be assigned. The second region in 7q22 defined by the CUTL1 (CCAAT displacement protein and its splice variant) locus is 416 kb in length and contains three known genes, including PMSL12, APS, CUTL1, and a novel gene (CDS5). The CUTL1 locus, consisting of two splice variants (CDP and CASP), occupies >300 kb. Based on the G, C profile an isochore switch can be defined between the CUTL1 gene and the APS and PMSL12 genes.

Repressor elements in the coding region of the human histone H4 gene interact with the transcription factor CDP/cut.

The coding region of the human histone H4 gene FO108 undergoes dynamic changes in chromatin structure that correlate with modifications in gene expression. Such structural alterations generally reflect transcription factor interactions with gene regulatory sequences. To test for regulatory elements within the coding region, we performed transient transfection experiments in HeLa cells using constructs with histone H4 sequences fused upstream of a heterologous thymidine kinase promoter and CAT reporter gene. H4 gene sequences from -10 to +210 repressed transcription 4.8-fold. Further deletion and mutational analysis delineated three repressor elements within this region. Using oligonucleotide competition analysis and specific antibody recognition in electrophoretic mobility shift assays, as well as methylation interference and DNase I footprinting analyses, we have identified the CCAAT displacement protein (CDP/cut) as the factor that interacts with these three repressor elements. CDP/cut binding to these repressor sites is proliferation-specific and cell-cycle-regulated, increasing in mid to late S phase. Our results indicate that the proximal 200 nucleotides of the histone H4-coding region contain transcriptional regulatory elements that may contribute to cell-cycle control of histone gene expression by interacting with repressor complexes containing CDP/cut homeodomain transcription factors.

Isolation and Characterization of the cDNA for Mouse Neutrophil Collagenase: Demonstration of Shared Negative Regulatory Pathways for Neutrophil Secondary Granule Protein Gene Expression

AbstractA characteristic of normal neutrophil maturation is the induction of secondary granule protein (SGP) mRNA expression. Several leukemic human cell lines mimic normal morphologic neutrophil differentiation but fail to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase (NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to differentiate into neutrophils and induce expression of LF and NG. Therefore, to study the normal regulation and function of these genes, the corresponding murine homologs must be isolated. Using cDNA representational difference analysis (RDA) to compare a committed myeloid progenitor cell line (EPRO) with the multipotent stem cell line from which it was derived (EML), we isolated a fragment bearing homology to human neutrophil collagenase (hNC). Here, we describe the cloning and characterization of a full-length (∼2 kb) clone that exhibits nearly 65% nucleotide and 73% amino acid identity to hNC. Ribonuclease protection analysis (RPA) of the tissues and cell lines shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the mouse homolog of hNC. Furthermore, we have demonstrated a shared negative regulatory pathway for this and other SGP genes. We have previously shown that CCAAT displacement protein (CDP/cut) binds to a specific region of the LF promoter, and overexpression of CDP blocks G-CSF–induced upregulation of LF gene expression in 32D C13 cells. We show here that in these cells, upregulation of both NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.

Isolation and Characterization of the cDNA for Mouse Neutrophil Collagenase: Demonstration of Shared Negative Regulatory Pathways for Neutrophil Secondary Granule Protein Gene Expression

A characteristic of normal neutrophil maturation is the induction of secondary granule protein (SGP) mRNA expression. Several leukemic human cell lines mimic normal morphologic neutrophil differentiation but fail to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase (NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to differentiate into neutrophils and induce expression of LF and NG. Therefore, to study the normal regulation and function of these genes, the corresponding murine homologs must be isolated. Using cDNA representational difference analysis (RDA) to compare a committed myeloid progenitor cell line (EPRO) with the multipotent stem cell line from which it was derived (EML), we isolated a fragment bearing homology to human neutrophil collagenase (hNC). Here, we describe the cloning and characterization of a full-length (∼2 kb) clone that exhibits nearly 65% nucleotide and 73% amino acid identity to hNC. Ribonuclease protection analysis (RPA) of the tissues and cell lines shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the mouse homolog of hNC. Furthermore, we have demonstrated a shared negative regulatory pathway for this and other SGP genes. We have previously shown that CCAAT displacement protein (CDP/cut) binds to a specific region of the LF promoter, and overexpression of CDP blocks G-CSF–induced upregulation of LF gene expression in 32D C13 cells. We show here that in these cells, upregulation of both NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.

CCAAT Displacement Protein (CDP/cut) Recognizes a Silencer Element Within the Lactoferrin Gene Promoter

Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non–LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing basepair (bp) −387 to bp −726 of the LF promoter, but not in a −916-bp plasmid. Transfection of a −916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the −916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non–LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non–LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor–induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

CCAAT Displacement Protein (CDP/cut) Recognizes a Silencer Element Within the Lactoferrin Gene Promoter

AbstractExpression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non–LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing basepair (bp) −387 to bp −726 of the LF promoter, but not in a −916-bp plasmid. Transfection of a −916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the −916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non–LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non–LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor–induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

Phylogenetic Footprinting of Hypersensitive Site 3 of the β-Globin Locus Control Region

AbstractHypersensitive site 3 (HS3) of the β-like globin locus control region has been implicated as an important regulator of the β-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two γ genes (galago γ is embryonic; human γ is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of γ in adult life).

The regulation of human CFTR gene transcription by the CCAAT-displacement protein repressor. † 619

The regulation of human CFTR gene transcription by the CCAAT-displacement protein repressor. † 619

CCAAT displacement protein, a regulator of differentiation-specific gene expression, binds a negative regulatory element within the 5' end of the human papillomavirus type 6 long control region.

We have reported previously that a 636-bp fragment spanning the 5' two-thirds of the human papillomavirus type 6 (HPV6)-W50 long control region (LCR) functions as a transcriptional silencer (A. Farr, S. Pattison, B.-S. Youn, and A. Roman, J. Gen. Virol. 76:827-835, 1995). We have utilized nested deletion analyses to implicate a 66-bp sequence which appears to be critical for this activity. A comparison of the transcriptional regulatory activities of the LCRs of HPV6-W50 and HPV6b (which has a 94-bp deletion, resulting in the elimination of the 66-bp sequence) indicates that sequences within the 94-bp region negatively regulate the activity of the intact HPV6 LCR. Two sequence-specific DNA-protein interactions were visualized via electrophoretic mobility shift assays. One of the binding events is mediated by the transcriptional repressor CCAAT displacement protein (CDP), a factor which is active in undifferentiated cells but inactive in terminally differentiated cells. This conclusion is based on the following three lines of evidence: (i) a consensus CDP binding site oligonucleotide serves as a competitor in band shift assays, (ii) the band shift complex is not seen when a CDP-negative nuclear extract is used, and (iii) anti-CDP antiserum specifically inhibits the binding. These studies identify a DNA-protein interaction occurring within the 5' end of the LCR which may be important in maintaining the tight link between keratinocyte differentiation and HPV gene expression.

CCAAT Displacement Protein Competes with Multiple Transcriptional Activators for Binding to Four Sites in the Proximal gp91phox Promoter

CCAAT displacement protein (CDP) competes with transcriptional activating proteins for binding to each of four elements within the myeloid-specific gp91(phox) promoter. CDP exhibits the strongest affinity for a site centered at -110 base pairs (bp) of the promoter and progressively weaker affinities for three more distal binding sites. CDP binding to each site is down-regulated during terminal phagocytic differentiation, coincident with induction of gp91(phox) expression. Deletion of the high affinity CDP-binding site at -110 bp leads to inappropriate gp91(phox) promoter activity in HeLa, K562, and HEL cells. An overlapping binding site for the CCAAT box-binding factor CP1 is required for derepressed promoter activity in HeLa and K562 cells, but is dispensable in HEL cells, indicating that different cell types require distinct cis-elements for gp91(phox) promoter activity. Derepressed gp91(phox) promoter activity is further increased upon removal of a second CDP-binding site centered at -150 bp, revealing that CDP represses gp91(phox) expression via multiple cis-elements. We present a model in which restriction of gp91(phox) expression to mature myeloid cells involves competition between transcriptional activators and repressors for binding to multiple sites within the promoter.

Molecular characterization of the 7q deletion in myeloid disorders.

Deletion of the long arm of chromosome 7 is a common karyotypic finding in myeloid disorders and in particular is found in association with secondary leukaemias. We have used restriction fragment length polymorphisms and gene dosage experiments to assess the loss or retention of sequences localized to chromosome 7q in five patients with clonal myeloid disorders and a 7q deletion. The deletion was interstitial in all cases with retention of the anonymous marker pS194 located at 7q36-qter. Three out of five cases also retained the more proximal gene T-cell receptor beta (TCR beta) located at 7q35. The proximal breakpoints of all five cases were localized to 7q22 by cytogenetic analysis. In two cases the proximal breakpoint lay between the genes for elastin (ELN) and collagen type 1 alpha (COL1A2) and in three cases distal to this region between the genes for erythropoietin (EPO) and acetylcholinesterase (ACHE). The genes of ACHE, plasminogen activator inhibitor 1 (PLANH1), CCAAT displacement protein (CUTL1) and Met proto-oncogene (MET) were deleted in all cases. Molecular analysis of the 7q deletion in myeloid leukaemias demonstrates heterogeneity of the breakpoints, supporting a recessive mechanism of tumourigenesis.

Interaction of the CCAAT displacement protein with shared regulatory elements required for transcription of paired histone genes.

The H2A and H2B genes of the Xenopus xlh3 histone gene cluster are transcribed in opposite directions from initiation points located approximately 235 bp apart. The close proximity of these genes to one another suggests that their expression may be controlled by either a single bidirectional promoter or by separate promoters. Our analysis of the transcription of histone gene pairs containing deletions and site-specific mutations of intergenic DNA revealed that both promoters are distinct but that they overlap physically and share multiple regulatory elements, providing a possible basis for the coordinate regulation of their in vivo activities. Using the intergenic DNA fragment as a probe and extracts from mammalian and amphibian cells, we observed the formation of a specific complex containing the CCAAT displacement protein (CDP). The formation of the CDP-containing complex was not strictly dependent on any single element in the intergenic region but instead required the presence of at least two of the three CCAAT motifs. Interestingly, similar CDP-containing complexes were formed on the promoters from the three other histone genes. The binding of CDP to histone gene promoters may contribute to the coordination of their activities during the cell cycle and early development.

Repressor Activity of CCAAT Displacement Protein in HL-60 Myeloid Leukemia Cells

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.

Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein.

CCAAT displacement protein (CDP), a nuclear protein of 180-190 kDa, contains a triplicated motif, the cut domain, similar (80-90% conserved) to three repeats of 60-65 amino acids first identified in Drosophila cut, a homeo-domain protein involved in cell-fate decisions in development. Cut repeats bind DNA and exhibit subtle differences in target-site recognition. DNA sequences specifically bound by cut repeats were isolated by PCR-mediated DNA target-site selection. Sequences selected for cut repeat 2 and 3 (CR2 and CR3) binding are A+T-rich and favor an ATA motif with similar, but not identical, flanking base preferences. CR2 and CR3 discriminate among similar target sequences. CR1, which is more divergent from CR2 and CR3, displays the most restricted pattern of DNA sequence recognition. Methylation interference analysis demonstrates different protein-DNA contacts for CR1 and CR3 binding to a target sequence. Thus, CDP/cut is a complex protein whose DNA-binding properties reflect the combinatorial interaction of four domains (three cut repeats and one homeodomain) with target DNA sequences.

A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins.

The recently cloned 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 (mClox, for mammalian Cut-like homeo box). Although these proteins reportedly bind to apparently unrelated DNA sequences, we show by in vitro selection of optimal binding sites that both Clox and CDP have the same preferred DNA-binding specificity. The palindromic consensus target sequence, 5'-(t/a)(a/t)tATCGATTAt(t/c)(t/g)(t/a)-3', contains a bona fide homeo domain binding motif (ATTA). In addition, 37% of the in vitro-selected sequences have a CCAAT box, the canonical target for members of the family of CCAAT-binding factors. 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. We present evidence that the cut repeat II binds to mClox consensus targets independently of the DNA-binding activity of the homeo domain. 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 homeo domain, an ATTA core. These results indicate that the DNA-binding activity of the second cut repeat can account for the suggested role of CDP mClox as CCAAT displacement protein, a putative repressor of gene expression. We also report that the mClox 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. No cooperativity was observed between the cut repeat II and the homeo domains of Oct-1 and Gtx. Furthermore, the Drosophila cut repeat II, which does not appear to bind to DNA, also enhances the DNA-binding activity of the mClox homeo domain. Thus, the bifunctional cut repeat II, which defines a new family of bipartite DNA-binding proteins, is likely to play an important role in the function of the cut homeo proteins.

Regional Localization of the CCAAT Displacement Protein Gene (CUTL1) to 7q22 by Analysis of Somatic Cell Hybrids

CCAAT displacement protein (CDP) is a putative repressor of tissue-specific gene expression, implicated in regulation of expression of the human myeloid-specific gene, gp91-phox. The cDNA sequence of the human gene predicts a polypeptide of 1506 amino acids, with a homeodomain and three repeated motifs closely homologous to cut, a protein known to specify the cell fate of peripheral neurons and other tissues during embryonic development of Drosophila. Therefore, the human gene locus for CDP has been named CUTL1, for cut-like 1. Preliminary data revealed that CUTL1 mapped to human chromosome 7 (E.J.N., P.M.-J.L., and S.H.O., unpublished results). Since diseases with abnormalities of myeloid differentitation often have aberrations of chromosome 7, especially in patients previously exposed to mutagenic agents or radiation therapy, the cell-type specificity of CDP makes it an attractive candidate for involvement in these conditions. As a first step in addressing this question, we have examined the regional localization of CUTL1 using a panel of rodent-human somatic cell hybrids containing various portions of chromosome 7. The results of our hybridization analysis are summarized. The regional localization of CUTL1 to 7q22 was based primarily on the negative hybridization signal with the human/hamster hybrid 1EF2/3/K017 (7cen-q21) and the positive result withmore » 1CF2/5/K016 (7cen-q22). Since no hybridization signal was detected in GM1059Rag5 (a human-mouse hybrid carrying a single human chromosome 7 with an interstitial deletion 7pter-q22:q32-qter) and 2068Rag22-2 (7qter-q22), the regional assignment of CUTL1 could be narrowed to the distal boundary of 7q22.« less